**2. Genetic engineering for human benefit**

and transferred to the recipient where it can be transcribed into messenger RNA (mRNA) and translated to proteins by utilizing the recipient machinery. The donor protein in the recipient system performs its targeted function to modify the desired character of recipient plant, animal, or microorganism. Genomic DNA manipulations may involve addition of a foreign gene from another genome, deletion of an existing gene, or enhancing the expression of an indigenous gene. RNA interfering (RNAi) technology is used to silence the expression of an unwanted gene by inhibiting the mRNA availability for protein synthesis [1]. The genome-level genetic engineering approaches require an insight in the genome, transcriptome, and metabolome [2] of the organisms under study. Like all other applied fields, genetic engineering requires comprehensive information about the genome structure of the donor and recipient before genetic modification. Decision about the morphological character that needs to be improved, the choice of a particular donor and recipient species, genetic networks, and metabolic pathways involved

Transcriptome analysis is a robust and cost-efficient method which provides information about the internal biological processes, cellular biosynthesis, and metabolic functions of a cell, tissue, or living organism [3]. This technique can be utilized by the genetic engineering scientists for the identification and quantification of genetic factors which positively or negatively regulate a particular trait of interest [4]. Comparison of gene expression profiles of an organism exhibiting the desired traits with the genetically similar organism lacking that trait can help in the identification of genetic factors involved in the development of that trait [5, 6]. These genetic factors might affect that trait positively or negatively. Enhanced accumulation of a particular transcript in the organism with desired phenotype as compared to the reference organism indicates that overexpression of that transcript is required for the exhibition of that trait. This phenomenon is called as positive regulation. In negative regulation, reduced

Positively regulated genes serve as genetic engineering tools for overexpression of a gene regulating a particular trait resulting in the introduction of that trait in genetically modified organism (GMO). For example, in transgenic cotton, expression of crystal protein (Cry10Aa) is responsible for resistance against boll weevil [9]. Advances in gene silencing technology through RNAi have led to utilization of genes which are negatively correlated with the desired traits. In cotton plant, seed-oil content increased by 16.7% by silencing GhPEPC1 gene

Transcriptome analysis and genetic engineering go hand in hand in the modern era of genetic improvements. Comparative transcriptional studies using single gene approaches or high-throughput approaches are used to identify the differentially expressed genes in a specific condition/organism as compared to reference. In single gene approaches, the expression of a gene of interest is quantified in different sets of conditions/tissues using northern blotting or reverse transcriptase polymerase chain reaction (RT-PCR). Northern blotting technique utilizes the gene-specific probes for comparative quantification of mRNAs of the target gene, whereas RT-PCR uses gene-specific primers to amplify and subsequently quantify the mRNA molecules. High-throughput technologies have the power to measure and analyze the expression of all the genes in a set of conditions. Differential display reverse

expression of a gene is responsible for the exhibition of a desired trait [7, 8].

in the expression of a specific trait need to be explored.

214 Applications of RNA-Seq and Omics Strategies - From Microorganisms to Human Health

through RNAi technology [8].

Genetic engineering is the field of science which is revolutionizing the world by manipulating the genome and transcriptome of living organisms to introduce desired traits in them. Since the commercialization of "Flavr Savr" tomato in 1994 [10], 357 GM crops belonging to 27 species all over the world have been commercialized [11], and this number is increasing day by day. Genetic engineering is widely being used for the improvement of crops, animals, fungi [12], bacteria [13], and other organisms to benefit mankind. Insect resistance, herbicide resistance, disease resistance, and abiotic resistance are being incorporated in the industrially important crops to make them tolerant to stresses. Yield and nutritional content of food crops are being modified to improve the feed for humans and animals. Scientists [14] produced transgenic maize with overexpressing *Oryza sativa* myeloblastosis 55 (OsMYB55) gene and found that the transgenic maize became more tolerant to heat and drought stress through activating the expression of stress-responsive genes. Microorganisms (bacteria and fungi) are being genetically engineered for the production of useful enzymes [13], secondary metabolites, beneficial oils [12], and antibiotics on commercial scale to be utilized in the pharmaceutical, food, and medical industry.

In 2010, 29 countries were growing genetically modified crops, and 31 countries had the approval to import GM crops. In USA, more than 94% of the cultivated soybean and cotton while 92% of corn is genetically modified [15]. The commercialization of the first genetically modified animal "AquAdvantage Salmon" for food was approved recently in 2015 [16]. RNA-based genetic engineering technology is becoming more attractive after the approval of white button mushroom for commercialization without stringent testing by the USDA, as this technology does not involve the introduction of foreign DNA [17].

limit the entry of approved varieties across the borders of a country, proper monitoring of

Transcriptome Analysis and Genetic Engineering http://dx.doi.org/10.5772/intechopen.69372 217

The first step in the monitoring of GMO is the detection of transgene in an organism under question. Many methods are being used to detect the genetically modified varieties. GMOs produced by insertion of DNA fragments can be detected by protein-based assays [Enzymelinked immunosorbent assay (ELISA), Western blotting, etc.] or nucleotide-based assays including PCR. PCR-based detection is the most sensitive method which makes use of sequence-specific primers [28]. Due to the abundance of GMOs in the market, it has become very difficult to keep the sequence information of all the transgenes. The advanced highthroughput technologies for GMO detection/monitoring are developed to detect multiple transgenes or related nucleotide components (promoter, enhancer, and terminator) of the cassette [29, 30] in a single experiment. For rapid PCR at atmospheric temperature, various methods have been developed [31]. DNA microarray chips are being developed which contain the probes against all the transgenes present in the commercial varieties [32]. Sampling and hybridization of DNA of a variety under question can detect the presence of any transgene. More efficient, sensitive, and robust methods are required for proper monitoring.

All the above methods are used for the detection of DNA insertion in the transgenic organisms. However, in the RNA-based GMOs, the detection of transgene requires transcriptomic approaches. Transcriptional methods including RT-PCR, gene expression microarray, and RNA-seq can detect all types of GMOs produced through RNA- or DNA-based methods. In transcriptomic approaches, RNA is isolated from the sample and reverse transcribed to produce complementary DNA (cDNA). Due to resemblance in the biochemical properties of RNA and DNA, DNA is often present in the RNA preparations which is eliminated by treating the sample with DNase enzyme. By avoiding this step of DNase treatment, we get both RNA and DNA in the sample. This crude RNA is transcribed and RT-PCR is used for the

The developers of GMOs are required to assess the phenotypic and molecular characteristics of modified organisms. Many countries have adopted regulations for commercialization of GMOs which mainly include the comprehensive risk assessment of the new organism before field trials, to be used as feed/food or before release to the environment. These risk assessment methods mainly involve the comparison of the agronomic traits, composition, animal nutrition, and production of toxins of the new product with commercially available for multiple years and at multiple sites. But these assessments are targeted and require the prior information about the risk. The untargeted risks can be left without evaluation with the potential to

During the screening and selection of a GMO, the emphasis is given to the insertion of the transgene as a single copy without disruption of an endogenous gene, preserving the gene

GMOs is required.

detection of RNA or DNA of the transgene.

harm the environment and health.

**5. Validation of genetically modified organisms**
