Table 7.
The advantages and disadvantages of genetic manipulation tools applicable to ovine and caprine genomes
| Tool | Uses | Advantages | Disadvantages | References |
|---|---|---|---|---|
| PNI | Insertional transgenesis | The first tool to be applied for the generation of transgenic animals | Random integration, variable transgene copy number, low efficiency | Hammer et al., 1985; Clark, 2002 |
| SCNT | Gene targeting, editing | An alternative that facilitated the implementation of HR gene targeting in species that lack ESCs, a low-level mosaicism | Development of a small proportion of reconstructed embryos that become live offspring, potential complications at birth of offspring as a result of developmental abnormalities | Schnieke et al., 1997; Wilmut et al., 1997; Wilmut et al., 1999 |
| SMGT | Gene transfer, integration | Simple, cost-effective, minimal embryo handling required | Initial doubt with regard to its repeatability, variable results, low incorporation of the exogenous gene | Lavitrano et al., 1989; Wall, 2002; Lavitrano et al., 2006 |
| VMGT | Gene transfer, integration | Able to infect germline cells and dividing or non-dividing somatic cells, delivery of the system to the egg/zygote is less damaging compared to pronuclear injection, high integration | Variability of transgenic expression, potential health risks, limited DNA capacity | Whitelaw et al., 2008; Modric and Mergia, 2009 |
| Recombinases | Integration, selectable cassette excision | Increased gene integration efficiency, offer different forms of modifications including the removal of unwanted DNA | Conservative specificity, in specific cases, pre-introduction of specific target sites within the host genome is required which is an inefficient and time-consuming process, potential toxicity | Xu et al., 2008; Gaj et al., 2014; Olorunniji et al., 2016 |
| Transposons | Integration | Able to integrate transgenes and RNAi-expressing constructs for the mediation of knockdown expression, lower immunogenicity and larger DNA capacity compared to viral systems | Classical transposons are less efficient for gene transfer compared to viral systems, potential cytotoxicity | Muñoz-López and García-Pérez, 2010; Meir and Wu, 2011; Hudecek et al., 2017 |
| RNAi | Gene knock down | Targeting gene expression at mRNA level, useful tool to elucidate gene functions | Variability and incompleteness of knockdowns, potential off-target | Boutros and Ahringer, 2008; Boettcher and McManus, 2015; Bradford et al., 2017 |
| ZFNs | Gene editing | First “practical” endonuclease that has been applied to mediated gene-editing events | Difficult to design, potential off-target, mosaicism in offspring generated from microinjected embryos | Gaj et al., 2013; Gupta and Musunuru, 2014; Oliver et al., 2015; Zhang et al., 2019a |
| TALENs | Gene editing | A simplified alternative of the previously emerged ZFNs | Moderate difficulty in design, potential off-target, mosaicism in offspring generated from microinjected embryos | Bedell et al., 2012; Gaj et al., 2013; Gupta and Musunuru, 2014; Zhang et al., 2019a |
| CRISPR/Cas9 | Gene editing | Simple, cost-effective, customizable, precise compared to other endonucleases, able to mediate multiplex editing | Potential off-target, mosaicism in offspring generated from microinjected embryos | Gaj et al., 2013; and Gupta and Musunuru, 2014; Mehravar et al., 2019; Zhang et al., 2019a |
PNI, pronuclear injection; SCNT, somatic cell nuclear transfer; SMGT, sperm-mediated gene transfer; VMGT, virus-mediated gene transfer; RNAi, RNA interference; ZFNs, zinc finger nucleases; TALENs, transcription activator-like effector nucleases; CRISPR/Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9.