Kevin A. Ward

Transgene-mediated modifications to animal biochemistry.

The application of gene-transfer technology to domestic animals provides a way for the introduction of genes encoding biochemical pathways that are currently nonfunctional in these animals. This might provide a mechanism for increasing the availability of specific substrates that currently limit certain production characteristics, such as the production of wool. The progress and problems associated with recent attempts to transfer a cysteine biosynthetic pathway and a glyoxylate cycle to sheep are discussed, in addition to the extension of this concept to other biochemical pathways.

Phosphorus-friendly transgenics

Transgenic animals engineered to express a bacterial enzyme that liberates phosphate from animal feed may provide a solution to a common form of environmental pollution.

Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-aceB gene-ovine growth hormone (GH) gene (3′ GH sequence) construct was fused to the ovine MT-Ia promoter-aceA gene-ovine GH gene (3′ GH sequence). Therefore, in this single DNA sequence, bothaceA andaceB are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of theaceB-aceA gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressedEscherichia coli cells.

The commercial and agricultural applications of animal transgenesis.

The commercial potential for transgenesis techniques is substantial, particularly in the fields of animal and plant agriculture. This results from productivity being a function of genetic potential and interaction with the environment, but environmental factors being only partially subject to influence by the farmer. Thus, concentrating on genotype improvement becomes an important goal if substantial cumulative gains in productivity are to be made. Historically, the genetic potential associated with important animal production traits, such as wool growth, milk yield, and body growth, has been improved by selective breeding, whereby phenotypically superior animals are used as parental stock for following generations. The high quality of the domestic animals in use in farming today compared with those of earlier centuries is witness to the success of the approach, but nevertheless, the method has significant limitations that have frustrated animal breeders for many years. The complex genetic interactions that combine to produce a particular animal phenotype result in slow genetic gain, averaging at best about 1-3% per year. In addition, separating a desired production trait from one or more undesirable traits is often very difficult. However, the most important of these limitations is the inability to transfer genetic information between species, because of the biological barrier that prevents interspecific breeding.

The application of transgenic techniques for the improvement of domestic animal productivity

The application of transgenesis techniques to domestic animals has now been achieved in all the major species of domestic animals. In this review, the progress towards genuine practical applications of the technique is examined. Areas which appear to have made progress during the past several years include the evaluation of animals with elevated growth hormone levels, the introduction of novel metabolic pathways, the expression of transgenes in the mammary glands of pigs and sheep, and the real possibility that functional immunoglobulin genes might be used to confer genetically-inherited disease resistance to commercially-valuable animal breeds.

Altering nutrient utilization in animals through transgenesis

Improved domestic animal productivity is necessary in order to provide for an increasing world population over the next two to three decades and such improvement would be aided by an increase in the efficiency of nutrient utilization. This can be achieved by conventional genetic selection protocols but progress by this approach is slow. A more rapid but as yet largely unproven technique is the direct modification of the genome which can be achieved by the transfer of recombinant DNA to the nuclei of early embryos. This new technology is potentially powerful because it allows the direct transfer of genes without regard to inter-species barriers to breeding. However, it raises a new set of problems associated with the integration and expression of the foreign genetic information in the new genome. In this review the application of the technology to increasing nutrient utilization and increased productivity are discussed. Two areas have received substantial attention in the 15 years since the technique was first applied to domestic animals. First, the current status of the modification of growth hormone levels to improve productivity and feed utilization efficiency is reviewed, with current results suggesting that several of the projects may soon be approaching field trial status. Second, the introduction of novel biochemical pathways to domestic animals to provide them with different sources of the substrates required for growth and production is discussed. Recent results obtained in the introduction of a cysteine biosynthetic pathway to animals is reviewed. While this line of research remains some distance from commercial application, it provides a useful example of the powerful possibilities inherent in the new technology. However, it also serves to highlight some of the difficulties that might be expected as new genes are expressed to produce enzymes that must fit compatibly with existing animal biochemistry.

Production of Transgenic Sheep

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 metallothioneinovine growth hormone fusion gene, GH9, 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.