Development of Germline Manipulation Technologies in Livestock

Abstract

Breeding, based on conventional selection, has been the mainstay for livestock genetic improvement for more than 70 years, and is still so today Sophisticated statistical and computing tools now enhance conventional genetic selection, nevertheless traits such as fertility and disease resistance have still proved difficult to improve Gene transfer technology (transgenesis) offers the potential, as yet unproven, to modify these types of traits A transgenic animal carries integrated DNA sequences in its genome The introduced DNA can be derived from species other than the host and can be modified in vitro prior to being introduced into the germline Therefore, transgenic livestock overcome some of the limitations of classical animal breeding regimes, where importation of genes by crossbreeding is limited to those traits already present within a given species The most used method for introducing genes into the germline of animals involves the direct microinjection of DNA into the pronuclei of fertilised eggs Pronuclear microinjection, although conceptually simple - a fine needle is used to pierce the pronucleus and the DNA is injected - requires special equipment and considerable dexterity on behalf of the person involved By adapting the techniques employed for gene transfer in mice, pronuclear microinjection has been used to generate transgenic farm animals The first attempts to genetically modify livestock owe much to pioneering experiments in mice, where the introduction of growth hormone gene dramatically increased the growth rate and final size of the animals By contrast, the same approaches in livestock did not prove successful Indeed, in terms of modifying livestock for agricultural purposes, most of the early expectations were not realised Rather, it has been the development of novel uses of livestock, particularly for human medicine, that has led the way and advanced this technology The majority of transgenic livestock have been produced using this method but it only allows gene addition For gene removal the integration of the introduced transgene has to be targeted to the gene of interest This requires a relatively high frequency of homologous recombination that occurs in embryonic stem (ES) cells Thus the desired genetic modification can be identified and selected for while the cells are grown in culture This enables vastly more sophisticated genetic changes to be engineered, including gene knock-out Unfortunately, ES cells have only been isolated for mice and even in this species there are only a few permissive strains The lack of methods for gene knock-out in livestock was the driving force leading to the development of nuclear transfer technology This technique was made famous through the generation of 'Dolly' It is fair to say that although catching both the scientific and media in a frenzy of cloning issues, perhaps the greatest legacy of nuclear transfer will be the development of cell based therapeutic strategies based on stem and somatic cells to treat human genetic diseases Although 'Dolly' is not herself transgenic, this technique does offer the potential to make transgenic animals more efficiently than by using the pronuclear microinjection method This is primarily because all founder animals are transgenic and a flock/herd of clonal animals can be produced within one generation More importantly, nuclear transfer uses cells grown in culture therefore, for the first time, allows precise changes to the germline of ruminants to be attempted This has now been shown to be possible, a sheep carrying a disruption of the PrP gene, a targeted insertion into the collagen gene and pigs that have a deletion of the galactosyltransferase gene having been produced However, the generation of knockout transgenic livestock is a hugely demanding technical and financial undertaking First, the techniques utilised efficiently in mice, do work in livestock cells but are considerably less effective Second, the stringent selection and extended in vitro culture required for targeting somatic cells (the target for the genetic modification prior to nuclear transfer) reduces their developmental potential, compounding the high cost and low efficiency of nuclear transfer Finally, nuclear transfer in livestock is beset by the losses in utero and after birth, having both a welfare and economic cost In summary, yes we can generate gene knock-out livestock using nuclear transfer but unless there is a conceptual leap in our understanding of the technique it will not become common place At the beginning of last year a new approach to transgenesis was reported for the generation of transgenic mice and rats Two groups demonstrated that lentivirus (specialised retrovirus) vectors can be used to efficiently introduce foreign DNA into the germline There appears two dramatic advantages to this technology which make it very appealing for use in livestock Only a fraction of the resources needed for conventional pro-nuclear injection would be required, given the DNA transfer efficiencies reported in the mouse It is over 18 years since the first demonstration that transgenic livestock can be produced Subsequently, the development of nuclear transfer technology was set to revolutionise this area of biotechnology, since it overcame the lack of livestock ES cells Certainly it does enable gene-targeting approaches to the generation of transgenic livestock to be performed; although this produces a recessive mutation in the first instance Both pronuclear injection and nuclear transfer are inefficient methods for modifying the germline of animals The recent development of new methods of transgenesis based on viral vectors again offers an avenue to overcome the current restricted application of transgenesis in livestock