Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm-mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ-line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.

We studied the effect of murine leukaemia inhibitory factor (LIF), human basic fibroblast growth factor (bFGF) and porcine stem cell factor (SCF) on the survival and/or proliferation of porcine primordial germ cells (PGCs) obtained from 27-day-old embryos in vitro. PGCs were cultured in embryonic stem cell (ESC) medium supplemented with or without either LIF (1000 IU/ml) alone or LIF together with bFGF (10 ng/ml). They were seeded on mitotically inactivated feeder cells, either STO or transfected STO cells (STO#8), expressing the membrane-bound form of porcine SCF. PGCs were identified by their alkaline phosphatase (AP) activity and counted after 1, 3 and 5 days in culture. After 1 day of culture, PGCs cultured on STO#8 cells showed significantly higher survival than PGCs cultured on STO cells (p < 0.05). The combined effect of SCF and LIF caused a significant increase in PGC number by day 3 of culture when PGCs were cultured on either STO cells (p < 0.01) or STO#8 (p < 0.001). When SCF and LIF were used together with bFGF no increase in the PGC number was observed. Our results suggest that the membrane-bound form of porcine SCF plays a pivotal role in the primary culture of porcine PGCs and that bFGF is not required in vitro.

The generation of transgenic pigs is an entirely new way of breeding. In contrast to classical breeding techniques the objects of manipulation in this case are individual genes rather than the entire genome of an organism. In pigs DNA-microinjection into the pronuclei of zygotes is the only available technique of transferring genetic material developed so far. The process involves collection, manipulation, microinjection, cultivation, and transfer of early embryos and also molecular-biological techniques allowing cloning of gene constructs, preparation of suitable injection solutions, and techniques allowing detection of integrated and expressed transgenes in transgenic animals. Gene transfer in pigs usually yields less than 1% transgenic piglets per injected zygote. Our own experiments have shown that simultaneous transfer of untreated control embryos increases yields from 0.5% to 1%.

Gene transfer in pigs can be employed in particular to increase growth performance and carcass composition by using genes encoding hormones of the growth hormone cascade (GHRH, GH, IGF-I). So far, the effects already known from experiments in mice have not been reproduced in pigs.

We are currently investigating whether the transfer of the influenza resistance gene Mx+ of mice will yield disease-resistant pigs.

Breeding with transgenic animals must take into account that approximately 30% of the primary transgenic animals will be mosaics which will not pass on the transgene to their offspring. Unwanted side effects may also occur during gene transfer. Most important examples are instability of integrated transgenes and variability of gene expression over many generations.

In about 5% of all primary transgenic animals integration of the transgene can be assumed to lead to the generation of insertion mutations. Animals carrying these mutations should not be used for breeding. Furthermore severe health problems may be caused by uncontrolled over-expression of the transgene.

Much more work will be necessary in future before we will be able to employ gene transfer techniques in practical breeding programmes.