Over the past two decades, many approaches for transferring genes into the genome of domestic animals have been devised. The main purposes of transgenesis are: to increase animal capabilities, to knock down or silence both onco-genes and deleterious genes, and to produce a pharmaceutical protein. Transgenesis techniques include pronuclear microinjection (PNM), somatic cell nuclear transfer (SCNT), viral infection (VI) and sperm-mediated gene transfer (SMGT). The first transgenic mouse was produced by using the PNM technique and transgenic animals from other species (rabbit, sheep, pig and cattle) have been produced thereafter. However, the PMN technique had certain drawbacks: low efficiency, random integration site of the transgene and a high mosaic rate. For this reason, other alternative techniques have been devised to overcome its drawbacks. The most reliable method for transgenesis which bypasses mosaics is SCNT. However, this method is complicated and tedious; with multiple stages that need setting up. VI has been used to transfer genes into the oocytes and zygotes with high efficiency and versatility. In spite of its simplicity, the maximum transgene length should be less than 8.5 kb. Currently, spermatozoa are considered as an alternative method of carrying transgenes into the oocytes with minimum technical demands. In contrast to VI, SMGT is being used successfully to transfer different kinds of BACs with more than 200 kbp into mouse oocytes. The present review summarizes the methods by which transgenes can be introduced into zygotes of domestic animals.

Over millenia humans have shaped the genetic composition of today’s livestock. Traditional breeding schemes and, more recently, marker assisted selection strategies have been successfully used for incremental genetic improvement of livestock. In principle, transgenic technology, which creates the potential to enhance existing characteristics at unprecedented magnitude and speed, can be seen as the logical progression from these more traditional efforts to modify livestock genomes. Unlike traditional breeding and selection, the technology is not restricted by the species barrier and can utilise the gene pool of other species to introduce entirely novel and unique characteristics. The recent development of cell-mediated transgenesis for livestock based on somatic cell nuclear transfer allows for the introduction of a wide repertoire of genetic modification. This includes not only additive strategies to introduce a new gene function (gain of function) but also the deletion of gene functions (knockout, loss of function), replacing a gene function with a different one (knockin, exchange of function) or the transfer of genes in a spatial-temporal manner (conditional knockout). The use of the mammary gland’s high protein production capacity in dairy animals for the production of biopharmaceutical proteins in the milk has so far been the main driver for this technology platform due to strong economic incentive, ethical justification, greatest public acceptance and relative simplicity. The first biomedical product arising from a transgenic goat has recently been approved bringing transgenic technology into commercial reality. The genetic engineering of livestock provides also exciting opportunities to incorporate additional health benefits into important foods, such as milk, thereby creating entirely novel foods with unique properties not achievable by conventional means. Such food applications are however much more complex than biopharming and while presently in the research and development stage, their eventual introduction into the food chain remains some distance in the future.

Objective: The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose ofin vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection.Materials and Methods: In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches.Results: PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique.Conclusion: The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperminjection (ICSI) method.