**Table 2.**

*Transgenesis in aquaculture.*

#### *Applied Molecular Cloning: Present and Future for Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.88197*

*Synthetic Biology - New Interdisciplinary Science*

environment.

*2.2.14 Transgenesis*

*2.2.15 Gene therapy*

Grass carp (*Ctenopharyngodon* 

Nile Tilapia (*O. niloticus)* and Redbelly Tilapia (*Tilapia zillii)*

*Transgenesis in aquaculture.*

*idellus)*

production and consumption, transport and higher resistance. The other important feature of these vaccines is the ability to put several antigens in the plasmid, resulting in immunization against all agents [43]. In 2005, APEX-IHN (Novartis/Elanco) became the first DNA vaccine licensed for commercial use in aquaculture for protection of Atlantic salmon against Infectious Hematopoietic Necrosis Virus (IHNV) in British Colombia. In 2017, the European Commission through the European Medicines Agency (EMA) granted marketing authorization of CLYNAV (Elanco), a polyprotein-encoding DNA vaccine against Salmon Pancreas Disease Virus (SPDV) infection in Atlantic salmon (*Salmo salar*) for use within the EU. However, administration of vaccines typically requires individual handling and treatment of all production fish, which can be expensive and impractical in a large-scale production

Transgenics are those genetically engineered organisms which have heterologous DNA (transgene) integrated stably into their genome through artificial means like microinjection, electroporation, sperm mediated transfer, lipofection, retrovirus, etc. The transgene construct carries a target gene, encoding product of interest and regulatory elements that regulate the expression of the gene in a spatial, temporal and developmental manner [44]. Since the development of the first transgenic fish in 1984, a wide number of transgenic fish species have been produced (**Table 2**) to

In the mid-twentieth century, researcher demonstrated that the rate of mutagenesis could be enhanced with radiation or chemical treatment [46, 47]. Later with the help of transposons, targeted genomic changes were made in various model organism including medaka and zebrafish [48–50]. But due to prevalence of transposon machinery in these fish, longer time requirement for generating particular line and

**Species Foreign gene Desired effect** Striped bass (*Morone saxatilis)* Insect genes Disease resistance

hLF

promoter

+ CMV

IgM genes

Atlantic Salmon (*Salmo salar)* Mx genes Potential resistance to

rainbow trout GH

hLF + common carp β-actin

*cecropia)* cecropin genes

cecropin-like peptide genes

Shark (*Squalus acanthias* L.)

Improved disease resistance

Increased disease resistance to

Increased disease resistance to grass carp hemorrhage virus

Enhance bactericidal activity

Enhanced bactericidal activity against common fish

pathogens following treatment with poly I:C

Enhanced immune response

pathogens

bacterial pathogen

improve growth, disease resistance, cold resistance, etc. [45].

Common carp (*Cyprinus carpio)* Salmon and human GH;

Channel catfish (*Ictalurus punctatus)* Silk moth (*Hyalophora* 

Japanese Medaka (*Oryzias latipes)* Insect cecropin or pig

**60**

**Table 2.**

concerns about transgenics associated wild genepool contamination and biodiversity degradation has led aquaculture researchers to focus on other knockdown and knockout technologies.

In fish, antisense morpholinos, small interfering RNA (siRNA) and PNAs (peptide nucleic acid) are widely used to transiently interfere with gene function. Morpholinos are typically 25 bp long oligos that specifically interfere with gene function based on their complementarity to the target sequence either by blocking translation initiation or by interfering with splicing. The non-ribose-based backbone renders morpholinos insensitive to enzymatic degradation. PNAs have a higher affinity for RNA, yet they are less soluble and therefore the *in vivo* use is limited. Dorn et al. [51] changed the chemical composition of the PNA backbone to increase solubility and showed efficient knockdown of the *six3* gene in medaka. In most cases, the chemical/RNA is micro-injected or electroporated into fertilized eggs at early cleavage stages to ensure a ubiquitous distribution to all cells of the developing embryo. If thus applied, they interfere with gene function during early development. To study gene function during later stages, morpholinos can be activated conditionally by light-induced uncaging. However, recent results in zebrafish indicate that morpholino-based gene knockdown often results in unspecific off-target effects [52].

To overcome abovementioned complications advanced genome editing techniques were developed, in which, no genetic material from another species is introduced and thus the genome remains untainted. Although tilling (target induced local lesion in genome) was first of this kind, it mostly creates single point mutation and requires large screening. Some of the next generation gene editing tools used in fish are zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs) and CRISPR/Cas system. Mutations can be achieved by introducing double strand breaks into the target gene and non-homologous end joining (NHEJ) repair mechanism is used to produce insertions or deletions in a site-specific manner resulting in permanent disruption of the function of the target gene. On the other hand, exogenous gene sequence can be introduced into the genome by co-delivering the targeted nucleases along with a target vector containing the DNA homologous to the break site for gene correction (**Figure 1**).


#### **Figure 1.**

*Comparative evaluation of various knockout technologies used in fish manipulation.*


*M, morpholino; Z, zinc finger nuclease (ZFN); T, transcription activator-like effector nucleases (TALEN), C, clustered regularly interspaced short palindromic repeats (CRISPR).*

#### **Table 3.**

*Genome editing using ZFN, TALEN and CRISPR system in varies model and non-model fish species.*

Theoretically, ZFN is an ideal tool for inducing mutations at target DNA sites in any organisms [53]. However, its application has been constrained by limitations in zinc finger domain design and construction as well as low efficiency [54]. Compared with ZFN, the recently emerged TALEN provides us a more advanced approach for genome editing; it is much easier to construct plasmids for expressing TALE proteins, making this technology easily available to most molecular biology laboratories. Because of this and its high specificity and efficiency, TALEN has quickly replaced ZFN as a dominant platform for genome editing since its establishment in 2011 [55]. Unlike ZFN and TALEN, the nuclease Cas9 is guided towards the target DNA site by a small guide RNA followed by random cleavage of the DNA. Particularly, the rapid emergence of CRISPR/Cas9 caused a paradigm shift in the research community [56]. There is complementary usage of these two technologies in recent years, as CRISPR/Cas9 works as monomer, it consists of protein and RNA and produces blunt end, while TALEN works as dimer, it consists of protein only and produces cohesive ends [57]. Although each one has its associated pros and cons [58], TALENs and CRISPR technologies have comparatively high specificity and efficiency with low off target effect [59]. Not only the methodology, but selection of delivery methodology (microinjection, electroporation, etc.), target tissue, and host is critical for ensured success in

**63**

**Table 4.**

*Applied Molecular Cloning: Present and Future for Aquaculture*

**Category Type of approach Popular methods**

Forward genetics Chemical mutagenesis ENU mutagenesis Transposon mutagenesis

analysis

Genomics High throughput

Reverse genetics Antisense and small

*Summary of molecular biology application in fish.*

RNA

Conditional knockdown

Transgenesis Meganuclease ISecI

Recombinases (site specific)

Molecular genetics Reporter cell line Promoter analysis,

aquaculturable strain production. Numerous genes are being knocked out using various techniques and some of them are already adapted for commercial aqua-

With the continual growth of global aquaculture, fish production continues to grow globally and till date only a small proportion of the aquatic animals come from managed breeding especially through applied molecular cloning and genomics (**Table 4**). The molecular biology of aquatic organisms offers many opportunities for rapid genetic gains as new genetic techniques make the improvement feasible in a wider range of model and non-model species. The future of molecular biology in aquaculture is bright with the technologies mentioned above being cheaper than ever, widely available and easily applicable in laboratories. However, the results obtained from these methods should not be conclusive without additional information, such as clinical diagnosis, as the mere detection of a certain pathogen does not

Microarray, NGS, whole genome bisulfate

Sleeping beauty, AcDs, Tol2, EnSpm-N6

Morpholino, PNA, SiRNA, shRNA

PhiC3, Cre-loxP, BAC, Fosmid, YAC

CDNA library, RNA-seq, microarray, QPCR, PCR

sequencing

RFLP

Marker based analysis Rad sequencing, microsatellite, SNP, AFLP, RAPD,

Micro RNA miRNA sponges, miRNA knockdown, miRNA mimics

Tet on/off

Transposon Sleeping beauty, AcDs, Tol2, EnSpm-N6

Transactivation LexPR, Gal4, tet on/off, heat shock protein

spectrophotometry

Tilling ENU mutagenesis

Cell lineage Gaudi toolbox

Transcriptomics RNA detection In situ hybridization, expressed sequence tagging,

Proteomics Protein detection Antibody based analysis, chromatography and

Genome editing ZFN, CRISPR/Cas9, TALEN

*DOI: http://dx.doi.org/10.5772/intechopen.88197*

culture (**Table 3**).

**3. Conclusions**

aquaculturable strain production. Numerous genes are being knocked out using various techniques and some of them are already adapted for commercial aquaculture (**Table 3**).
