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## Meet the editors

For the past 35 years, Prof. Sadık Dincer has been involved in teaching, research, and academic work in numerous distinguished universities in Turkey. Currently, he is working at the Biology and Biotechnology Departments, Cukurova University, Adana, Turkey. His manuscripts and book chapters have been published in national and international journals and his works has been cited 1167 times. To date, he has trained twenty-six

MSc and eleven Ph.D. students. He received the Technology Development Award from the Scientific and Technological Research Council of Turkey (TÜBİTAK) in 2013 and a national study patent in 2019. His research is focused on bacteriology, microbial ecology, industrial biotechnology, and microbial genetics.

Associate Prof. Dr. Hatice Aysun Mercimek Takci received her MSc and Ph.D. in Biotechnology and Biology from Cukurova University, Adana, Turkey in 2007 and 2011, respectively. Since 2009, she has worked at Kilis 7 Aralik University, Kilis, Turkey. Her teaching interests include microbiology, biotechnology, enzymology, microbial genetics, and bacteriology. She has published forty-six manuscripts in national and international

journals and her works have been cited 243 times. Her research interests focus on multiple antibiotic and heavy metal resistance in bacteria, production and characterization of bacterial enzymes, bioremediation by bacteria, and aquatic bacterial contamination. Related to these research areas, she has seventeen projects supported by the Scientific and Technological Research Council of Turkey (TÜBİ-TAK) and is the coordinator of scientific research projects.

Associate Prof. Dr. Melis Sumengen Ozdenefe received her BSc, MSc, and Ph.D. in Biology from Cukurova University, Adana, Turkey in 2009, 2011, and 2014, respectively. During her MSc, she was an international exchange student and a researcher at Anhalt University, Germany for six months. She has been working in the Department of Biomedical Engineering, Near East University, Northern Cyprus since 2014. Her teaching interests

include industrial microbiology, bacteriology, biotechnology, enzymology, and environmental microbiology. Her research areas involve enzymes and biosurfactants, which are produced from various bacteria and fungus, for industrial applications, the production and characterization of bacterial enzymes and bacteriocins, the antimicrobial and antioxidant activity of various plant structures, multiple antibiotic resistance, and heavy metal resistance in Gram-negative bacteria. Dr. Ozdenefe's works have been published in national and international journals, conferences, congresses, and symposiums and cited 140 times.

### Contents


## Preface

Molecular cloning refers to the creation of recombinant DNA molecules. With the discovery of restriction and ligase enzymes, recombinant DNA technology emerged in the 1970s and spurred great advances in molecular biology, particularly in DNA manipulation. This technology, which enables detailed molecular studies to understand the structure and functions of genes, allows researchers to isolate large DNA molecules from different origins, cutting, pasting, reproducing, assembling, recognizing, changing their structure, and recovering them to living organisms. The cloning of genes, which has become a standard laboratory technique, has significantly increased the understanding of gene function in recent years. This book discusses the fundamentals of molecular cloning with chapters on tools for molecular cloning, molecular cloning for medicine, molecular cloning for food and feed, molecular cloning for the environment, molecular cloning methods, and the future of molecular cloning. It is an intended useful resource for graduate and postgraduate students as well as researchers and industry experts in the domains of biotechnology, ecology, enzymology, food engineering, medicine, microbiology, and molecular biology.

> **Sadık Dincer** Cukurova University, Adana, Turkey

### **Hatice Aysun Mercimek Takci**

Kilis 7 Aralik University, Kilis, Turkey

#### **Melis Sumengen Ozdenefe**

Near East University, Nicosia, Turkish Republic of Northern Cyprus

### Section 1

## Genetic Transformation - Prokaryotic and Eukaryotic Cells

#### **Chapter 1**

## High Throughput Methods to Transfer DNA in Cells and Perspectives

*Colin Béatrice and Couturier Cyril*

#### **Abstract**

Genome sequencing led to thousands of genes to study and their molecular cloning to provide ORF collection plasmids. The main approach to study their function involves analysis of the biological consequences of their expression or knockdown, in a cellular context. Given that, the starting point of such experiments is the delivery of the exogenous material, including plasmid DNA in cells. During the last decades, efforts were made to develop efficient methods and protocols to achieve this goal. The present chapter will **first give a rapid overview of the main DNA transfer methods** described so far: physical, chemical, and biological. Secondly, it will focus **on the different methods having reached high-throughput nowadays**. Finally, it will discuss **the perspectives of this field in terms of future enhancements**.

**Keywords:** cell nanoconstriction, cell-penetrating peptide, DNA, electroporation, high-throughput transfection, lipofection, microfluidic, nano-acoustic dispensing, nucleofection, viral transduction

#### **1. Introduction**

The most used approach to decipher proteins' function or their interactome is to study the effects induced by the delivery of exogenous materials in living cells (deoxyribonucleic acid: DNA, ribonucleic acid: RNA, oligonucleotides, proteins, and ribonucleoproteins). Coding sequence overexpression, then gene silencing, and genome editing approaches offer a panel of induced biological modifications within cells that allowed us to increase our knowledge of most cellular processes. However, in a post-genome era, thousands of genes must be studied and exogenous material transfer into cells, including DNA, became a limiting factor. Indeed, available technologies predominantly allowed analysis at a gene-by-gene scale, and new approaches were developed to reach higher throughput. Libraries of material such as small interfering RNA (siRNA) [1, 2] and Open Reading Frame (ORF) expressing plasmids collection were developed [3, 4] to cover all proteome. To take advantage of these, concomitant High-Throughput (HT) technologies are pointed out for their transfer in cells. Plasmid DNA (pDNA) transfer in cells (by transfection or transduction) plays a central role when studying the precise biological role of proteins. For pDNAs, several efficient transfection methods were pushed to higher throughput. All these induced

changes performed in cells allow not only our understanding on the biological processes of cells' life but also have therapeutic applications [5, 6]. The huge interest in gene and cellular therapy approaches is indeed a motor in the development of highly efficient gene delivery strategies.

In this chapter, we will first give a brief overview of DNA transfer methods in cells, then a more detailed part will focus on those that reached higher throughputs and we will conclude with future expected enhancements.

#### **2. The different methods to transfer DNA in live cells**

To promote a biological effect in the cell, exogenous DNA must face several levels of pitfalls starting from the outside of the cell. First, it must cross the plasma membrane composed of a hydrophobic lipid bilayer which naturally prevents hydrophilic material such as DNA from entering the cells. In addition, DNA and the plasma membrane carry a general negative charge that impedes DNA transfer into cells by electromagnetic repulsion. Furthermore, once entered, the DNA has to face degradation mechanisms that occur in the cells. Finally, if part of the exogenous DNA succeeds in passing all these steps, the expected biological effect would be measurable. To circumvent all these, a range of approaches to transfer DNA has been developed. DNA delivery to cells can be divided into three main categories: physical, chemical, and biological methods. Among all approaches, viral ones are the most efficient but present some limitations such as the transgene size and biosafety issues. Physical and chemical methods were developed to circumvent these limitations and are not limited in the size and number of genes to be transferred. Some of these are still largely used whereas some were more a proof of concept. In this section, we briefly describe the physical, chemical, and biological methods.

#### **2.1 Physical methods**

As mentioned above, due to the plasma membrane and DNA respective properties, the transfer of DNA into cells is impaired. All the physical methods aim to directly circumvent the hydrophobic and electrochemical repulsion parameters by disrupting the integrity of the membrane and promoting a transient permeability. Physical methods then do not have a limit in cargo size and do not depend on biological mechanisms as a direct material delivery is performed [7].

An evident method is the direct microinjection of DNA in cells, which was performed in early-stage embryo [8], and then human cell lines [9]. It implies micromanipulation of a single cell under a microscope to bypass the membrane barrier using a thin glass needle to inject DNA directly into its cytosol or compartments [8, 10]. This approach is reproducible but tedious due to the need to inject each cell individually.

Electroporation method has emerged when it was shown that an electric field could promote a loss of membrane permeability by transient pore formation [11] thus allowing DNA delivery in cells [12]. DNA, target cells, and electroporation buffer laying between two electrodes are submitted to an electric pulse [13]. This pulse is divided into a high-voltage stage to create temporary pores, and a low-voltage one to allow electrophoresis of DNA through these pores [14]. Extensive optimizations (pulse voltage and duration, buffer composition) were done to balance transfection efficiency with cell viability as the requested high voltage promotes cell death [15, 16].

#### *High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

"Electroporators" devices are nowadays available with many predefined settings to achieve efficient transfection in almost all cell types, even hard-to-transfect ones [17].

Biolistic or micro-projectiles bombarded to the cells represent another delivery mode. The projectiles made of gold-covered by nucleic acids, penetrate into cells by high-speed bombardment [18]. First developed for plants, this approach is also efficient in mammalian cells/tissues [19]. However, the method suffers the cost of particles. Nanoparticles that can bind nucleic acids, and whose small size allows them to pass cell membranes with high efficiency, represent a cheaper alternative [20].

Femtosecond laser optoporation consists in focusing ultrashort laser pulses on a cell membrane to induce a transient perforation. This membrane perturbation allows the pDNA transfer [21]. Many cell types can be transfected using a variety of laser sources [22, 23]. Despite efficient, due to the needed laser focusing on a single cell level, its throughput is limited.

Acoustoporation or sonoporation uses ultrasounds to induce a transient plasma membrane disruption promoted by bubbles cavitation phenomenon and thus allowing gene transfer [24, 25]. The method was enhanced by the use of high-frequency waves creating reversible nanopores and furthermore promoting "molecular bombardment" on the bilayer membranes that enhances DNA delivery while limiting cell mortality [26].

Passing constriction or nano-constriction is an approach based on the mechanical deformation of cells as they pass through micro constrictions channels [27]. This controlled compression induces transient pores formation into the cell membrane and allows DNA entry from the surrounding buffer [27]. This method is expected to be universal and showed efficiency in easy and hard-to-transfect cell lines like primary and stem cells [28].

The last method, magnetofection, has been classified as a physical method. It is based on magnetic nanoparticles (MNPs) coated with transfection reagents that bind nucleic acids and promote cell entry [29]. Indeed, MNP only induces the concentration of the MNP on the cells mat when a magnetic force is applied but is per se not able to transfer DNA into cells. However, it enhances DNA delivery up to several hundred and allows to lower DNA consummation, and is furthermore efficient in hard-to-transfect cells [30, 31].

#### **2.2 Chemical methods**

Interest to develop non-viral and reproducible gene delivery methods has led to the use of chemical reagents. Chemical transfection methods represent an alternative way to bypass the membrane barrier and furthermore try to protect DNA from degradation within cells [32]. These reagents promote DNA compaction, negative charge neutralization, and cell interaction for later entry into cells. These reagents are briefly summarized here after.

Calcium phosphate co-precipitation is the cheapest method and was first described in 1973 [33]. It relies on the formation of a precipitate when the negatively charged DNA binds to calcium ions (Ca2+) [34]. This precipitate interacts with the plasma membrane and enters the cell by endocytosis [35]. This widely used method reaches up to 90% efficiency for easy-to-transfect cells but is impaired by the need for fresh preparation, avoiding any storage of ready-to-transfect plates [36]. The formation of an efficient precipitate depends on several parameters and this method can be toxic for certain cells such as primary ones [37]. Calcium was also shown to enhance gene delivery by other methods [38] and was then tested alone as a transfection

reagent (calfection) [39]. The mechanism does not rely on the formation of a precipitate and do not need fresh preparation. Furthermore, the Ca/DNA mixture can be stored for a long period without any loss in efficacy. Intended for batch transfection of the high number of cells, it worked in a 12-wells plate format for adherent or non-adherent cell lines. The easy use, storage ability, and low cost make this method interesting whereas it was not tested so far in higher throughput.

The diethylaminoethyl-dextran (DEAE-dextran) is another reagent that showed efficiency [40]. This polycationic derivate of dextran compacts DNA to form a positively charged complex that later interacts with the plasma membrane to enter cells by endocytosis [41]. The method is simple, low cost, and efficient for many cell types however, new enhanced approaches surpassed it.

Lipofection method is based on the use of lipids and cationic lipids [42, 43]. When mixed with DNA solution, these lipids form liposomes, a kind of vesicular structure with the same composition as cellular membranes and entraps DNA in solution [44]. The formed complexes (lipoplexes) allow DNA delivery through binding to the cell membrane (due to electrostatic forces), cell entry, mainly by endocytosis [45], and release of the DNA for expression. Lipids-based transfection reagents are efficient and mostly insensitive to serum so that medium has not to be removed before transfection. Furthermore, lipofection can be used efficiently in forward or reverse mode transfection in numerous cell lines [46]. Cationic lipids are more and more efficient in DNA delivery, and furthermore efficient on suspension or adherent cells, and for increasing number of cell types, and even hard-to-transfect ones [47].

Cationic polymers are non-lipidic as deprived of a hydrophobic moiety and are then soluble in water. They use a similar mechanism: being positively charged, they interact and compact DNA under the form of polyplexes [48]. They enter the cell by endocytosis, and traffic through endosomes and cytoplasm to finally deliver DNA to the nucleus [49]. This class of reagent has the advantage to limits DNA degradation in lysosomal compartments, increasing delivery efficiency [50].

#### **2.3 Biological methods**

Biological approaches to transfer DNA are inspired by natural mechanisms. The most potent of these approaches is gene transfer by viruses. Other methods represent fields in expansion: cell-penetrating peptides or the use of exosomes or vesicular transfer. These approaches do not rely on natural products but on diverted forms to allow the transfer of a gene of interest.

Viral approaches are the highest efficient among all, even in hard-to-transfect cells [51]. To be permissive, the cells must express the receptor interacting with the virus envelope proteins. To enter in almost all cell types, a ubiquitous and widely expressed receptor is preferred. The Vesicular Stomatitis Virus G (VSV-G) protein promotes entry in almost all cell types as the Low-Density Lipoprotein Receptor family is its ubiquitously expressed receptor [52]. Its interaction with the VSV-G protein promotes membrane fusion and allows virus content to be delivered to the cells [53]. The use of a viral vector is however limited in throughput as viral particles have to be produced for each different DNA to transfer. This production involves the cloning of the gene of interest in a viral vector backbone that is later transfected into a packaging cell line to be integrated into pseudo-viral particles. Pseudo-virus are then recovered from the cell's supernatant, concentrated, and titrated before their use for transduction of the target cells. Despite lower throughputs, viral delivery remains the most powerful way to transfer DNA in cells, even in primary cells (90% efficiency).

#### *High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

The fusiogenic envelope G glycoprotein of the VSV-G was also used as a reagent for gene transfer when mixed with plasmid DNA [54]. The resultant product termed "Gesicles" showed 55% transfection efficiency in HeLa cells, and 22% for hard-totransfect human myoblast cells [55]. Whereas promising, this method did not reach HT yet.

Another interesting biological derivative used for DNA transfer is represented by proteins having natural properties to enter the cells by surface receptors dependent [56] or independent mechanisms [57]. Some natural peptides derived from these proteins, the cell-penetrating peptide (CPP) are able to enter the cell through the membrane [58, 59]. These peptides have short lengths and a global positive charge. Involved mechanisms are still unclear and depend on the CPP (direct penetration, endocytosis, or translocation via intermediate structure in the membrane lipid bilayer). Peptide from the Trans-Activator of Transcription (TAT) protein of the Human Immunodeficiency Virus (HIV) was efficiently used as a DNA carrier in HeLa cells [60]. Some others have been modified and their properties mixed with each other to promote efficient delivery of exogenous nucleic acid into cells [61]. CPP can be engineered by multiplexing peptides with distinct properties or by modifying their composition. One of the engineered CPP is the pepFect14 [62] which showed efficiency for DNA delivery in several cell types such as CHO, HEK293, U2OS, or U87 cells [63].

One last example of naturally occurring biological derivatives is the use of exosomes. First described in 1977, these nano-sized vesicles derived from plasma membrane elements, are involved in mediating messages to proximal and distant cells [64, 65]. This natural process is found in normal or pathological cells [66] and can be turned around to deliver DNA of interest [67].

#### **3. High-throughput batch DNA transfection**

To enhance the throughput of the experiments performed on cells transfected by exogenous DNA, it is interesting to do it in a HT way. However, a distinction must be done between experiments performed at HT using transfected cells, and HT transfection of cells. Indeed, depending on assay requirements, transfection of a single condition may be performed using a large volume of suspended cells that are then distributed among several individual wells for subsequent treatments and assays [68]. Alternatively, it is interesting to transfect many different plasmids, each well of transfected cells expressing different transgenes [69, 70]. This difference is generally concomitant with the way the transfection is performed: batch protocol or not.

Batch protocol allows to transfect a large number of cells that are then dispatched in separate wells for further experiments. In this case, all transfected cells in the batch share the same conditions of transfection. This protocol is generally used to limit variability in HT assays for monitoring the effect on a biological parameter under a unique transfection condition. Typically, it can be performed on adherent cells in a forward-protocol modus: cells are plated and transfected 24 h later, according to the transfection reagent's manufacturer instructions. The day or several hours after transfection, adherent cells are suspended and dispatched in multi-well plates (96, 384, or even 1536) for further HT treatments and analysis [71]. Depending on the cells used, the batch transfection is also compatible with the suspended cells that are then directly dispatched on separate wells after transfection.

The batch protocol is not per se a HT transfer of different biological materials in cells, but rather a way to perform HT assays and treatments in separate wells. On the opposite, HT protocols can achieve a true HT transfection in which each well receive a different DNA or transfection conditions.

### **4. High-throughput transfection protocols**

To be able to determine the behavior of cells or biological effects induced by the transfer of many different pDNAs in the cells, a real HT transfection becomes interesting. Several methods allow the management of numerous pDNA or different conditions when transfecting the cells, but to reach HT, good efficiencies are almost necessary. HT transfection can be achieved using several methods that are presented below.

#### **4.1 High-throughput transfection using physical approaches**

#### *4.1.1 High-throughput electroporation-based transfection*

As described before, electroporation is performed with buffer diluted cells and DNA, subjected to an electrical pulse that promotes membrane destabilization. Many devices, protocols, and dedicated buffers have been implemented to reach universal use. However, it seemed incompatible for HT as each separate transfection must be performed one by one in micro cuvettes. This problem has been solved by the development of new devices able to deliver an electrical pulse simultaneously in several wells on dedicated plates. Harvard Apparatus/BTX developed an up to 96 wells approach using plates with embedded aluminum electrodes. Used with the plate handler Model HT-200, it allows transfection in 8 wells simultaneously and was shown efficient in neurons [72].

Another approach based on an array of 96 suspended electrode pairs fitting on top of standard 96-well plates represents a less expensive approach [73]. Each pair of electrodes can be loaded and held 10–20 μL of transfection mixture by the surface tension. After pulse delivery, the direct addition of the cell culture medium into the array allows the electroporated cells to drop and seed into the underlying microplate. The array is reusable, and uses standard microplates and inexpensive standard buffers, reducing the cost of this approach. In addition, common liquid handling robots can achieve a 96-well transfection time of approximately 1 min. This technology could be adapted to the 384-well plate format using a more sophisticated electrode array design and concomitant robotics.

Whereas successful in almost all cell types, the electroporation method has some limitations. First, it is the most DNA-consuming one of all the HT transfection approaches described so far. Secondly, as a cell suspension is required during the electrical pulse delivery, it avoids its use on adherent differentiated cells mat. Nevertheless, its advantage in terms of success in almost all cell types, and its versatility concerning the material to transfer (not only efficient for DNA) promises electroporation to further future enhancements and use.

#### *4.1.2 High-throughput nucleofection-based transfection*

In 2001, Amaxa™, (now owned by Lonza™) launched an electroporation-derived method termed nucleofection as DNA is transferred directly into the nuclei of the

#### *High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

cells. It shortens the time of experimentations, by suppressing the necessary nuclear import step of DNA and ensure proper expression of transgene [74]. It relies on an electroporation-based device (Nucleofector) and the use of dedicated buffer solutions to ensure nuclear transfer. The exact mechanism allowing nucleus targeting and buffer composition is kept proprietary. However, since the first published results on natural killer cells transfection [75], it has been widely used in many hard-to-transfect cells with efficiency ranging from 25 to 70% [76]. First, nucleofector devices used nucleocuvettes and were then limited in throughput. New apparatus and dedicated consumables were developed to reach higher throughput: the 96-well Shuttle® device (amaxa AG), in which cells are plated on 96 wells "nucleocuvette plates" and pulsed using Nucleofector™ programs. These plates, made of conductive polymers, allow the current delivery in each well individually. It takes less than 10 min in an automated way to process the entire plate [77]. Many optimized conditions have already been defined using nucleocuvettes depending on cell types (programs for the electrical pulses, cells number, and optimal buffer conditions) and as an advantage, these settings are transposable to nucleocuvette plates [17]. Numerous successful examples have been published ranging from 35 to 70% efficiency: primary chondrocytes [78]; dendritic cells [79], and even H9 hESC [80].

To push further the throughput a 384-well Nucleofector™ requiring 384-well Nucleocuvette™ plates was launched. The complete electrical pulse delivery process takes just one minute, and several wells are processed at the same time [81]. However, the overall process is the same as for the previous model, mixing of cells with buffer and DNA in the wells, delivery of the electric pulse, the addition of fresh medium in the 384-well Nucleocuvette™ for cell recovery, and then their dispense in a cell culture plate for later experimentations.

Whereas versatile and being efficient as electroporation in many cell types, nucleofection is still restricted to suspended cells, impairing its use on morphologically differentiated and adherent cells. Furthermore, the need to transfer transfected cells to a standard plate for further experimentation is a limiting step of the method. The cost of such approaches broadens their wide use in the scientific community due to the price of transfections kits, containing ready-to-use buffers, and nucleocuvette plates.

#### *4.1.3 High-throughput adherent cells electroporation-based transfection*

As explained above, electroporation or nucleofection are restricted to cell suspensions. To circumvent this limitation, new kind of electrodes able to deliver the electric pulse on a cell mat was developed.

One of the simplest developed approaches is an electrode device that takes place on top of standard culture cell dishes. The PetriPulser™ (from BTX) consists of 13 gold plated electrodes embedded in an isolating holder placed above the Petri dish containing the cell mat to electroporate [82]. This model fits 35 mm Petri dishes but a scaledup model, the "Petri dish electrode" made of stainless steel electrodes, fit 100 mm diameter dishes [83]. The 2 mm distance between electrodes is the same as in most cuvettes. A model for transwell cultured cells electroporation: the BTX™ Adherent Cell Electrodes [84] presents a 5 mm distance inter electrodes and may engender adverse effects on cell viability. All these devices are reusable, lowering the cost of this approach that has however not been used so far in published works.

A sophisticated version was launched by Cellectricon™: the Cellaxess®HT. It uses dedicated 384-wells microplates and a capillary embedded microelectrodes array.

Using a platform device, adherent cells seeded in 384-wells plates are washed, electroporated using transfection mixture (loaded from side donor plates), and allowed to recover with fresh medium addition. 96 wells are simultaneously electroporated by the device and throughput of 50,000 wells per day is announced by the manufacturer [85]. However, it was not really used in the academic laboratories as no work has been published except the proof of concept of the manufacturer. They simplified the method by launching the Cellaxes Elektra-Adherent Cell Electroporation System. It is also an electrode-based electroporation system optimized for the *in-situ* transfection of all adherent cell types, which offers superior efficiency and cell viability due to minimal cell processing and the low voltages enabled by the use of capillary electrodes laying above the cell mat. It uses 384-wells plates and delivers the electrical pulses in 96 wells simultaneously thus allowing the rapid management of the entire plate. However, whereas fully automated, the protocol is not homogeneous: some medium must be discarded from the wells to add DNA diluted in the electroporation buffer (Cellaxess Elektra Accelerator Solution). Once the pulse delivered, some fresh medium is added to the wells before returning the plate to the incubator. Such inhomogeneous protocol would render reproducibility harder to achieve. Cellaxess Elektra transfection system allows rapid optimization of the protocol as different pulse protocols can be applied in a single 384-well plate. This approach has not been widely used yet, probably due to the cost of consumables and devices, but was able to transfect primary neurons with an efficiency of up to 50% [86].

#### *4.1.4 High-throughput electroporation-based microarray in situ transfection*

Array approaches are based on spotting an array of transfection reagents and material to transfer on a planar slide where cells are later plated. Using such approaches with electroporation method was unimaginable. However, several teams pushed down this restriction by developing custom-made devices to electroporate adherent cells in a microarray manner. Two technologies are suitable for adherents cells: the delivery of the electrical pulse between the bottom and top of chip microwells; or between interlaced microelectrodes laying on the bottom of the dishes under the seeded cells [87, 88].

In the HT *in situ* cell electroporation (HiCEP) method, a microarray electroporation chip composed of 13 × 13 microelectrodes have been developed [89]. The electrodes lay under the cell cultured in a superhydrophobic microwell array chip (SMAR-chip) developed for this purpose. The electrical pulse is delivered simultaneously in the 169 wells, using for each, ten interdigital electrodes covering a 500-μm-diameter area [90]. The approach requires a dedicated platform to assemble the chip before covering it with the cells solution in a Petri dish. Before delivering the electrical pulse, the medium is removed, allowing 24 nL medium nanodrops to stay in each well of the hydrophobic matrix chip. Electroporation buffer is added and rests as nanodrop in each well after aspiration. The material to transfer is deposited by a standard microarrayer, on the top cover slide. Once reversed and placed on top of the wells using a micromanipulator under the microscope, the drops mix with the underlying buffer before electrical pulse delivery. Whereas this method is successful and promising in terms of throughput, it is not affordable for non-specialists, as many skills and specialized materials are required.

Another method was able to electroporate adherent cells, based on a glass gold electrode coated with PEI for pDNA loading [91]. Cells are plated on this electrode and the electrical pulse can be delivered using an additional top cover electrode up to 3 days post-seeding. Transfection efficiency reached 90% in HEK but was also efficient in primary fibroblasts. Although electroporation was performed in 13 mm square areas, this method allowed HT transfection using up to 169 plasmids microarrayed on the electrode. This method seems affordable, as it only requires a gold vaporized electrode.

#### *4.1.5 High-throughput electroporation-based microfluidics transfection*

Whereas it remains a field of specialists, microfluidic applications increased in the last decade due to their low-cost advantage, as it can be in-house designed using affordable technologies, and it deals with low quantities of reagents. Microfluidic can manipulate different solutions and mix them, and lead to cell culture and transfection chips design [92]. However, in-house designs might be difficult to reproduce, even more, if highly specialized skills are required. Furthermore, most biological experiments require a subsequent amount of transfected cells, harder to achieve using microfluidic. Despite these limitations, success in microfluidic transfection applications has been published for a wide variety of cells, and even at the single-cell level [93, 94]. First devices lacked the necessary throughput to test numerous transfections conditions in parallel, but recent advances pushed it further. In the field of transfection, two main approaches have been used with microfluidics: electroporation and nano-constriction.

Electroporation in standard 2 mm cuvettes requires high voltage that promotes cell death by a joule heating effect, a local pH change due to water electrolysis, that in turn induces the formation of bubbles promoting cells aggregation and impairing the DNA delivery efficiency [95]. Due to its efficiency, electroporation was used in microfluidic derivatives trying to circumvent some of its limitations. Embedding electrodes in a microfluidic channel can limit adverse effects on cell viability [92]. The diameter of the channel allows the electrodes to be closer to each other's and the use of voltages as low as 1 volt [96], reduces the heating joule effect, electrolysis, and bubbles. pH modifications are still present but enhanced buffer composition improved it [97]. These microfluidics devices mostly use flowing cells transfected in a semi-continuous way [98], avoiding testing many different conditions in parallel and lowering throughput. Some devices allow transfection of adherent cells in micro-chambers using a porous substrate on which cells are seeded. The electric field is then applied through the cells (under/upper compartment). This has been successfully performed on stem cells differentiated in neurons [99]. Despite the latest improvements, microfluidic-based approaches still lack HT. However, due to the booming application of microfluidic, reaching higher throughput would be achievable and a promising way to perform transfection.

#### **4.2 High-throughput transfection using chemical reagents**

Most of the chemical transfection reagent allows two kinds of protocols: the forward and the reverse protocol. In forward protocols, DNA and transfection reagent are mixed to form transfection complexes and then distributed on previously seeded cells. Such an approach is harder to manage in a HT way as each different mixture condition implies a different container (tube or wells of multiplate wells) and necessary tedious pipetting steps. However, this kind of protocol can be manually achievable with standard molecular biology material such as multichannel micro-pipettors. An experimented user can transfect one to four 96 well plate manually in 2 h with

up to 3 different pDNAs per condition [100, 101]. However, to our knowledge, the forward approach has not been automated so far to reach HT.

The forward mode has been surpassed by the reverse protocol mode. The DNA (eventually with the transfection reagent) is directly dispatched on the final wells (i.e., of a multi-well plate) or a glass slide, and cells are added directly on these deposits. This mode of transfection has several advantages: first it shortens the overall experimental time, second, it can easily be automated allowing to reach HT and good reproducibility. Suitable for such an application, liquid handling devices enable the dispense of low liquid volumes for the multiplexing of different solutions whose concentration and ratio are tightly controlled in each well. Such protocols have been developed for most of the biological material to transfer which includes DNA and follow the technological developments available to do it. An overview of these methods used for DNA transfection in a HT manner is detailed below.

#### *4.2.1 Chemical-based high-throughput transfection*

As mentioned before, lipidic transfection reagents are eligible to reverse protocol, making them suitable for potential HT approaches. This reverse mode was shown efficient on CHO cells grown in suspension in a 96 wells-plate format using PerFect Lipids (pFx-6 form lnvitrogen) as reagent [100] and even adherent cells using Lipofectamine (Invitrogen). Higher Throughput was reached using Turbofectin8 as reagent (Origene) and plasmids coding 704 different transcription factors dispensed in 384-wells plates [102].

The SMAR-chip described before in the HiCEP method [89], was also applied to HT reverse transfection but using Lipofectamine 2000 as a transfection way instead of electroporation. It allowed the efficient transfection of HEK293 (up to 65% transfected cells) in the 169 wells of the matrix. The authors aimed at producing viral particles using co-transfection of the necessary plasmids with 169 genes of interest. Proper viral packaging and sufficient viral production were shown by successful transduction of side cultured 3T3L1 cells using the supernant of the HEK producing cells.

Tavernier's group reached a much higher throughput in 2002 using reverse transfection for HT transfection of HEK293 cells in its MAmmalian Protein–Protein Interaction Trap (MAPPIT) Arrays approaches to study protein–protein interactions [103]. Effecten reagent was used in a reverse mode protocol to transfect prey expressing plasmids in up to 384-wells plate format using classical liquid handling facilities and a mammalian ORF collection plasmids.

With the emergence of such collection, examples of microplate-based arrays of the huge collection of plasmids have grown. One of the highest throughput was reached using 6049 different human cDNA expression plasmids to study their effect on the promoter activation of the zinc-finger protein RP58, using a luciferase reporter gene [104]. 50 ng plasmid/wells were loaded on sets of 384-well plates and a HT reverse transfection of HEK293 was successfully performed using Lipofectamine 2000.

A HT transfection protocol was reached in 384-wells plates format using nonliposomal polymers (Mirus TransitX2) as transfection reagent [101]. A reverse protocol led to about 90% transfection efficiency (even in cotransfection assay). The originality of this work is the use of a tips-free acoustic delivery of reagent and DNA (Echo nanodispencer from Labcyte™). This device sends multiple droplets of 2.5 nL from a 384-wells source plate to a destination one up to 1536-wells plates. Starting from unique diluted plasmids solutions, the overall process takes less than 20 min

#### *High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

for one plate, and transfection ready plates can be stored dry or frozen without loss of efficiency. Cells are seeding on dry or freshly dispensed plates in a reverse mode transfection. The optimized protocol would allow 20,000 human genes transfection in about 18 h on a dedicated automated platform. Nano-quantities of DNA and reagents should render this approach low cost if the nanoaccoustic dispenser was not such expensive. Nevertheless, this protocol renders transfection affordable for newbies as the tedious work of DNAs and reagents combining in each well is controlled by spreadsheet driven software [105].

#### *4.2.2 Chemical-based microarray transfection*

In 2001, DNA transfection throughput was pushed further by the use of a microarayer for the generation of transfection ready arrays of DNA [106]. In this study, 140 different plasmids DNA/gelatin mixture were deposited on glass microscope slides as 1 nL spots (of about 150 μm diameter). Effecten, a lipid transfection reagent was used to transfect cells seeded on the overall slide. Each spot led to the transfection of 30–80 HEK cells, in a DNA dose-dependent manner from 10 to 50 pg. Storage of the dried glass slides for more than 3 months did not affect transfection efficiency, allowing the matrix to be prepared in advance of use. Since this princeps study, other groups have successfully used this approach. Using the same reagent, one study transfected 16 different plasmids expressing proteins to study their cellular localization [107]. Another group used this approach for the HT screening of potential therapeutic membranedisplayed single-chain antibodies [108]. A true HT attempt was reached by the use of 1959 un-tagged ORF taken from the Mammalian Gene Collection (MGC) and expressed in HEK cells to identify genes implicated in apoptosis [109]. One similar array approach showed efficiency using Lipofectamine 2000 directly in the DNA mixture before arraying [110]. However, whereas simplified by combining the transfection reagent with DNA before dispensing, it requires about 10-fold more DNA to reach the same efficiency as the above protocols. A throughput of 2880 conditions on a complete 96-wells plate to study v-Src Mutant Protein Function was reached in HEK cells, using 30 spots of pDNAs mixtures per well of 96-wells plate [111]. Lipofectamine 2000 also showed efficiency in another microarray approach testing 600 cDNA spots on a single glass slide using reverse transfection [112]. Authors showed high efficiency in many cell types such as mouse preadipocytes (3T3L1), muscle myoblasts (C2C12), liver hepatoma (Hepa1c1c7), or macrophage (RAW-164.), human cervix epithelia adenocarcinoma (HeLa), or at bone osteosarcoma (UMR-108).

Tavernier's group also pushed further its MAPPIT and MAmmalian Small molecule-Protein Interaction Trap (MASPIT) microplate-based array to microarrays using attractene (Qiagen), a non-liposomal lipid, as transfection reagent and a fluorescent reporter gene in place of the initial luciferase reporter [113]. Here, the ORFeome derived prey plasmid collection (15,000 cDNA) and a fluorescent reporter plasmid was mixed in 384-wells plates used as a matrix for further depositing by a microarrayer on polystyrene plates, to reach an industrial scale.

All these arrays' methods are impressive in terms of throughput as many conditions, or different expressed genes, can be tested simultaneously in parallel cells. However, they require a consequent preparation time. DNA dilution, most of the time with gelatin, and optimally with the transfection reagent are generally performed in 96 or 384-wells plates. Once done, an arrayer robot is then plunging its tips for deposition of the DNA on several slides. The tips must be washed with detergent and then sonicated or heated to avoid cross contaminations before arraying the next DNA

mixture. Finally, when the full array is printed, the slides have to be dried for 12 h to 2 days before later use and cell seeding. At the end of the experiment, a slide scanner became necessary to analyze transfected cells. The real throughput of such methods is then truly high once the arrays are ready to be incubated with the cells. Once the reagent used is efficient with the cell type requested, the throughput becomes only dependent on the liquid handling facility available in the lab. However, the method needs a certain financial investment for robotics, microarrayer platform and a scanner as the spots size and inter-distance need high resolution scanning to be analyzed.

#### *4.2.3 Chemical-based high-throughput microfluidic transfection*

As previously mentioned, microfluidic is now widely used due to the miniaturized scale it allows. Whereas it was applied to transfection using nanoconstriction or electroporation, it can also be used as a liquid and cell manipulation tool to perform transfection using chemical reagents. Schudel et al. first developed an inexpensive microfluidic-based miniaturized RNAi screening platform [114]. It relies on the use of a lipid-based transfection mixture and is low throughput as a maximum of 8 parallel transfections can be performed on this chip.

In another study, a two microchannel irrigating 8-chambers was designed on a glass slide [115]: 10 nL of a reverse transfection mixture containing gelatin, fibronectin, Lipofectamine 2000, and plasmid DNA were arrayed on a coated glass slide. This slide is mounted under the microscope, to face the microfluidic embedded chambers and showed successful transfection of Cancer LBT-N2b cells with almost no induced mortality, but the throughput was still clearly limited.

Based on the same kind of chambers design, the highest throughput was reached with a microfluidic chip of 1.6 × 5.8 cm containing 280 separate chambers. In about 10 min, the complete chip is loaded with about 600 cells per chamber of 500 μm diameter [116]. A set of valves allows the loading of different cell densities or even cell types. Once cells are loaded, the functional chip is obtained by alignment of the chambers to 280 DNA arrays (Lipid-DNA transfection mixtures) spotted on polylysine matrixes in an automated manner (about 2 h to complete). The assays showed a high transfection rate (99% efficiency) using an optimized condition but a cell linedependent optimization is necessary. Whereas feasible, microfluidic managed reverse transfection still seems to have a long road to meet the scientific community mostly due to its required skills in the field, to be able to reproduce or use such devices.

#### **4.3 High-throughput transfection using biological derivatives**

As discussed before, some natural biologicals materials, viruses, proteins, peptides, or macromolecules have shown cell-penetrating properties and their ability to deliver different molecules to target cells either in their natural form, modified, and sometimes multiplexed by engineering. Here are some examples of such approaches that reached HT in the delivery of DNA into cells.

#### *4.3.1 High-throughput DNA transfer using viral approaches*

The main limiting factors to reach HT with viral delivery is the ability to produce these particles (i.e., biosafety cabinets class 2 or 3), in a HT manner (one independent viral production for each cDNA to transduce), and at a sufficient titer to promote efficient transduction of target cells. This production step has been shown feasible

#### *High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

at HT in a pilot study with 1990 ORFs from the mammalian ORFeome collection [117]. In an automated platform, HEK cells were reverse co-transfected with these "gene of interest" plasmids and viral packaging ones to allow the production of the corresponding lentivirus in a 96-wells plate format (viruses transferring one ORF per wells). Supernatants (cDNA containing viral particles) were used to transduce target cells seeded in 96-wells plate format. In a similar manner, up to 16,000 cDNA were pushed to HT lentiviral production in 96-wells plates for later HT expression in target cells [118].

The previously developed SMAR-chip [89] was also used for viral particles production on the 169 matrixes embedded microwells, using reverse lipofection [119]. The method showed sufficient production to transduce 3T3L1 cell cultured in parallel to the producing cells array.

Despite these advances, such methodologies remain difficult to settle routinely due to the required material, specific skills, knowledge, and adequate biosafety facilities. To render it more accessible, some companies now propose ready-to-use kits in 96-wells microplate format to produce viruses in high titers from lab collection of cDNA [120]. Despite these limitations, this approach is highly promising as being universal for almost all cell types with high efficiency and furthermore efficient on suspension or adherent differentiated cells.

#### *4.3.2 Protein-derivatives based HT transfection*

One example of protein derivatives used is collagen derivatives, which are produced by collagen treatment or digestion. Atelocollagen, is a polymer obtained by pepsin treatment of type I collagen that shows various effects in cell and animals. Atelocollagen condenses and delivers DNA, antisense oligodeoxynucleotides, or siRNAs into cells on its own [121]. Protocol based on this polymer reached a HT microplate array level in 2001, with a collection of pDNA showing a long-term gene expression in HEK cells [122]. The array can handle long storage without loss of efficiency. Another study reached HT transfection in PC-12 cells using Atelocollagen and 288 different plasmids dispensed in 96-wells microplate arrays [123]. The advantages of these last approaches remain in the fact that atelocollagen intrinsically regroups two properties in a single bio-product: DNA condensation and cell entry of the formed complexes into cells. Furthermore, it is derived from a biocompatible natural material and per se is rarely cytotoxic for cells.

#### *4.3.3 High-throughput cell-penetrating peptides-based transfection*

Due to their potential, the use of CPP was pushed to HT transfection. The surface transfection and expression protocol (STEP method) is the only biological derivatives-based DNA transfer approach that reached such HT. It relies on the use of transferrin receptor, polylysine, adenoviral penton protein, and the HIV Tat protein to engineer some chimeric proteins. These combine functional motifs: binding of the DNA, binding to cell-surface receptors, the facilitated passage across membranes, the DNA targeting to the nucleus, and also adhesion and survival of the target cells on the arrayed spots [124]. The DNA/recombinants proteins mixtures are loaded in 384-wells source plates for standard arraying. Optimized conditions showed efficient GFP plasmid transfection efficiency (50–80%) and transgene expression in several cell types from easy to transfect HEK cells to more difficult ones such as SH-SY5Y neurons, N2A neurobalstoma cells, or PC-12 pheocromocytose cells. This method is

promised for future enhancements accompanying the study of new CPPs. Indeed, many CPP have already been identified and validated leading to the creation of a dedicated database in 2012 referencing 843 CPP identified so far [125]. However, an exhaustive list is impossible to give as some are still identified nowadays and the developed database now contains 1700 unique CPPs 10 years later [126]. Some of them may represent better candidates for DNA transfer. This DNA transfection approach is also of great interest for gene therapy as it enables a kind of transduction, efficient like viral particles but without all the safety concerns for their production and use [127].

#### **5. Conclusions and perspectives**

Last human genome sequencing assembly led to more than 24,000 genes to study [128]. Many approaches to transfer DNA in cells were then pushed to HT to interrogate each gene function. While still in progress with developments of new reagents and methods, HT DNA transfer approaches are already available. The main remaining challenge is to render them cheaper and affordable for non-specialists.

Among physical approaches, electroporation methods surpass the others being efficient in all cell types. The suspended electrodes array design represents several advantages: it is low cost, usable with standard electroporators and liquid handling devices, but is currently limited to 96-wells plate format [73]. Due to the technical design, it should be amenable to a 384-wells plate format. However, it is still restricted to suspended cells. Electroporation approaches for adherents' cells have also been developed in 384-wells plate format, but suffer from their cost and their need for expensive consumables [86].

Microfluidics devices suffer from the required skills and technologies to be assembled and used. Microfluidics combined with electroporation appears as a solution to some limitations but chamber-based devices seem too far from the standard assays format to be widely used. Applicable to all cells, microfluidic devices based on semi-continuous electroporation of flowing cells currently lack the necessary throughput [98]. The same concern is pointed out for nanoconstriction-based transfection designs [28]. However, higher throughputs would be amenable as microfluidic manipulation of cells and solutions in an automated way is possible at a high rate. Such a device would advantageously require an automated loading of pDNA from a source plate to the chip, transfection of the expected amount of cells and their dispensing on microplate wells, and then a rinsing step of the chip before starting a new cycle with the next pDNA. Indeed, these technologies are readily available and just need to be combined [129].

Methods combining microfluidic electroporation and DNA arraying seem at that time more difficult to be widely used. Indeed, many skills are necessary to prepare the functional chip: design of microfluidic device, micro arraying of the DNA, and even a micromanipulation platform to mount the complete functional chip [116]. This and the cost of the required material will limit its use in the scientific community.

Chemical-based transfection is readily available and represents the methods that reached the highest throughputs. The reverse protocol is the preferred mode with the use of lipids or cationic polymers and achieved a throughput of several thousand independent points [104]. A major limitation is that transfection occurs after a suspension step when cells are seeded. The use of the same approaches but in a microarray manner, also showed HT being possible to perform transfection on

#### *High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

adherent differentiated cells. However, in this case, the use of microfluidics and their inconvenients impair its wide use.

Biological approaches also reached HT. Viral transduction is the most powerful tool to transfer DNA. However, biosafety concerns, and furthermore difficulties to produce viruses in arrays format avoid its wide use. CPP-based delivery is of great potential and a more important use should be expected in the next decade with the advance of our knowledge in this field.

In order to deliver an easy way to perform transfection even by novices, a fully automated transfection protocol was developed using a tipless nano-acoustic dispenser device [101]. Users just have to indicate amounts of DNA and transfection reagent to be delivered in each well using a custom spreadsheet and prepare the requested source plate. The device-controlled software performs the tedious dispensing from the source plate to destination one, based on the spreadsheet [105]. The method could be applicable to any chemical reagents and even to CPP-based approaches. This approach could also be performed in forward mode then allowing adherent differentiated cells transfection. Newer versions of the device allow 1536 wells plates as the source and can now dispense in 3456-wells plates. It then becomes possible to regroup the human ORFeome collection plasmids on less than 15 sources plates, and their transfer to about seven 3456-wells plates only. The method allows preloading of the plates and long-term storage before cell dispensing. However, the cost of the dispenser is extremely huge and still impairs its use. The future end of the patented technologies protection, expected in 2025–2030, should induce a price drop due to competitors' and wider the use of such an approach.

#### **Acknowledgements**

The authors thank the National Institute of Health and Medical Research (INSERM) for its financial support for the publication of this chapter.

### **Author details**

Colin Béatrice1 and Couturier Cyril2 \*

1 LIttoral, ENvironment et Sociétiés (LIENSs), UMR CNRS 7266, La Rochelle University – BCBS Group (Biotechnologies et Chimie des Bioressources pour la Santé), La Rochelle, France

2 INSERM, Institut Pasteur de Lille, U1177—Drugs and Molecules for Living Systems, University of Lille, Lille, France

\*Address all correspondence to: cyril.couturier@univ-lille.fr

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Kaykas A, Moon RT. A plasmid-based system for expressing small interfering RNA libraries in mammalian cells. BMC Molecular and Cell Biology. 2004;**5**:1-11

[2] Sen G, Wehrman TS, Myers JW, Blau HM. Restriction enzyme–generated siRNA (REGS) vectors and libraries. Nature Genetics. 2004;**36**:183-189

[3] Bechtel S, Rosenfelder H, Duda A, Schmidt CP, Ernst U, Wellenreuther R, et al. The full-ORF clone resource of the German cDNA consortium. BMC Genomics. 2007;**8**:1-12

[4] Lamesch P, Li N, Milstein S, Fan C, Hao T, Szabo G, et al. hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes. Genomics. 2007;**89**:307-315

[5] Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD. siRNA, miRNA, and shRNA: In vivo applications. Journal of Dental Research. 2008;**87**:992-1003

[6] Modell AE, Lim D, Nguyen TM, Sreekanth V, Choudhary A. CRISPRbased therapeutics: Current challenges and future applications. Trends in Pharmacological Sciences. 2022;**43**:151-161

[7] Wells DJ. Gene therapy Progress and prospects: Electroporation and other physical methods. Gene Therapy. 2004;**11**:1363-1369

[8] Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980;**22**:479-488

[9] Diacumakos EG, Holland S, Pecora P. A microsurgical methodology for human cells in vitro: Evolution and applications. Proceedings of the National Academy of Sciences of the United States of America. 1970;**65**:911-918

[10] Lin TP. Microinjection of mouse eggs. Science. 1966;**151**:333-337

[11] Benz R, Beckers F, Zimmermann U. Reversible electrical breakdown of lipid bilayer membranes: A chargepulse relaxation study. The Journal of Membrane Biology. 1979;**48**:181-204

[12] Wong T-K, Neumann E. Electric field mediated gene transfer. Biochemical and Biophysical Research Communications. 1982;**107**:584-587

[13] Fajrial AK, He QQ, Wirusanti NI, Slansky JE, Ding X. A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing. Theranostics. 2020;**10**:5532-5549

[14] Shigekawa K, Dower WJ. Electroporation of eukaryotes and prokaryotes: A general approach to the introduction of macromolecules into cells. BioTechniques. 1988;**6**:742-751

[15] Sherba JJ, Hogquist S, Lin H, Shan JW, Shreiber DI, Zahn JD. The effects of electroporation buffer composition on cell viability and electro-transfection efficiency. Scientific Reports. 2020;**10**:3053

[16] Li S. Optimizing electrotransfection of Mammalian cells in vitro. Cold Spring Harbor Protocols. 2006;**1**:pdb-prot4449

[17] Flanagan M, Gimble JM, Yu G, Wu X, Li S. Competitive electroporation formulation for cell therapy. Cancer Gene Therapy. 2011;**18**:579-586

*High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

[18] Ye X, Brown SK, Scorza R, Cordts J, Sanford JC. Genetic transformation of peach tissues by particle bombardment. Journal of the American Society for Horticultural Science. 1994;**119**:367-373

[19] Uchida M, Li XW, Mertens P, Alpar HO. Transfection by particle bombardment: Delivery of plasmid DNA into mammalian cells using gene gun. Biochimica et Biophysica Acta (BBA) - General Subjects. 2009;**1790**:754-764

[20] Svarovsky SA, Gonzalez-Moa MJ, Robida MD, Borovkov AY, Sykes K. Self-assembled micronanoplexes for improved biolistic delivery of nucleic acids. Molecular Pharmaceutics. 2009;**6**:1927-1933

[21] Tsukakoshi M, Kurata S, Nomiya Y, Ikawa Y, Kasuya T. A novel method of DNA transfection by laser microbeam cell surgery. Applied Physics B: Lasers and Optics. 1984;**35**:135-140

[22] Rhodes K, Clark I, Zatcoff M, Eustaquio T, Hoyte KL, Koller MR. Cellular laserfection. Methods in Cell Biology. 2007;**82**:309-333

[23] Krasieva TB, Chapman CF, LaMorte VJ, Venugopalan V, Berns MW, Tromberg BJ. Cell permeabilization and molecular transport by laser microirradiation. In: Optical Investigations of Cells In Vitro and In Vivo. Bellingham, Washington, United States: SPIE; 1998. pp. 38-44

[24] Pislaru SV, Greenleaf JF, Miller DL. Sonoporation: Mechanical DNA Delivery by Ultrasonic Cavitation. Kluwer Academic Publishers-Plenum Publishers; Plenum Publishing Corporation ; Springer Science+Business Media; 2002

[25] Mignet N, Marie C, Delalande A, Manta S, Bureau M-F, Renault G, et al. Microbubbles for nucleic acid delivery in liver using mild Sonoporation. Methods in Molecular Biology. 1943;**2019**: 377-387

[26] Zhang Z, Wang Y, Zhang H, Tang Z, Liu W, Lu Y, et al. Hypersonic Poration: A new versatile cell Poration method to enhance cellular uptake using a piezoelectric Nano-electromechanical device. Small. 2017;**13**

[27] Sharei A, Zoldan J, Adamo A, Sim WY, Cho N, Jackson E, et al. A vector-free microfluidic platform for intracellular delivery. PNAS. 2013;**110**:2082-2087

[28] Chakrabarty P, Gupta P, Illath K, Kar S, Nagai M, Tseng F-G, et al. Microfluidic mechanoporation for cellular delivery and analysis. Materials Today Bio. 2022;**13**:100193

[29] Huth S, Lausier J, Gersting SW, Rudolph C, Plank C, Welsch U, et al. Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer. The Journal of Gene Medicine. 2004;**6**:923-936

[30] Scherer F, Anton M, Schillinger U, Henke J, Bergemann C, Krüger A, et al. Magnetofection: Enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy. 2002;**9**:102-109

[31] Kami D, Takeda S, Itakura Y, Gojo S, Watanabe M, Toyoda M. Application of magnetic nanoparticles to gene delivery. IJMS. 2011;**12**:3705-3722

[32] Kim TK, Eberwine JH. Mammalian cell transfection: The present and the future. Analytical and Bioanalytical Chemistry. 2010;**397**:3173-3178

[33] Graham FL, van der Eb AJ. A new technique for the assay of infectivity

of human adenovirus 5 DNA. Virology. 1973;**52**:456-467

[34] Guo L, Wang L, Yang R, Feng R, Li Z, Zhou X, et al. Optimizing conditions for calcium phosphate mediated transient transfection. Saudi Journal of Biological Sciences. 2017;**24**:622-629

[35] Batard P, Jordan M, Wurm F. Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection. Gene. 2001;**270**:61-68

[36] Pick HM, Meissner P, Preuss AK, Tromba P, Vogel H, Wurm FM. Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human rhesus-D IgG1 synthesis. Biotechnology and Bioengineering. 2002;**79**:595-601

[37] Schenborn ET, Goiffon V. Calcium phosphate transfection of mammalian cultured cells. In: Tymms MJ, editor. Transcription Factor Protocols [internet]. Totowa, NJ: Humana Press; 2000. p. 135-145 10.1385/1-59259-686-X:135

[38] Haberland A, Knaus T, Zaitsev SV, Stahn R, Mistry AR, Coutelle C, et al. Calcium ions as efficient cofactor of polycation-mediated gene transfer. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1999;**1445**:21-30

[39] Lindell J, Girard P, Müller N, Jordan M, Wurm F. Calfection: A novel gene transfer method for suspension cells. Biochimica et Biophysica Acta. 2004;**1676**(2):155-161

[40] Sussman DJ, Milman G. Shortterm, high-efficiency expression of transfected DNA. Molecular and Cellular Biology. 1984;**4**8:1641-1643

[41] Kumar P, Nagarajan A, Uchil PD. DEAE-Dextran Transfection. Cold

Spring Harbor, New York, United States: Cold Spring Harbor Laboratory Press; 2018

[42] Chesnoy S, Huang L. Structure and function of lipid-DNA complexes for gene delivery. Annual Review of Biophysics and Biomolecular Structure. 2000;**29**:27-47

[43] Kumar P, Nagarajan A, Uchil PD. Lipofection. Cold Spring Harbor, New York, United States: Cold Spring Harbor Laboratory Press; 2019

[44] Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, et al. Lipofection: A highly efficient, lipidmediated DNA-transfection procedure. Proceedings of the National Academy of Sciences of the United States of America. 1987;**84**:7413-7417

[45] El Ouahabi A, Thiry M, Pector V, Fuks R, Ruysschaert JM, Vandenbranden M. The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Letters. 1997;**414**:187-192

[46] Stürzl M, Konrad A, Sander G, Wies E, Neipel F, Naschberger E, et al. High throughput screening of gene functions in mammalian cells using reversely transfected cell arrays: Review and protocol. Combinatorial Chemistry & High Throughput Screening. 2008;**11**:159-172

[47] Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, et al. Advanced transfection with lipofectamine 2000 reagent: Primary neurons, siRNA, and high-throughput applications. Methods. 2004;**33**:95-103

[48] Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in

*High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

culture and in vivo: Polyethylenimine. PNAS. 1995;**92**:7297-7301

[49] Merdan T, Kunath K, Fischer D, Kopecek J, Kissel T. Intracellular processing of poly(ethylene imine)/ ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments. Pharmaceutical Research. 2002;**19**:140-146

[50] Sonawane ND, Szoka FC, Verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes\*. Journal of Biological Chemistry. 2003;**278**:44826-44831

[51] Gilboa E. Retrovirus vectors and their uses in molecular biology. BioEssays. 1986;**5**:252-257

[52] Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein M. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proceedings of the National Academy of Sciences of the United States of America. 2013;**110**:7306-7311

[53] Yamauchi Y, Greber UF. Principles of virus uncoating: Cues and the snooker ball. Traffic. 2016;**17**:569-592

[54] Miyanohara A. Preparation of vesicular stomatitis virus-G (VSV-G) conjugate and its use in gene transfer. Cold Spring Harbor Protocols. 2012;**2012**:453-456

[55] Mangion M, Robert M-A, Slivac I, Gilbert R, Gaillet B. Production and use of Gesicles for nucleic acid delivery. Molecular Biotechnology. 2022;**64**:278-292

[56] Balistreri G, Yamauchi Y, Teesalu T. A widespread viral entry mechanism: The C-end Rule motif– neuropilin receptor interaction. PNAS. 2021;**118**(49):e2112457118

[57] Wadia JS, Dowdy SF. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Advanced Drug Delivery Reviews. 2005;**57**:579-596

[58] Dupont E, Prochiantz A, Joliot A. Penetratin story: An overview. In: Langel Ü, editor. Cell-Penetrating Peptides: Methods and Protocols. New York, NY: Springer; 2015. pp. 29-37. DOI: 10.1007/978-1-4939-2806-4\_2

[59] Derakhshankhah H, Jafari S. Cell penetrating peptides: A concise review with emphasis on biomedical applications. Biomedicine & Pharmacotherapy. 2018;**108**: 1090-1096

[60] Ignatovich IA, Dizhe EB, Pavlotskaya AV, Akifiev BN, Burov SV, Orlov SV, et al. Complexes of plasmid DNA with basic domain 47-57 of the HIV-1 tat protein are transferred to mammalian cells by endocytosismediated pathways\*. Journal of Biological Chemistry. 2003;**278**: 42625-42636

[61] EL Andaloussi S, Lehto T, Mäger I, Rosenthal-Aizman K, Oprea II, Simonson OE, et al. Design of a peptidebased vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Research. 2011;**39**:3972-3987

[62] Ezzat K, EL Andaloussi S, Zaghloul EM, Lehto T, Lindberg S, Moreno PMD, et al. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation. Nucleic Acids Research. 2011;**39**:5284-5298

[63] Veiman K-L, Mäger I, Ezzat K, Margus H, Lehto T, Langel K, et al. PepFect14 peptide vector for efficient gene delivery in cell cultures. Molecular Pharmaceutics. 2013;**10**:199-210

[64] Ronquist G, Hedström M. Restoration of detergent-inactivated adenosine triphosphatase activity of human prostatic fluid with concanavalin A. Biochimica et Biophysica Acta (BBA) - Enzymology. 1977;**483**:483-486

[65] Waldenström A, Ronquist G. Role of exosomes in myocardial remodeling. Circulation Research. 2014;**114**:315-324

[66] Nilsson BO, Lennartsson L, Carlsson L, Nilsson S, Ronquist G. Expression of prostasome-like granules by the prostate cancer cell lines PC3, Du145 and LnCaP grown in monolayer. Upsala Journal of Medical Sciences 1999;**104**(3):199-206

[67] Munagala R, Aqil F, Jeyabalan J, Kandimalla R, Wallen M, Tyagi N, et al. Exosome-mediated delivery of RNA and DNA for gene therapy. Cancer Letters. 2021;**505**:58-72

[68] Inoue N, Hirouchi T, Kasai A, Higashi S, Hiraki N, Tanaka S, et al. Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response. Biochemical and Biophysical Research Communications. 2018;**495**:1992-1997

[69] Arena TA, Harms PD, Wong AW. High throughput transfection of HEK293 cells for transient protein production. Methods in Molecular Biology. 1850;**2018**:179-187

[70] Fiscella M, Perry JW, Teng B, Bloom M, Zhang C, Leung K, et al. TIP, a T-cell factor identified using highthroughput screening increases survival in a graft-versus-host disease model. Nature Biotechnology. 2003;**21**:302-307

[71] Madoux F, Simanski S, Chase P, Mishra JK, Roush WR, Ayad NG, et al. An ultra-high throughput cell-based screen for Wee1 degradation inhibitors. The Journal of Biomolecular Screening. 2010;**15**:907-917

[72] Buchser WJ, Pardinas JR, Shi Y, Bixby JL, Lemmon VP. 96-well electroporation method for transfection of mammalian central neurons. BioTechniques. 2006;**41**:619-624

[73] Guignet EG, Meyer T. Suspendeddrop electroporation for highthroughput delivery of biomolecules into cells. Nature Methods. 2008;**5**:393-395

[74] Martinet W, Schrijvers DM, Kockx MM. Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnology Letters. 2003;**25**:1025-1029

[75] Trompeter H-I, Weinhold S, Thiel C, Wernet P, Uhrberg M. Rapid and highly efficient gene transfer into natural killer cells by nucleofection. Journal of Immunological Methods. 2003;**274**:245-256

[76] Mellott AJ, Forrest ML, Detamore MS. Physical non-viral gene delivery methods for tissue engineering. Annals of Biomedical Engineering. 2013;**41**:446-468

[77] SF Cell Line 96-well Nucleofector™ Kit (96 RCT)|Lonza. 2022. Available from: https://bioscience.lonza. com/lonza\_bs/FR/en/Transfec tion/p/000000000000198709/ SF-Cell-Line-96-well-Nucleofector-Kit

[78] Haag J, Voigt R, Soeder S, Aigner T. Efficient non-viral transfection of primary human adult chondrocytes in a high-throughput format. Osteoarthritis and Cartilage. 2009;**17**:813-817

*High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

[79] Bowles R, Patil S, Pincas H, Sealfon SC. Validation of efficient highthroughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation. Journal of Immunological Methods. 2010;**363**:21-28

[80] Moore JC, Atze K, Yeung PL, Toro-Ramos AJ, Camarillo C, Thompson K, et al. Efficient, highthroughput transfection of human embryonic stem cells. Stem Cell Research & Therapy. 2010;**1**:23

[81] 384-well Nucleofector™ System|Lonza. 2022. Available from: https://bioscience.lonza. com/lonza\_bs/FR/en/Transfection/ p/000000000000198370/384-well-Nucleofector-System

[82] Electrode, Petri Pulser™. VWR. 2022. Available from: https://ru.vwr. com/store/product/10597200/ electrode-petri-pulsertm

[83] Petri Dish Electrode. VWR. 2022 Available from: https://ru.vwr.com/store/ product/10597253/petri-dish-electrode

[84] BTX Adherent Cell Electrodes - Clinical Analyzers and Instruments, Electroporators. 2022. Available from: https://www. fishersci.com/shop/products/ adherent-cell-electrodes-2/p-7222369

[85] Pihl J, Johansson M-L, Granfeldt D, Tokarz M, Karlsson M, Sinclair J. Cellaxess®HT: high-throughput transfection for genome-wide RNAi. Nature Methods. 2008;**5**:i-ii

[86] Marine S, Freeman J, Riccio A, Axenborg M-L, Pihl J, Ketteler R, et al. High-throughput transfection of differentiated primary neurons from rat forebrain. Journal of Biomolecular Screening. 2012;**17**:692-696

[87] Fujimoto H, Kato K, Iwata H. Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells. Analytical and Bioanalytical Chemistry. 2008;**392**:1309-1316

[88] Jain T, Papas A, Jadhav A, McBride R, Saez E. In situ electroporation of surfacebound siRNAs in microwell arrays lab chip. The Royal Society of Chemistry. 2012;**12**:939-947

[89] Bian S, Zhou Y, Hu Y, Cheng J, Chen X, Xu Y, et al. High-throughput in situ cell electroporation microsystem for parallel delivery of single guide RNAs into mammalian cells. Scientific Reports. 2017;**7**:42512

[90] Zhang P, Zhang J, Bian S, Chen Z, Hu Y, Hu R, et al. High-throughput superhydrophobic microwell arrays for investigating multifactorial stem cell niches. Lab on a Chip. 2016;**16**:2996-3006

[91] Yamauchi F, Kato K, Iwata H. Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes. Nucleic Acids Research. 2004;**32**:e187

[92] Fox MB, Esveld DC, Valero A, Luttge R, Mastwijk HC, Bartels PV, et al. Electroporation of cells in microfluidic devices: A review. Analytical and Bioanalytical Chemistry. 2006;**385**:474-485

[93] Huang Y, Rubinsky B. Flowthrough micro-electroporation chip for high efficiency single-cell genetic manipulation. Sensors and Actuators A: Physical. 2003;**104**(3):205-212

[94] Hur J, Chung AJ. Microfluidic and nanofluidic intracellular delivery. Advanced Science. 2021;**8**:2004595

[95] Kim JA, Cho K, Shin MS, Lee WG, Jung N, Chung C, et al. A novel electroporation method using a capillary and wire-type electrode. Biosensors & Bioelectronics. 2008;**23**:1353-1360

[96] Li Y, Wu M, Zhao D, Wei Z, Zhong W, Wang X, et al. Electroporation on microchips: The harmful effects of pH changes and scaling down. Scientific Reports. 2015;**5**:17817

[97] He H, Chang DC, Lee Y-K. Using a micro electroporation chip to determine the optimal physical parameters in the uptake of biomolecules in HeLa cells. Bioelectrochemistry. 2007;**70**:363-368

[98] Wang S, Zhang X, Wang W, Lee LJ. Semicontinuous flow electroporation chip for high-throughput transfection on mammalian cells. Analytical Chemistry. 2009;**81**:4414-4421

[99] Kang W, Giraldo-Vela JP, Nathamgari SSP, McGuire T, McNaughton RL, Kessler JA, et al. Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons. Lab on a Chip. 2014;**14**:4486-4495

[100] Nasoff M, Bergseid M, Hoeffler JP, Heyman JA. High-throughput expression of fusion proteins. Methods in Enzymology. 2000;**328**:515-529

[101] Colin B, Deprez B, Couturier C. High-throughput DNA plasmid transfection using acoustic droplet ejection technology. In: SLAS DISCOVERY: Advancing the Science of Drug Discovery. Vol. 24. Thousand Oaks, CA: SAGE Publications Inc STM; 2019. pp. 492-500

[102] Sichero L, Sobrinho JS, Villa LL. Identification of novel cellular transcription factors that regulate early promoters of human papillomavirus types 18 and 16. The Journal of Infectious Diseases. 2012;**206**:867-874

[103] Lievens S, Vanderroost N, Van der Heyden J, Gesellchen V, Vidal M, Tavernier J. Array MAPPIT: High-throughput interactome analysis in mammalian cells. Journal of Proteome Research. 2009;**8**:877-886

[104] Yokoyama S, Ito Y, Ueno-Kudoh H, Shimizu H, Uchibe K, Albini S, et al. A systems approach reveals that the myogenesis genome network is regulated by the transcriptional repressor RP58. Developmental Cell. 2009;**17**:836-848

[105] Colin B, Rocq N, Deprez B, Couturier C. High-throughput DNA plasmid multiplexing and transfection using acoustic nanodispensing technology. JoVE (Journal of Visualized Experiments). 2019;**150**:e59570

[106] Ziauddin J, Sabatini DM. Microarrays of cells expressing defined cDNAs. Nature. 2001;**411**:107-110

[107] Palmer E, Freeman T. Investigation into the use of C- and N-terminal GFP fusion proteins for subcellular localization studies using reverse transfection microarrays. Comparative and Functional Genomics. 2004;**5**:342-353

[108] Delehanty JB, Shaffer KM, Lin B. Transfected cell microarrays for the expression of membrane-displayed single-chain antibodies. Analytical Chemistry. 2004;**76**:7323-7328

[109] Palmer EL, Miller AD, Freeman TC. Identification and characterisation of human apoptosis inducing proteins using cell-based transfection microarrays and expression analysis. BMC Genomics. 2006;**7**:145

[110] Konrad A, Jochmann R, Kuhn E, Naschberger E, Chudasama P, Stürzl M. Reverse transfected cell microarrays in

*High Throughput Methods to Transfer DNA in Cells and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.104542*

infectious disease research. Methods in Molecular Biology. 2011;**706**:107-118

[111] Walczak W, Pipalia NH, Soni M, Faruqi AF, Ralph H, Maxfield FR, et al. Parallel analysis of v-Src mutant protein function using reverse transfection cell arrays. Combinatorial Chemistry & High Throughput Screening;**9**:711-718

[112] Rajan S, Djambazian H, Dang HCP, Sladek R, Hudson TJ. The living microarray: A high-throughput platform for measuring transcription dynamics in single cells. BMC Genomics. 2011;**12**:115

[113] Lievens S, Van der Heyden J, Masschaele D, De Ceuninck L, Petta I, Gupta S, et al. Proteome-scale binary Interactomics in human cells. Molecular & Cellular Proteomics. 2016;**15**:3624-3639

[114] Schudel BR, Harmon B, Abhyankar VV, Pruitt BW, Negrete OA, Singh AK. Microfluidic platforms for RNA interference screening of virus–host interactions. Lab Chip. 2013;**13**:811-817

[115] Enomoto J, Takagi R, Onuki-Nagasaki R, Fujita S, Fukuda J. Reverse transfection in microchamber arrays for cell migration assays. Sensors and Actuators B: Chemical. 2014;**190**:896-899

[116] Woodruff K, Maerkl S. A highthroughput microfluidic platform for mammalian cell transfection and culturing. Scientific Reports. 2016;**6**:23937

[117] Škalamera D, Ranall MV, Wilson BM, Leo P, Purdon AS, Hyde C, et al. A High-throughput platform for lentiviral overexpression screening of the human ORFeome. PLOS ONE, Public Library of Science. 2011;**6**:e20057

[118] Yang X, Boehm JS, Yang X, Salehi-Ashtiani K, Hao T, Shen Y, et al. A public genome-scale lentiviral expression library of human ORFs. Nature Methods. 2011;**8**:659-661

[119] Zhang J, Hu Y, Wang X, Liu P, Chen X. High-throughput platform for efficient chemical transfection, virus packaging, and transduction. Micromachines. 2019;**10**:387

[120] Streamlined Production of High-titer Lenti Virus in a 96-well format. 2022. Available from: https://www.takarabio.com/ learning-centers/gene-function/ viral-transduction/lentivirus/ high-throughput-lentivirus-production

[121] Ochiya T, Takahama Y, Nagahara S, Sumita Y, Hisada A, Itoh H, et al. New delivery system for plasmid DNA in vivo using atelocollagen as a carrier material: The Minipellet. Nature Medicine. 1999;**5**:707-710

[122] Honma K, Ochiya T, Nagahara S, Sano A, Yamamoto H, Hirai K, et al. Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions. Biochemical and Biophysical Research Communications. 2001;**289**:1075-1081

[123] Saito S, Honma K, Kita-Matsuo H, Ochiya T, Kato K. Gene expression profiling of cerebellar development with high-throughput functional analysis. Physiological Genomics. 2005;**22**:8-13

[124] Redmond TM, Ren X, Kubish G, Atkins S, Low S, Uhler MD. Microarray transfection analysis of transcriptional regulation by cAMP-dependent protein kinase\*. Molecular & Cellular Proteomics. 2004;**3**:770-779

[125] Gautam A, Singh H, Tyagi A, Chaudhary K, Kumar R, Kapoor P, et al. CPPsite: A curated database of cell penetrating peptides. Database (Oxford). 2012;**2012**:bas015

[126] Agrawal P, Bhalla S, Usmani SS, Singh S, Chaudhary K, Raghava GPS, et al. CPPsite 2.0: A repository of experimentally validated cell-penetrating peptides. Nucleic Acids Research. 2016;**44**:D1098-D1103

[127] de Figueiredo IR, Freire JM, Flores L, Veiga AS, Castanho MARB. Cell-penetrating peptides: A tool for effective delivery in gene-targeted therapies. IUBMB Life. 2014;**66**:182-194

[128] Cunningham F, Amode MR, Barrell D, Beal K, Billis K, Brent S, et al. Ensembl 2015. Nucleic Acids Research. 2015;**43**:D662-D669

[129] Alam MK, Koomson E, Zou H, Yi C, Li C-W, Xu T, et al. Recent advances in microfluidic technology for manipulation and analysis of biological cells (2007- 2017). Analytica Chimica Acta. 2018;**1044**:29-65

**Chapter 2**

## Genetic Transformation in Prokaryotic and Eukaryotic Cells

*Endang Semiarti, Yekti Asih Purwestri, Saifur Rohman and Wahyu Aristyaning Putri*

#### **Abstract**

Improving the quality and quantity of an organism and its products can be approached by molecular characters enhancement through the insertion of a gene of interest into cells of the desired organism. Genetic transformation of an organism involves isolation, identification, cloning a gene of interest into a vector, and transferring the gene to the target organism. This chapter reviews the process of genetic transformation into the organism's cell from bacterial (*Escherichia coli*), yeast, plant (Onion, Tobacco, and Orchids), and mammalian. The discussion will be focused on the introduction of DNA molecules into plant cells and protoplast mediated by polyethylene glycol (PEG), electroporation, and gene gun using particle bombardment. Further discussion on the transient protein expression system of plant-based on protoplast, onion cell, and tobacco will also be covered in this chapter as well. The systems have been proven as a powerful tool for determining subcellular protein localization, protein-protein interactions, identifying gene function, and regulation. Finally, it can be clearly seen, the differences and similarities in the mechanism of genetic transformation both in prokaryotic and eukaryotic systems.

**Keywords:** eukaryotic cells, genetic transformation, molecular character enhancement, prokaryotic cells, transient expression

#### **1. Introduction**

To improve the quality and quantity of an organism, both prokaryotes and eukaryotes, it can be approached by molecular character enhancement through the insertion of interest genes or superior genes into the cells of the desired organism. The process of genetic transformation of an organism involves the isolation and identification of the gene of interest, the technique of cloning the gene on a plasmid vector until the process of transferring the gene to the target organism's cell. One of the important genes in the growth of organisms is the homeobox gene, which is a gene that regulates the growth and development of organisms in a very early stage. Homeobox genes were first discovered in the *Drosophila melanogaster*. These homeobox genes have been also found in all multicellular organisms from fungi to plants, and vertebrate animals [1].

In plants, overexpression of the homeobox gene at an early stage of growth will activate the formation of apical buds from apical meristems that will produce shoots. The addition of exogenous cytokinin and auxin growth regulators will activate the homeobox genes to induce cell division genes that in turn will produce somatic embryos. Theoretically, each somatic cell can grow and transform itself into somatic


#### **Table 1.**

*The homeobox gene in plants.*

embryos, therefore it can produce plant seeds in large quantities and uniform phenotypic characters. This is very profitable for agriculture and industry, especially for the mass production of identical plant seeds using tissue culture techniques.

In the model plant, *Arabidopsis thaliana,* it has been reported that the homeobox genes always maintain the growth of meristem cells in Shoot Apical Meristem (SAM) [2]. Overexpression of the homeobox gene in Arabidopsis has shown that the cells can convert from a determinate state to the meristematic indeterminate state, depending on the levels of expression of the gene (s) (**Table 1**) [23].

#### **2. Transformation for transient expression in onion, tobacco leaves, and protoplast**

Transient expression become a powerful tool in functional genomics study for detecting gene expression in a short time and the inserted gene do not integrate into the plant genome. A transient expression system has been developed in planta using different cells or tissues, including protoplast, onion cells, and tobacco (*Nicotiana benthamiana*) leaves (**Table 2**). A transient expression system using protoplasts has proven to be a good experimental tool in molecular biology. This approach is an efficient technique to study subcellular protein localization, protein complexes, *in vivo* gene silencing, and promotor activity [24, 25].

The advantages of the transient expression system compared to stable expression are that it does not require regeneration of transformed cells, does not affect the stability of the host genome, and is independent of the effect of T-DNA integration site position [28]. Protoplast transfection can be performed using a variety of procedures commonly used for the transfection of animal cell cultures. The procedures that are often used to insert DNA into protoplasts are polyethylene glycol (PEG) and electroporation [29].

Polyethylene glycol (PEG)-mediated transformation plant cells can be transformed through certain chemicals, namely PEG (polyethylene glycol). PEG is an oligomer or hydrophilic polymer synthesized from ethylene oxide, containing repeating units of -(O-CH2-CH2)-. Polyethylene oxide (PEO) is another name for PEG. Typically, ethylene oxide macromolecules with a molecular weight of less than 20,000 g/mol are called PEGs, while macromolecules with values above 20,000 g/molar are called PEOs [29]. PEG is soluble in acetonitrile, benzene, water, ethanol, and dichloromethane, while it is insoluble in diethyl ether and hexane (**Figures 1** and **2**).

PEG is available in various structures, such as branched, stellar, and comb-like macromolecules. PEG can bind various reactive functional groups to the PEG polymer site. Homo and heterobifunctional PEG derivatives are particularly suitable as agents


**Table 2.**

*Transient expression system and its purposes in planta.*

#### **Figure 1.** *Agroinfiltration in tobacco (Nicotiana benthamiana) leaves for protein-protein analysis.*

#### **Figure 2.**

*Transient expression in onion cell and protoplast for determining the subcellular localization of the protein. (a) Subcellular localization of OsKAN1-GFP fusion protein in the nucleus of onion cell transformed using particle bombardment [30]. (b) Transient expression of GFP-GF14c and Hd3a-mCherry in rice protoplast was driven by the 35S promoter of cauliflower mosaic virus and ubiquitin promoter, respectively, Bar = 10 μm.*

or spacers of two chemical entities, whereas mono-functional PEGs prevent linking reactions that can affect the PEGylation of certain compounds with bifunctional PEGs. PEGylation is an interesting process in which PEG is bound to other molecules [31, 32].

PEG was used to increase DNA uptake into the protoplast during transfection. Very high concentrations of PEG can reduce transfection efficiency because it is toxic to protoplasts [33]. PEG-mediated DNA uptake is a direct gene transfer method that utilizes the interaction between PEG, naked DNA, salts, and protoplast membranes to influence the transport of DNA into the cytoplasm. The advantage of PEG-mediated transformation is that it does not require special equipment and can be carried out in

the laboratory under sterile conditions [34]. Compared to *Agrobacterium tumefaciens*mediated transformation, PEG-mediated transformation was not species-specific. In addition, PEG-mediated transformation is also useful for functional analysis of genes through transient expression, a technique that is often used for promoter analysis [35].

Particle bombardment particles are coated in DNA and can penetrate plant cells without killing the plant cells themselves. Previous experiments have shown that particle bombardment has been successfully used to insert DNA into rice callus and seedlings grown in dark conditions but has the disadvantage of low efficiency and reliance on expensive equipment [36].

#### **3. Expression of recombinant psychrophilic RNase III in** *Escherichia coli*

To understand the mechanism of how the transformation and expression of recombinant protein in a prokaryotic system, *Escherichia coli* BL21(DE3) have been used as host and recombinant RNaseIII as a model protein. Ribonuclease III is an enzyme that specifically cleaves the double-stranded RNA molecules. It functions for ribosomal RNA maturation; therefore, RNase III is indispensable for the survival of cells. Here, the production of recombinant psychrophilic RNase III from *Shewanella* sp. SIB1 in the *Escherichia coli* system was reported. As a psychrophilic enzyme, recombinant RNase III was produced in the form of inclusion bodies. To produce the soluble recombinant psychrophilic RNase III, co-expression with FKBP22 from the same bacteria was carried out. The result showed that FKBP22 significantly improved the solubility of recombinant psychrophilic RNase III. It strongly suggested that FKBP22 assists the proper folding of recombinant psychrophilic RNase III when it was overproduced in the *Escherichia coli* system.

Ribonuclease III (RNase III) is an enzyme that specifically cleaves double-stranded RNA [30, 37–40]. RNase III has an important role in both the RNA transcript maturation and decay of diverse cellular and viral RNA. A primary function of RNase III, however, is the maturation of ribosomal RNA (rRNA) [30, 37, 38, 40, 41]. RNase III has been known to be widely distributed across the living kingdom of life, from bacteria to higher eukaryotes. RNase III family has common features in their molecular organization, by which it consists of catalytic domain with the common feature of HNERLFGDS located at the N-terminus and double-stranded binding domain (dsRBD) that located on their C-terminus [39]. RNase III exhibited enzymatically active in homodimeric form, by which each monomer has its catalytic mechanism and therefore the cleavage product of the RNase III exhibits a very regular length of short double-stranded RNA [39]. By such properties, RNase III can be manipulated to produce short dsRNA that can be implemented for the RNA interference technology in combination with Argonaute, Drosha, and Dicer [42]. Therefore, the production of recombinant RNase III is necessary from the scientific and technological point of view.

Production of recombinant proteins could be done in either bacterial or mammalian cells as a host. The choice of the host to produce recombinant protein may be the subject of proteins of interest. It depends on whether further processing of the proteins of interest is necessary or not. However, the bacterial cell is the most prominent host for recombinant protein production. *Escherichia coli* is the most common bacterial cell that is generally used as a host organism because of the following advantages— (a) it has unparalleled fast growth kinetics, (b) high cell density cultures are easily achieved, (c) the growth media are easily prepared and inexpensive, and (d) transformation with exogenous DNA is fast and easy [43]. There are several commercially

available *Escherichia coli* appropriate for the expression host of recombinant proteins, such as *Escherichia coli* BL21(DE3) and its derivatives. *Escherichia coli* BL21(DE3) is carrying the T7gene1 from the lysogens DE3, a derivative of bacteriophage lambda, that encodes for T7 RNA polymerase under the control of *lac*UV5 promoter [44]. T7 RNA polymerase is a polymerase that can recognize T7 promoter, a strong promoter appropriate for the high-level expression of proteins. Such promoter is commonly used in several commercially available expression vectors, such as pET series, pRSET, and pACYC-Duet. These vectors contain a regulatory system in the form of *lac*I in which the gene product suppresses the expression of recombinant proteins.

This report will discuss the production of recombinant RNase III from a psychrotrophic bacterium, *Shewanella* sp. SIB1. *Shewanella* sp. SIB1 is a psychrotrophic bacterium that grows most rapidly at 20°C [45]. This strain can grow even at 0° but cannot grow higher than 30°C. Phylogenetic analysis indicates that *Shewanella* sp. SIB1 is closely related to the *Shewanella* sp. AC10 isolated from the Antarctic ocean [44]. Interestingly, protein from psychrotrophic bacterium exhibits distinct properties compared to the mesophilic counterparts by their ability to adapt to cold temperatures [45].

Protein adaptation in such low temperatures requires a strategy that is not commonly found in mesophilic, for example, psychrophilic proteins must be flexible enough to avoid the problem in protein folding and to perform the optimum catalytic activity if it is an enzyme. Therefore, the production of psychrophilic protein would be interesting due to their properties to adapt to such low temperatures. Although the production of recombinant protein in bacterial host seems to be straightforward, several difficulties that arise and how to solve the problems during the production of recombinant psychrophilic protein will be discussed.

#### **3.1 Localization of Shewanella sp. SIB1 RNase III encoding gene (***Sh-rnc***)**

To localize the *Sh-rnc* gene from the *Shewanella* sp. SIB1 genome, as well as to obtain the full length of the RNase III open reading frame, the inverse PCR was carried out in this work. Previously, the partial *Sh-rnc* gene was amplified by using a pair of primers constructed based on the sequence of open reading frames of the *rnc* gene from *Shewanella oneidensis* MR1. Once the fragment of the *Sh-rnc* gene was amplified then it was used to construct new primers for the inverse PCR. For the inverse PCR, the SIB1 genome was digested by the *Dra*I restriction enzyme and then the digestion product was then allowed to perform self-ligation to form small circular products. Since the *orf* of the *Sh-rnc* gene contains a recognition site for *Dra*I, therefore, the PCR was conducted by using two pairs of primers. By such a strategy, the two PCR products were obtained and were then cloned into pUC18 for sequencing. The sequencing results indicated that the two fragments corresponded to the *lep*B and *era* genes, which means that the *rnc* gene was flanked by the *lep*B and *era* genes at the upstream and downstream regions, respectively (**Figure 3**) [46]. It seems that the three genes are organized in one operon, since there was no promoter detected in the upstream of every orf of *lep*B, *rnc,* and *era* genes. The gene organization was similar to that of *Rhodobacter capsulatus* [41]. Based on the information of *rnc* gene organization in *Shewanella* sp. SIB1 genome, the full length of orf of *rnc* gene could be isolated and then used for the expression of recombinant psychrophilic RNase III. The length of the orf of the *rnc* gene was determined to be 678 bp, which produced the recombinant RNase III with a molecular weight of ±24.8 kDa.

*Genetic Transformation in Prokaryotic and Eukaryotic Cells DOI: http://dx.doi.org/10.5772/intechopen.103839*

#### **Figure 3.**

*Molecular organization of rnc gene in Shewanella sp. SIB1 genome. The rnc gene is flanked by lepB and era genes at the upstream and downstream regions. It seems that lepB-rnc-era is organized in one operon since there was no promoter sequence was found at the upstream of each gene. Moreover, the rnc-era sequence overlaps with each other (hatched area), while lepB-era (white area) is separated only by one base. Arrows indicate the expression direction [46].*

#### **3.2 Expression of recombinant psychrophilic RNase III**

To overexpress the recombinant psychrophilic RNase III from *Shewanella* sp. SIB1, the pET28a expression vector, and *Escherichia coli* BL21(DE3) as a host were used in this work. Insertion of the orf of *rnc* gene into the multiple cloning sites of pET28a produces the recombinant protein that is fused with the hexahistidine tag. The resultant plasmid, pET-*rnc,* was then used to transform *Escherichia coli* BL21(DE3). Expression of the recombinant psychrophilic RNase III was induced by isopropyl thio-b-D-galactopyranoside (IPTG).

The result showed that the recombinant psychrophilic RNase III was accumulated in inclusion body form, although the overproduction was shifted at 20°C (**Figure 4**). Several attempts have been implemented to improve the solubility of recombinant

#### **Figure 4.**

*SDS-PAGE of recombinant psychrophilic RNase III overproduced in Escherichia coli BL21(DE3). Samples were subjected to 15% SDS-PAGE and stained with Coomassie brilliant blue (CBB). Low molecular weight kit (GE Healthcare) (lane M); cell pellet of cell harboring pET-rnc without co-expression with FKBP22 (lane 1); soluble part of cell harboring pET-rnc without co-expression with FKBP (lane 2); cell pellet of cell harboring pET-rnc and FKBP22 (lane 3); and soluble part of cell harboring pET-rnc and FKBP22 (lane 4). Recombinant psychrophilic RNase III was indicated by arrow [46].*

psychrophilic RNase III in the *E. coli* system. Shifting of the expression temperatures to 15 and 10°C and adjustment of pH of growing media also did not significantly improve the solubility of recombinant proteins (data not shown).

Another strategy that has been carried out to improve the recombinant psychrophilic RNase III was by co-expression with the chaperone or chaperone-like proteins. Chaperon is a protein that functions for assisting another protein folding. Two types of assisting folding proteins used were GroEL-ES from *Escherichia coli* and FKBP22 from *Shewanella* sp. SIB1 [47]. Among them, co-expression with FKBPP22 successfully improved the solubility of recombinant psychrophilic RNase III (**Figure 2**). FKBP22 belongs to the group of peptidyl-prolyl isomerase (PPIase) that functions for switching *cis*- to *trans*-configuration of proline during polypeptide biosynthesis [47]. This result indicated that strong induction to produce recombinant psychrophilic RNase III might cause the misfolding of the protein. Therefore, during co-expression with FKBP22, it helps to assist the proper folding of the psychrophilic RNase III. Although co-expression with FKBP22 only partly solubilizes the recombinant psychrophilic RNase III, it is sufficient for the biochemical characterization of the recombinant proteins.

Psychrophilic enzymes have unique properties in their folding and activity. Expression of such recombinant psychrophilic enzymes in mesophilic host generally produces misfolding recombinant protein represented by the inclusion bodies formation. Overexpression of recombinant psychrophilic RNase III in *Escherichia coli* has been improved when it was expressed with chaperone-like protein, FKBP22. It is apparently that FKBP22 assists the proper folding of recombinant psychrophilic RNase III.

#### **4. Eukaryote model organism and animal gene transformation Yeast genetics**

The yeast *Saccharomyces cerevisiae* is an essential option for expanding breakthrough research in gene cloning in *E. coli*, including eukaryotes. It can be manipulated and cultured using standard techniques applied to unicellular microorganisms. Yeast is a eukaryotes cell whose genetic material is packed into the chromosomes of the membrane-enclosed cell nucleus. In addition, extensive knowledge has been accumulated over the years that yeast has been used as a model system for genetic and biochemical studies. A comprehensive map showing the 17 chromosomes and more than 400 genes is available. The discovery first drove research in this area that yeast genes can be reliably expressed in *E. coli*. Yeast DNA fragments, when cloned into *E. coli* can restore histidine-independent growth of the mutant strain. In another case, a fragment of the yeast chromosome carries the gene for the enzyme that corresponds to the defect in the bacterial strain. Therefore, the yeast HIS3 gene can be expressed in bacterial cells and produce the yeast gene. Usually, wild-type alleles are specified in uppercase, and mutant ones are set in lowercase. Therefore, HIS3 is a wild-type allele, and his3 is a mutant allele that causes histidine dependence. Other yeast genes isolated and used as markers include TRP1, LEU2, URA3, and ARG4. In general, eukaryotic genes have more complex functions than bacterial genes due to introns. Due to the lack of introns, yeast genes may develop easier than other animal cells. An important marker of wild-type yeast attempts to insert exogenous DNA into yeast cells.

#### **4.1 Yeast transformation**

Yeast cells are protected by a thick cell wall, a potential barrier to DNA invasion. Removing the cell wall to create protoplasts or spheroplasts increases the chances of genetic transformation. Reseachers adopted this method was adopted and widespread used by these researchers, but some changes have since been have been reported to improve efficiency. This method is based on the technique described initially for protoplast fusion yeast. Yeast cells are recovered in the late stage of growth, the cell wall is weakened with a reducing agent such as mercaptoethanol, and the wall is removed by incubation with an enzyme such as glucanase. Various formulations, such as glucanase enzyme and actinomycete extract have been successfully used. Spheroplasts were then carefully washed with an osmotically equivalent solution of the free buffer and suspended in a solution-containing polyethylene glycol (PEG) and CaCl2 [48]. DNA was added at this stage. For cells to divide, the walls need to be rebuilt. This case requires the cells to be placed in osmotically stabilized agar.

#### **4.2 Gene recognition and gene number regulation**

Both plasmid vectors and chromosomal integration are widely used to introduce genes and control copy numbers into *S. cerevisiae*. Each has an important role, and the choice depends on the overall goal (overexpression, tight control of gene number, etc.) [49]. The plasmids used in yeast are far more limited than the *E. coli.* However, plasmids with little copy number control and isolation stability can be a significant problem even in selective media. Homologous recombination is so efficient in *S. cerevisiae* that integrating genes into the genome provides an alternative and simple mechanism for introducing genes. Chromosomal integration also allows the insertion of several identical or different genes. It is critical for the gene expression of regulated metabolic pathways. There are classes of plasmids that replicate independently in yeast: YIp, YAC, YRp, Yep, and YCp [50–53]. *S. Sacevisiae* has a multi-cloning site (MCS) for inserting expression cassettes. The YRp vector originates from replication such as Autonomously Replicating Sequence (ARS) without partition control. However, this plasmid is extremely unstable and is not widely used in metabolic engineering applications. In contrast, the widely used YCp and YEp vectors have been demonstrated in many applications. The YCp vector (centromere/CEN) has an origin of replication; the centromere sequence is maintained at 12 copies per cell and exhibits high isolation stability in selective media. Strong constitutive promoter expression can significantly affect plasmid stability, reduce average copy counts, and overwhelm intracellular metabolic pathways [54]. In extreme cases, the CEN/ARS vector provides overproduction. Due to the general lack of yeast plasmids, very high copy counts were maintained. On the other hand, defective marker promoters lead to increased copy counts [55]. Hundreds of copies have been reported on selective media, but this high copy count is not essential for survival [54]. Generally, such vectors help with the overexpression of product genes rather than metabolic engineering applications [49]. There are 11 classes of animals' homeobox that share homology and function among yeast and animal (**Table 3**). Today, the use of model organisms to replace animal cells is increasing more rapidly due to animal-free thinking in social development. However, cloning and transformation in mammals remain important [51].



**Table 3.** *Homeobox gene in animal.*

#### **4.3 Cloning in animal cells**

The development of a vector system for gene transformation in animal cells is under consideration [71]. These vectors are required in biotechnology to synthesize recombinant proteins from genes that are not correctly expressed when cloned in *E. coli* or yeast. Human cloning techniques are sought after by clinical molecular biologists seeking to develop gene therapy techniques: Diseases are treated by introducing the cloned genes into patients [71]. The clinical aspect means that the most excellent attention is paid to the mammalian cloning system, but significant advances have also been made in insects. Cloning insects is fascinating because it uses a new type of vector that we have never encountered.

#### **4.4 Cloning in mammals**

Currently, gene cloning in mammals is performed for one of three reasons: (1) To produce recombinant proteins in mammalian cell culture and related farming techniques. Milk. (2) In gene therapy, human cells are manipulated to treat diseases. (3) Achieve gene knockout, an important technique used to determine the function of unknown genes. These experiments are usually performed on rodents, such as mice. Viruses as a mammalian clone vector have been known to be the key to cloning mammals for many years. The first cloning experiment with mammalian cells was performed in 1970 using a vector-based on Simian virus 40 (SV40) [72, 73]. The virus can infect several mammalian species following a lysogenic cycle in some hosts and others. SV40 has the same problem as e and has a calicivirus embedded in it. This is because packaging restrictions limit the amount of new DNA inserted into the genome. Therefore, cloning with the SV40 requires replacing one or more of the existing genes with DNA to clone. The original experiment replaced the late gene region segment, but early gene replacement was also an option [73]. However, the discovery of CRISPR/Cas which is based on cloning technology is one of the essential techniques in gene therapy [74].

#### **5. Conclusions**

Genes are the universal language that controls the nature of all living things, shared homology among organisms. It is always interesting to reveal the evolution of cloning and gene expression in plant, bacteria, and animal cells. Therefore, with the discovery of genetic engineering, possible to exchange good genetic traits which beneficial for human life. In conclusion, genetic transformation is a genetic engineering technique that can be used to understand the function of a gene or several genes in various events in the life of an organism, both prokaryotes and eukaryotes, so that genetic transformation is carried out for two kinds of purposes, namely scientific purposes to determine the function of certain genes in an organism, and economic goals to improve the quality and productivity of an organism to increase the economic value of an organism. In the future, genetic engineering on prokaryotes and eukaryotes perspective can be used for various purposes in the fields of medicine, agriculture, horticulture, forestry, and food.

#### **Acknowledgements**

We thank Badan Penerbit dan Publikasi Universitas Gadjah Mada (BPP UGM) for supporting this publication.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Appendices and nomenclature**



### **Author details**

Endang Semiarti1 \*, Yekti Asih Purwestri1,2, Saifur Rohman3 and Wahyu Aristyaning Putri1

1 Biochemistry Laboratory, Department of Tropical Biology, Universitas Gadjah Mada. Jl. Teknika Selatan, Yogyakarta, Indonesia

2 Research Center for Biotechnology, Universitas Gadjah Mada, Yogyakarta, Indonesia

3 Laboratory of Agricultural Microbiology, Faculty of Agriculture, Department of Agricultural Microbiology, Universitas Gadjah Mada, Yogyakarta, Indonesia

\*Address all correspondence to: endsemi@ugm.ac.id

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Genetic Transformation in Prokaryotic and Eukaryotic Cells DOI: http://dx.doi.org/10.5772/intechopen.103839*

#### **References**

[1] Thesleff I. Homeobox genes and growth factors in regulation of craniofacial and tooth morphogenesis. Acta Odontologica Scandinavica. 1995;**53**(3):129-134

[2] Takada S, Hibara K, Ishida T, Tasaka M. The *CUP-SHAPED COTYLEDON1* gene of *Arabidopsis* regulates shoot apical meristem formation. Development. 2001;**128**:1127-1135

[3] Matsuoka M, Ichikawa H, Saito A, Tada Y, Fujimura T, Kano-Murakami Y. Expression of a rice Homeobox gene causes altered morphology of transgenic plants. American Society of Plant Physiologists. 1993;**5**:1039-1048

[4] Kusaba S, Kano-Murakami Y, Matsuoka M, Tamaoki M, Sakamoto T, Yamaguchi I, et al. Alteration of hormone levels in transgenic tobacco plants overexpressing the rice Homeobox gene *OSH1* [Internet]. Plant Physiology. 1998;**116**:471-476. Available from: www. plantphysiol.org

[5] Sato Y, Sentoku N, Miura Y, Hirochika H, Kitano H, Matsuoka M. Loss-of-function mutations in the rice homeobox gene *OSH15* affect the architecture of internodes resulting in dwarf plants. The EMBO Journal. 1999;**18**:992-1002

[6] Lincoln CL, Ong J, Judy Y, Serikawa K, Hakeaibi S. A knottedl-like Homeobox Gene in *Arabidopsis* is expressed in the vegetative meristem and dramatically alters L6af morphology when overexpressed in transgenic plants. The Plant Cell. 1994;**6**:581-590

[7] Byrne ME, Groover AT, Fontana JR, Martienssen RA. Phyllotactic pattern and stem cell fate are determined by the Arabidopsis homeobox gene *BELLRINGER*. Development. 2003;**130**: 3941-3950

[8] Kubo H, AJM P, MGM A, Pereira A, Koornneef M. *ANTHOCYANINLESS2*, a Homeobox gene affecting anthocyanin distribution and root development in Arabidopsis [Internet]. The Plant Cell. 1999;**11**:1217-1226. Available from: www. plantcell.org

[9] Ohgishi M, Oka A, Morelli G, Ruberti I, Aoyama T. Negative autoregulation of the Arabidopsis homeobox gene *ATHB-2*. The Plant Journal. 2021;**25**(4):389-398

[10] Dong Y-H, Yao J-L, Atkinson RG, Putterill JJ, Morris BA, Gardner RC. *MDH1*: An apple homeobox gene belonging to the *BEL1* family. Plant Molecular Biology. 2000;**42**:623-633

[11] Ejaz M, Bencivenga S, Tavares R, Bush M, Sablowski R. *Arabidopsis thaliana* Homeobox Gene *1* controls plant architecture by locally restricting environmental responses. PNAS. 2021;**18**:1-8

[12] Shen B, Sinkevicius KW, Selinger DA, Tarczynski MC. The homeobox gene *GLABRA2* affects seed oil content in Arabidopsis. Plant Molecular Biology. 2006;**60**(3):377-387

[13] Hendelman A, Zebell S, Rodriguez-Leal D, Dukler N, Robitaille G, Wu X, et al. Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection. Cell. 2021;**184**(7):1724-1739

[14] Hirakawa Y, Kondo Y, Fukuda H. *TDIF* peptide signaling regulates vascular stem cell proliferation via the *WOX4* homeobox gene in Arabidopsis. Plant Cell. 2010;**22**(8):2618-2629

[15] Janssen B-J, Lund L, Sinha N. Overexpression of a Homeobox Gene, *LeT6,* Reveals Indeterminate Features in the *Tomato Compound Leaf 1* [Internet]. 1998;**117**(3):771-786. Available from: www.plantphysiol.org

[16] Lee YH, Oh HS, Cheon CI, Hwang IT, Kim YJ, Chun JY. Structure and expression of the *A. thaliana* homeobox gene *Athb-12*. Biochemical and Biophysical Research Communications. 2001;**284**(1):133-141

[17] Belles-Boix E, Hamant O, Witiak SM, Morin H, Traas J, Pautot V. *KNAT6:* An Arabidopsis homeobox gene involved in meristem activity and organ separation. Plant Cell. 2006;**18**(8):1900-1907

[18] Wang Y, Henriksson E, Söderman E, Henriksson KN, Sundberg E, Engström P. The Arabidopsis homeobox gene, *ATHB16*, regulates leaf development and the sensitivity to photoperiod in Arabidopsis. Developmental Biology. 2003;**264**(1):228-239

[19] Dockx J, Quaedvlieg N, Keultjes G, Kock P, Weisbeek P, Smeekens S. The homeobox gene *ATK1* of *A. thaliana* is expressed in the shoot apex of the seedling and in flowers and inflorescence stems of mature plants. Plant Molecular Biology. 1995;**28**:723-737

[20] Ingram GC, Magnard J-L, Vergne P, Dumas C, Rogowsky PM. ZmOCL1, an *HDGL2* family homeobox gene, is expressed in the outer cell layer throughout maize development. Plant Molecular Biology. 1999;**40**:343-354

[21] Horst NA, Katz A, Pereman I, Decker EL, Ohad N, Reski R. A single homeobox gene triggers phase transition, embryogenesis and asexual reproduction. Nature Plants. 2016;**2**:1-6

[22] Carabelli M, Morellit G, Whitelamt G, Ruberti I, La R, Le SP, et al. Twilightzone and canopy shade induction of the

*Athb-2* homeobox gene in green plants. Developmental Biology Communicated by Walter Journal. 1996;**93**:3530-3535

[23] Bowman J, Eshed Y. Embryonic origin of the shoot apical meristem. Trends in Plant Science. 2000;**5**(3):110-115

[24] Pitzschke A, Persak H. Poinsettia protoplasts-a simple, robust and efficient system for transient gene expression studies [Internet]. Plant Methods. 2012;**8**:2-11. Available from: http://www. plantmethods.com/content/8/1/14

[25] Wang H, Wang W, Zhan J, Huang W, Xu H. An efficient PEG-mediated transient gene expression system in grape protoplasts and its application in subcellular localization studies of flavonoids biosynthesis enzymes. Scientia Horticulturae. 2015;**191**:82-89

[26] Sparkes IA, Runions J, Kearns A, Hawes C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature Protocols. 2006;**1**(4):2019-2025

[27] Xu K, Huang X, Wu M, Wang Y, Chang Y, Liu K, et al. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis. PLoS ONE. 2014;**9**(1):1-7

[28] Tyurin AA, Suhorukova A, Kabardaeva KV, Goldenkova Pavlova IV. Transient gene expression is an effective experimental tool for the research into the fine mechanisms of plant gene function: Advantages, limitations, and solutions. Plants. 2020;**9**:1-19

[29] Twyman RM, Christou P, Stoger E. Genetic Transformation of Plants and Their Cells. Marcel Dekker Inc.; 2002

*Genetic Transformation in Prokaryotic and Eukaryotic Cells DOI: http://dx.doi.org/10.5772/intechopen.103839*

[30] Purwestri YA, Ogaki Y, Tsuji H, Shimamoto K. Functional Analysis of *OsKANADI1*, A Florigen *Hd3a* Interacting Protein in Rice (*O. sativa* L.). Indonesian. Journal of Biotechnology. 2012;**17**(2):169-176

[31] Hutanu D. Recent applications of polyethylene glycols (PEGs) and PEG derivatives. Modern Chemistry & Applications. 2014;**02**(02):1-6

[32] Zarrintaj P, Saeb MR, Jafari SH, Mozafari M. Application of compatibilized polymer blends in biomedical fields. In: Compatibilization of Polymer Blends: Micro and Nano Scale Phase Morphologies, Interphase Characterization and Properties. Oxford: Elsevier; 2019. pp. 511-537

[33] Armstrong CL, Petersen WL, Buchholz WG, Bowen BA, Sulc SL. Plant Cell Reports Factors affecting PEG-mediated stable transformation of maize protoplasts. Plant Cell Reports. 1990;**9**:335-339

[34] Liu YC, Vidali L. Efficient polyethylene glycol (PEG) mediated transformation of the moss *Physcomitrella patens*. Journal of Visualized Experiments. 2011;**50**:1-4

[35] Johnson GCM, Carswell GK, Shillito RD. Direct gene transfer via polyethylene. Journal of Cell Culture Method. Springer Verlag; 1989;**12**:127-133

[36] Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, et al. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/ chloroplast-related processes [Internet]. Plant Method. 2011;**7**(30):1-14. Available from: http://www.plantmethods.com/ content/7/1/30

[37] Nicholson AW. Ribonuclease III mechanisms of double-stranded RNA cleavage. Wiley Interdisciplinary Reviews: RNA. 2014;**5**:31-48

[38] Conrad C, Rauhut R. Ribonuclease III: New sense from nuisance [Internet]. The International Journal of Biochemistry & Cell Biology. 2002;**34**(2):116-129. Available from: www.elsevier.com/locate/ ijbcb

[39] Altuvia Y, Bar A, Reiss N, Karavani E, Argaman L, Margalit H. In vivo cleavage rules and target repertoire of RNase III in *Escherichia coli*. Nucleic Acids Research. 2018;**46**(19):10380-10394

[40] Sun W, Nicholson AW. Mechanism of action of *E. coli* ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant. Biochemistry. 2001;**40**(16):5102-5110

[41] Wang Z, Hartman E, Roy K, Chanfreau G, Feigon J. Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs. Structure. 2011;**19**(7):999-1010

[42] Wilson RC, Doudna JA. Molecular mechanisms of RNA interference. Annual Review of Biophysics. 2013;**42**(1):217-239

[43] Rosano GL, Ceccarelli EA. Recombinant protein expression in *E. coli*: Advances and challenges. Frontiers in Microbiology. Frontiers Research Foundation. 2014;**5**:1-2

[44] Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Bioengineering. 1991;**219**:37-44

[45] Kato T, Haruki M, Imanaka T, Morikawa M, Kanaya S. Isolation and characterization of psychrotrophic bacteria from oil-reservoir water and oil sands. Applied Microbiology and Biotechnology. 2001;**55**(6):794-800

[46] Feller G. Molecular adaptations to cold in psychrophilic enzymes.

Cellular and Molecular Life Sciences. 2003;**60**:648-662

[47] Rohman S. Gene cloning and biochemical characterization of mesophilic and psychrophilic RNases III. [Osaka]; 2005 [Unpublished]

[48] Suzuki Y, Takano K, Kanaya S. Stabilities and activities of the Nand C-domains of *FKBP22* from a psychrotrophic bacterium overproduced in *E. coli*. FEBS Journal. 2005;**272**(3):632-642

[49] Seresht AK, Nørgaard P, Palmqvist EA, Andersen AS, Olsson L. Modulating heterologous protein production in yeast: The applicability of truncated auxotrophic markers. Applied Microbiology and Biotechnology. 2013;**97**(9):3939-3948

[50] Brown TA. Gene Cloning & DNA Analysis: An Introduction. 7th ed. Manchester: Wiley Blackwell; 2016. pp. 260-266

[51] Joska TM, Mashruwala A, Boyd JM, Belden WJ. A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile. Journal of Microbiological Methods. 2014;**100**(1):46-51

[52] van Mullem V, Wery M, de Bolle X, Vandenhaute J. Construction of a set of *Saccharomyces cerevisiae* vectors designed for recombinational cloning. Yeast. 2003;**20**(8):739-746

[53] Goudot C, Etchebest C, Devaux F, Lelandais G. The reconstruction of condition-specific transcriptional modules provides new insights in the evolution of yeast AP-1 proteins. PLoS ONE. 2011;**6**(6):1-12

[54] Benders GA, Noskov VN, Denisova EA, Lartigue C, Gibson DG, Assad-Garcia N, et al. Cloning whole

bacterial genomes in yeast. Nucleic Acids Research. 2010;**38**(8):2558-2569

[55] Bossier P, Fernandes L, Rocha D, Rodrigues-Pousada C. Overexpression of *YAP2,* coding for a new YAP protein, and YAP1 in *S. cerevisiae* alleviates growth inhibition caused by 1,10-phenanthroline. Journal of Biological Chemistry. 1993;**268**(31):23640-23645

[56] Brauchle M, Bilican A, Eyer C, Baily X, Martinez P, Ladurner P, et al. *Xenacoelomorpha* survey reveals that all 11 animal homeobox gene classes were present in the first bilaterians. Society for. Molecular Biology and Evolution. 2018;**10**(9):1-36

[57] Lawrence A, Sauvageau G, Humphries K, Largman C. The Role of *HOX* Homeobox genes in normal and leukemic hematopoiesis. STEM Cells. 1996;**14**:281-291

[58] Pei Z, Wang B, Chen G, Nagao M, Nakafuku M, Campbell K. Homeobox genes *Gsx1* and *Gsx2* differentially regulate telencephalic progenitor maturation. Proceedings of the National Academy of Sciences of the United States of America. 2011;**108**(4):1675-1680

[59] Lee YM, Park T, Schulz RA, Kim Y. Twist-mediated activation of the *NK4* Homeobox gene in the visceral mesoderm of Drosophila requires two distinct clusters of E-box regulatory elements\*. The Journal of Biological Chemistry [Internet]. 1997;**272**(July 11):17531-17541. Available from: http://www.jbc.org/

[60] Makarenkova HP, Meech R. Barx Homeobox family in muscle development and regeneration. In: International Review of Cell and Molecular Biology. Oxford: Elsevier Inc.; 2012. pp. 117-173

[61] Yoshihara SI, Omichi K, Yanazawa M, Kitamura K, Yoshihara Y. *Arx* homeobox

*Genetic Transformation in Prokaryotic and Eukaryotic Cells DOI: http://dx.doi.org/10.5772/intechopen.103839*

gene is essential for development of mouse olfactory system. Development. 2005;**132**(4):751-762

[62] Aldaz S, Morata G, Azpiazu N. The *Pax-homeobox* gene eyegone is involved in the subdivision of the thorax of Drosophila. Development. 2003;**130**(18):4473-4482

[63] Takahashi T, Holland PWH. Amphioxus and ascidian Dmbx homeobox genes give clues to the vertebrate origins of midbrain development. Development. 2004;**131**(14):3285-3294

[64] Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, et al. The *Mohawk homeobox* gene is a critical regulator of tendon differentiation. Proceedings of the National Academy of Sciences of the United States of America. 2010;**107**(23):10538-10542

[65] Serrano-Saiz E, Leyva-Díaz E, de La Cruz E, Hobert O. BRN3-type *POU* Homeobox genes maintain the identity of mature postmitotic neurons in nematodes and mice. Current Biology. 2018;**28**(17):2813-2823

[66] Kumar JP. The *sine oculis homeobox (SIX)* family of transcription factors as regulators of development and disease. Cellular and Molecular Life Sciences. 2009;**66**(4):565-583

[67] Ledford AW, Brantley JG, Kemeny G, Foreman TL, Quaggin SE, Igarashi P, et al. Deregulated expression of the homeobox gene *Cux-1* in transgenic mice results in downregulation of p27kip1 expression during nephrogenesis, glomerular abnormalities, and multiorgan hyperplasia. Developmental Biology. 2002;**245**(1):157-171

[68] Yaguchi J, Angerer LM, Inaba K, Yaguchi S. Zinc finger homeobox is required for the differentiation of

serotonergic neurons in the sea urchin embryo. Developmental Biology. 2012;**363**(1):74-83

[69] Yuan HX, Feng XE, Liu EL, Ge R, Zhang YL, Xiao BG, et al. 5,2′-dibromo-2,4′,5′-trihydroxydiphenylmethanone attenuates LPS-induced inflammation and ROS production in EA.hy926 cells via *HMBOX1* induction. Journal of Cellular and Molecular Medicine. 2019;**23**(1):453-463

[70] Markitantova Y, Simirskii V. Inherited eye diseases with retinal manifestations through the eyes of homeobox genes. International Journal of Molecular Sciences. MDPI AG. 2020;**21**:1-51

[71] Oliver G, Sosa-Pineda B, Geisendorf S, Spana EP, Doe CQ, Gruss P. *Prox 1*, *a prospero-related homeobox* gene expressed during mouse development. Mechanisms of Development. 1993;**44**(1):3-16

[72] Ayala FJ. Cloning humans? Biological, ethical, and social considerations. Proceedings of the National Academy of Sciences of the United States of America. 2015;**112**(29):8879-8886

[73] Herzberg M, Winocour E. Simian virus 40 deoxyribonucleic acid transcription *In Vitro*: Binding and transcription patterns with a *Mammalian Ribonucleic Acid Polymerase1*. Journal of Virology. 1970;6(5):667-676

[74] Mengstie MA, Wondimu BZ. Mechanism and applications of crispr/ cas-9-mediated genome editing. In: Biologics: Targets and Therapy. Vol. 15. Dove Medical Press Ltd.; 2021. pp. 353-361
