Colin D Nancarrow James T A Marshall and Kevin A Ward

1. Introduction

The production of transgenic sheep has proven difficult compared to the mouse and lower animals. The work load is far greater and the rates of success far less by most criteria. However, the benefits to human and animal health and agricultural productivity are potentially enormous (Ward and Nancarrow, Chapter 5) and support for the continuation of the work is assured. Unfortunately, the low rate of transgenesis for sheep, at about 1% of injected, transferred embryos, means that investigation of the regulation of expression of the transgenes, their phenotypic effects, and optimization of the fusion gene constructs, all of utmost importance to the agricultural industry, can seldom be addressed. We know now that the mouse may not be a good model for the sheep, an example being the ovine metallothionein-ovine growth hormone fusion gene, GH9 (1-3), for which expression and phenotypic effects were quite different for sheep and mice. In sheep, pronuclear microinjection of several hundred copies of the foreign gene into embryos is the only published method used to regularly produce transgenics and it will be the standard by which future methods for incorporation of the transgene are judged.

Here, we are considering the production of transgenic sheep, from preparation of the animals through to the establishment of pregnancies in recipient ewes. Particulars relating to the preparation of the fusion genes and the identification of transgenic progeny are common with other species and will be detailed in other chapters of this book. Readers are referred to an excellent review of the more general aspects of production of transgenic ruminants by Wilmut and Clark (4).

This chapter details methods that allow for the generation of transgenic sheep by the physical introduction of DNA into embryos. However, other methods for producing transgenic sheep are being developed and their potential is discussed below.

1.1. Sperm as Vector

The report of Lavitrano et al. (5) whereby transgenic mice were produced following in vitro fertilization with sperm that had been premcu-bated with DNA created great hope, particularly within those working with farm animals. Although Gandolfi et al. (6) have reported similar success in pigs, many other laboratories, including ours, have failed to repeat the phenomenon, particularly in mice. However, there is recent evidence that DNA can be carried into cattle eggs at fertilization and subsequently identified in blastocysts by the polymerase chain reaction (PCR) (7,8). The significance of these results is that treated spermatozoa may eventually be available frozen in straws for artificial insemination (AI), a far cry from microinjection. Perhaps with the appropriate genes, we will eventually see treated semen used in a "terminal" fashion, to produce a high proportion of transgenic animals that will be used for their immediate phenotypic effect and not for transmission of the gene through the germ line.

The problems with this approach have been the low rate of integration, the rearrangements that occur in the transgene, and the possibility of formation of nonintegrated, replicating episomes resulting from such sequences as the SV40 origin of replication. More research is needed to evaluate further this interesting method.

1.2. Retroviral Vectors

It is possible for recombinant, attenuated vectors to be integrated into chromosomes but the success in producing transgenics has been limited. Problems relate to size limitations on the transgene that can be carried, interference of transgene expression by viral sequences, the high chance of producing chimeric animals, and a slight but important possibility of recombination with disease viruses (4,9).

1.3. Embryo Stem Cells

Perhaps the most important of new approaches to the many problems of production of transgenic livestock is the use of cultured embryo stem cells. These cells can be prevented from differentiating in culture and when introduced into the blastocoele cavity, they can take part in formation of any or all parts of the conceptus. However, chimeras may still be obtained and germ-line transmission may not be possible in all offspring. Transgenes can be introduced into these pluripotent mouse cells by electroporation or chemical means, the integration can be site-directed if required and selection of only those cells transformed can be carried out (10). Despite encouraging results with porcine and, to a lesser extent, ovine lines (11), embryonic stem cells have not yet been established. These methods are so important to genetic engineering that it will only be a matter of time before ruminant stem cell lines are established and used for production of transgenics.

Microinjection remains as the method for production of transgenic sheep at present, despite large efforts being put into other areas. There has been little improvement for nearly a decade in this technique and like many groups we can only hope that our search for more efficient methods will be successful. The most encouraging appear to be the use of sperm as a vector and the establishment of readily accessible methods of producing embryo stem cells. These two methods can be seen to have differing uses in genetic engineering. Not until one of these is developed to the extent that the production of transgenic embryos becomes a matter of course will the scientific community be able to get on with the most important work of all, which is to investigate gene constructs and evaluate their phenotypic effects on animal health and production.

2. Materials 2.1. Animals

The breed of our choice has been, for practical reasons, either the Australian Merino or a Border Leicester x Merino (BLM) cross. In the ideal situation, one should use the breed for which the transgenes have been tailored (see Note 1).

The major sheep breeds have similar reproductive physiology and so it is expected that the methods discussed here will suit all, with little alteration being necessary. However, parameters such as the timing and efficiency of detection of pronuclear visibility appears to vary considerably from experiment to experiment and this aspect may need to be investigated when using new breeds and with different environmental conditions operating.

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