*2.2.15 Gene therapy*

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


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**Figure 1.**

*Applied Molecular Cloning: Present and Future for Aquaculture*

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

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 unspe-

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**).

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

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

knockout technologies.

cific off-target effects [52].
