Retroviral Mediated Gene Transfer

Retroviral methodology is an effectual method to integrate interested genes into animal genomes (Van et al., 1985). It is considered as a naturally occurring gene transfering system to integrate DNA into mammalian cells (Varmus, 1998). In 1974, the first healthy mice, that carry copies of foreign viral DNA was made by microinjecting SV40 DNA into the blastocysts (Jaenisch R, Mintz, 1974). Consequently, the germline transmission of viral DNA into the animal genome could be achieved by directly infecting preimplantation stage embryos with Moloney leukemia viruses (MLV) (Jaenisch , 1976). Therefore, the two types of retroviral vectors are established for creating transgenic animals; the first vectors can be derived from the genome of prototypic retrovirus (such as MLV) and the second vectors are derived from the genome of more complex retroviruses (such as lentiviruses). These virus holds the structural gene such as gag,pol and env which can be responsible for virulence characteristics of the virus. For safety reasons these structural genes of retroviruses are frequently modified by removing this part. Though, retroviral lines that used in transgenic animal experiments are ecotropic, they infect only mice or rats. This could be dangerous for human rather than rodent if correct precautions are not taken (Dunn et al., 2005)

Retroviral vectors transgenesis was fruitful in many animal species, including livestock although the technique has some serious drawbacks. The first one is the transgenes brought by a retroviral vector cannot be expressed in the transgenic animal. Silencing of the transgene has been revealed to be affected by the enrollment of gene suppression machinery in the host cells by the promoter and enhancer sequences of the retroviral long-terminal-repeats (LTRs) and the following hypermethylation of the viral promoter sequences by de novo DNA methylation (Jahner and Jaenisch, 1985). Second, it is difficult to package a transgene larger than 10 kb due to the smaller size capacity of such viruses, which can seriously act to limits its use for delivering large genes (Brem, 1993).Third, it was indicated that the retroviral long-terminal-repeats (LTRs) can hamper the mammalian promoters to overcome or directing the host gene expression wrongly (Wells et al., 1999). the last is the breakdown of the nuclear envelope throughout mitosis is necessitates for infection of early embryos and yet the viruses over and over again show a postponement in transgene integration, subsequently germline transmission of the transgene was very low in which mosaic animals were produced.

Replication defective adenoviruses (DNA viruses) were used as vectors to overcome the limitation set by retroviruses in infecting non-dividing cells, (Tsukui et al., 1996), rats and cattle (Kubisch et al., 1997). While transgenic mice, with a tendency for germline transmission were produced (Tsukui et al., 1996), similar success has not been shown in other species (Kubisch et al., 1997). The real innovation in the application of viral vectors in making transgenic animals came after the development of lentiviral vectors (Naldini et al 1996 and Poeschla et al 1996) and their following use in creating transgenic animals (Lois et al., 2002 and Pfeifer et al., 2002). For the reason that of their capability for infecting a large spectrum of cells and embryo, lentiviral transgenesis is now a day the most efficient approach for making transgenic animals, including poultry and livestock.

Livestock species from which transgenic animals were made by a lentiviral vector-based method is developing. It was informed recently that transgenic pigs could be produced with high efficiency using lentiviral vectors, 13 (Hofmann et al 2003) and 31% (Whitelaw et al 2004). Moreover, it was exposed that more than 90% of the genetically modified animals expressed the transgene at a high level. This methodology was also effective in cattle. All of the resulting calves from the transgenic positive embryos expressed the transgene when lentiviral vectors carrying transgenes were used for infjecting oocytes before fertilization however; no transgenic calves were born when preimplantation stage bovine embryos were infected with the same lentiviral vector, (Hofmann et al., 2004).

Regardless of the advantages in using lentiviral vectors, this technique has limitations. Alike to conventional prototypic retroviral vectors lentiviral vectors still have the limitation of about 10 kb of transgene DNA. The application of lentiviral vectors success in certain species seems not to secure the success of the other species. In spite of its achievement in mouse, rat, pig, cattle and chicken, no transgenic monkeys were made by lentiviral transfer (Wolfgang et al 2001). Another drawback, which is common to all transgenic approaches that encompass random insertion into an endogenous chromosome, it is possible for mutating an endogenous genome.

Somatic cell nuclear transfer

Nuclear transfer commonly represented to as cloning that involves the insertion of donor nuclei took from either differentiated adult cells or stem cells into enucleated egg. The enucleated oocyte becomes the host for a nucleus that is transplanted from another cell, such as a skin cell. In this manner of reprogramming future development in which the enucleated oocytes are then transferred to a foster mother for maintenance of gestation or used to generate embryonic stem cells with a genetic match to the nucleus donor for therapeutic cloning. The first mammalian nuclear transfer experiments were demonstrated in mice in early 1980s (Hoppe, and Illmensee, 1982), (McGrath and Solter, 1983). The application of Somatic cell nuclear transfer or cloning techniques may have a great potential to get higher number of offsprings from a single female (Bondioli etal., 1990). Meanwhile the famous cloned sheep “Dolly” was born (Wilmut et al., 1997). Somatic cell nuclear transfer technology has become another methodology available for the production of transgenic animals.

Cloning by nuclear transfer from adult somatic cells is a outstanding manifestation of developmental manipulability of the oocyt by placing nucleues in the cytoplasm that can alter the chromatin structure and control the development of oocyte. In SCNT, all of the donor cell’s genetic information is not transferred, as the donor cell’s left behind its own mitochondrial DNA in the original cell. The resultant hybrid cells utilize the mitochondrial structures of the egg in which it inoculated in. as a result the cloned sheep Dolly(Campbell et al., 1996) that are born from SCNT are not absolute duplicates of the donor of the nucleus. Yet it has a great benefit in transgenesis, rescuing endanger animal and gene therapy.

SCNT, in transgenesis is feasibly the one that has promoted the most increasing the efficiency and reducing costs. It can make genetically altered animals through the cloning of transgenic founder animals or transfection of nuclei with vectors of DNA expression (Baldassarre and Karatzas, 2004). In this method the exploitation of the transfection of exogenous DNA in the nuclei is randomly assimilated into the genome using selective pressure. Likewise, the transgenic cells can be absolutely characterized with respect to the number of copies integrated, integration region and integration of the transgene prior to the nuclear transfer step. Even though the capability for development of the reconstructed embryos is lower, the majority of the offspring are transgenic which makes this technology much more efficient than pronuclear microinjection (Behboodi et al. 2005). Somatic cell nuclear transfer is applied in the gene therapy by using gene targeting method some successes are there. The first production of gene- targeted sheep by somatic cells nuclear transfer that encodes the recombinant human a1-antitrypsin (rhAAT) (McCreath et al., 2000) was efficient and reproducible. In cattle, mastitis and bovine spongiform encephalopathy free cattles were produced (Wall et al., 2005) and (Kuroiwa et al. 2004).pigs that can produce synthesized polyunsaturated fatty acids produced Lai et al. (Lai et al., 2006).The aother application of somatic nuclear transfer is interspecies nuclear transfer is a used to enable the rescuing of extinct animal, or even to bring back species after their extermination. The cloning of highly endangered or extinct species involves the use of a unique method of cloning. Interspecies nuclear transfer operates with a recipient and donor of two different organisms which are closely related species and within the same genus. The Gaur ( Bos gaurus ) nucleus combining successfully with a domestic cow( Bos taurus.) can be a good example in this scheme (Lanza et al., 2000).

The negative side of this technique is the capability to come into the reproductive system after targeting modification, screening, and amplification. Somatic cells of domestic animals have a limited lifespan in vitro for cloning, while some of them maintain totipotency after gene targeting. These aged cells reduce the efficacy of this method, causing in a high incidence of aberrations in cloned embryos, fetuses, and offspring.for instant McCreath et al produced live targeted sheep at an efficiency of less than 4% (McCreath et al., 2002) and Lia et al produced live targeted pigs at an efficiency of less than 2% (Lai et al., 2002). Furthermore, in many species, especially large farm animals, ES cells have not been successfully established. The improvement and solving of these problems could permit the increasing of practical application of SCNT to the generation of transgenic animals for different agricultural, biomedical, and veterinary purposes.

RNA interference mediated gene knockdown

RNA interference (RNAi) is a natural post transcriptional gene regulatory process found in all organisms including animals, plants and fungi (Clark and Whitelaw, 2003. RNA interference (RNAi) is the silencing of specific gene expression facilitated by the accumulation of double-stranded RNA that marks in the suppression of gene expression by degrading mRNA (Rettig and Rice, 2009) Therefore, RNAi can achieve the spatiotemporality and reversibility of gene expression modulation.

Gene silencing become an essential methods of regulating gene expression. It is difficult to remove an existing gene from an animal genome than inserting a foreign gene, unless a knock-out technique is used. This gene knock-out is, challenging, complex and irreversible, if the gene is once knocked out, it cannot be recovered again. The development of RNA interference (RNAi) technology partially overcome this problem, through cleavage of the expressed mRNA or blocking of gene expression, it allows specific, partial and reversible knock-down of the desired gene besides can achieve spatio-temporal regulation of gene expression. Synthetic siRNAs can be transfected into cells or early embryos for transient gene knockdown, (Clark and Whitelaw, 2003; Iqbal et al., 2007). For stable gene suppression, the siRNA sequences must be integrated into a gene construct and constitutively expressed. In bovine embryos, siRNA mediated knockdown has been undertaken to suppress the prion protein (PRNP) gene. (Golding et al., 2006). Porcine endogenous retrovirus (PERV) has been demonstrated in porcine primary cells using RNAi knockdown (Dieckhoff et al., 2007) and in cloned piglets (Dieckhoff et al., 2008). In the case of fish , microinjected myostatin small interference RNA (siRNA) into fertilized eggs of zebra fish, which can eliminating myostatin mRNA and reducing the inhibitory effect of myostatin on muscle growth and generating muscle hyper growth fish (Acosta et al., 2005).

The most important issue in the application of RNAi is how to design effective RNAi sequence and produce constitutively stable expression in cells since all of siRNA do not have an inhibitory effect equally some of them do have such characters. Although the tool of RNAi is not well understood and many enzymes proteins and that are comprised an undefined function, this technique is still promising as the combination of siRNA and lentiviral vector technology offers a method for highly efficient targeted gene knock- down in farm animals (Lyall et al., 2011). It could be easily joined into existing breeding programs and it could be applicable in studies of disease therapy and genetic function analysis.

Zinc-finger nuclease gene targeting technique

The beginning of the zinc-finger nuclease (ZFN) technique, suggest a qualitative leap in gene targeting techniques. ZFN is involved one DNA binding domain and one non-specific endonuclease domain that bind and cut DNA at specific sites by introduce double-stranded DNA fragment. This extrinsic DNA transferred by the induction of the endogenous DNA repair procedure in which homology-directed repair or non-homology terminal junction then the modification of the cellular endogenous gene take place (Li MA, Bradley, 2011) by using Highly specific endonucleolytic enzymes to attain, such as the Fok1 endonuclease cleavage domain bonded with zinc-finger DNA binding domains (Urnov et al., 2005) and custom-designed homing endonucleases (Arnould et al., 2007) collectively known as “molecular scissors”. This technique overcomes the limitation of gene targeting efficiency, which is enhanced by five orders of magnitude (Luo et al., 2011). Genetically distorted zebra fish have been made by targeted gene inactivation directly in the zygote using mRNA coding zinc-finger nucleases (Meng et al., 2008; Doyon et al., 2008). Gene knock-out rats and pigs have been produced by this approach (Geurts et al., 2009; Whyte et al., 2011).

Targeted genetically modified animals were obtain in less than four months using the ZFN technique which can be able reduced the time of experiment with high the efficiency (Carbery et al., 2011) Microinjection of constructed zinc-finger nucleases (ZFNs) in embryos of mouse and rat was used to generate gene knockouts by introducing non homologous end joining (NHEJ)-mediated deletions or insertions at the target site. Using ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague–Dawley and Long-Evans hooded rats and FVB mice, enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins (Cui et al., 2011) Zinc-finger nucleases (ZFNs) are powerful technolgy for producing gene knockouts (KOs) with high efficiency and it has a possibility to apply to other species. Whereas the demonstration of ZFN-mediated gene disruption has been used in expermental animals such as rats, mice and fruit flies, ZFNs have not been used to disrupt an endogenous gene of large domestic species. In recent times, cloned pigs that carries a biallelic ZFN brought the swine a1,3-galactosyltransferase (GGTA1) gene knockout (Hauschild et al., 2011) This approach unlocks a new era for the creation of gene knockout pigs, which could important both agriculture and biomedicine.

The ZFN-gene targeting technique significantly improves the capacity of precise genetic modification of the animal and the efficiency of gene integration, and allows the establishment of large animal models for human diseases and xeno-transplantations. The research of gene function and agricultural breeding can be also beneficial by this approach since it has the ability to target a specific site. However, there are some imperfections in the ZFN as a result of its initial stage in progress.