In 1984, I was approached by conference organizers with the request that I give the banquet speech at the first international conference ever held on genetic engineering of animals. Specifically, I was to address the topic of social and moral issues raised by the advent of this new and powerful technology. Flattered, stimulated, challenged, and totally ignorant, I accepted, confident of my ability to rise to the occasion by standing on the shoulders of my predecessors. Unfortunately, a brief visit to the university library shattered my preconceptions – I had no predecessors! My talk, in its published version, would be the first paper ever done on this major topic. Suddenly, I saw my task under a new and harsher light. The buck stopped – and started – with me. Truly an academic's nightmare.

Seeking a purchase on the topic, I solicited dialogue from colleagues in my department. “Genetic engineering of animals,” mused one such partner in discussion, “You're talking about the Frankenstein thing.” His remark was largely ignored by me at first, as it seemed to me flippant and shallow. It was only later that I realized that he had opened a portal into the issue by forthrightly expressing what in fact rises to most people's minds when genetic engineering is mentioned.

A GRESHAM'S LAW FOR ETHICS

In the mid–nineteenth century, the British economist Thomas Gresham enunciated the brilliant observation that became known as Gresham's law, that bad money drives good money out of circulation. In other words, if people are faced with the option of paying a debt with either of two currencies of the same face value, they will pay with the one possessing lower intrinsic value. A similar law could be articulated regarding social-ethical questions – especially those associated with science and technology: Bad moral thinking tends to drive good moral thinking out of circulation.

As in the case of money, it is not difficult to see why this should happen. In our society, there is little occasion for reflection on moral questions. This is a fortiori the case regarding those questions associated with science and technology, for one must be scientifically literate before one can engage the ethical questions generated by science.

As I shall shortly discuss, scientists do little to educate the public about moral issues, because most are trained to ignore them. In practice, then, what emerges as “ethical or moral issues” is shaped by the media. The media, as one reporter told me during the Baby Fae case, are “primarily interested in selling papers.” What sells papers is what can be packaged in small, provocative bits and what can be dramatically presented as black-and-white extremes.

The explosion of knowledge in the biological sciences is now widely recognized. This intellectual revolution has involved some remarkable examples of lateral thinking, and the production of transgenic animals is one such application. The development of these new animal types has depended on the exploitation of cloned genes and techniques capable of achieving the integration of transgenes into the chromosal DNA of the relevant animals. Although the dream of directly modifying an animal's genetic endowment was long cherished, no one, at least until ten years ago, could have guessed how rich the benefits of the transgenic technology would be. We now stand at a scientific watershed, and interesting and productive applications have already appeared in genetics, medicine and agriculture. Obviously they are only the beginning: The technology is now proven and the future looks promising in innumerable ways.

This book attempts to provide the reader with a picture of what has been achieved and where the methodology is most likely to go from here. We have chosen to divide the topic by animal groups, partly because the technology varies from group to group, but even more because present and future applications differ strikingly among organisms. Thus, whereas the greatest appeal of transgenic insects is their power to throw light on problems like mutagenesis and gene regulation, the greatest appeal of transgenic mammals is their contribution to agricultural productivity and pharmaceutical research.

I am most grateful to the contributors to this volume for their expenditure of time and energy.

The introduction of novel genes into fish of many species has become a common procedure. It has been reviewed by Ozato et al. (1989), Chourrout et al. (1990), Cloud (1990), Maclean and Penman (1990), Fletcher and Davies (1991), Guise et al. (1991), Houdebine and Chourrout (1991), Powers et al. (1992a) and Hew and Fletcher (1992). A comprehensive review would now be a major task. Instead we attempt a shorter appraisal of certain critical aspects of this research area.

Fish species selection

The species of fish which have been subjected to transgenic induction include Atlantic salmon (Salmo Salar; see, e.g., Shears et al. 1991), rainbow trout (Oncorhynchus mykiss; e.g., Guyomard et al. 1989), tilapia (Oreochromis niloticus; Brem et al. 1988; Rahman & Maclean 1992a), carp (Cyprinus carpio; Zhang et al. 1990), channel cat-fish (Ictalurus punctatus; Dunham et al. 1987), African cat-fish (Clarias gariepinus; Muller et al. 1992) and northern pike (Esox lucius; e.g., Guise et al. 1992), and attempts have been made with sea bass (Sparus auratus; Cavari et al. 1993). In addition to these commercially important species, certain “model” fish have been used, notably goldfish (Carassius auratus; see, e.g., Yoon et al. 1990), loach (Misgurnus fossilis; e.g., Kozlov et al. 1988), medaka (Oryzias latipes; e.g., Ozato et al. 1986) and zebra fish (Brachydanio rerio; e.g., Stuart et al. 1990).

Unless there are compelling local or strategic reasons for choosing salmonids or other slow-maturing species, it seems that small, fast-maturing species make better initial choices in the present stage of development of this field. Zebra fish and medaka make excellent models, especially the former species, for which a substantial genetic background exists.

Introduction

For many years, transgenic research in eukaryotic organisms has been dominated by only three species, namely Drosophila, the mouse and yeast. Perhaps at the forefront, research with the fruit fly Drosophila melanogaster has, to an unprecedented degree, shown how transgenic technology can be used not only to manipulate genes, but also to extend our understanding of molecular genetics. We therefore feel it appropriate to devote the first half of this chapter to a description of the development of transgenic technology in Drosophila melanogaster and an overview of the ways in which this technology has been applied. The second section concentrates on attempts to apply the same technology to non-drosophilid insects. The future of transgenic insects is then briefly discussed in order to explore the potential that transgenic technology may have in insects of economic, agricultural and medical significance.

Drosophila melanogaster

Historical introduction

One chance event has, more than any other in recent times, generated an explosive increase in what is known of the molecular biology of Drosophila melanogaster. That chance event was the finding of male recombination among the progeny of crosses between wild caught males and laboratory stock females, reported by Hiraizumi in 1971. Male recombination is not normally seen in this species, and this aberration, once associated with work elsewhere, ultimately led to the unearthing of the P transposable element family. We now know that male recombination is one of a syndrome of germ-line-specific genetic aberrations, collectively referred to as ‘hybrid dysgenesis’ (Kidwell et al. 1977), which result from transposition of the P element.

Introduction

The possibility of expressing foreign genes in mammals by gene transfer has opened new dimensions in the genetic manipulation of animals. The basic techniques of gene transfer were developed in mice, which have been most extensively used in such experiments because they are ideal for studying gene expression during development and for establishing animal models of carcinogenesis and other diseases. Additional applications include analysis of mutations and the use of the transgene as a genetic marker (Jaenisch 1988).

Only a few years after the first successful gene transfer into mice, the new technique was used with farm animals, offering the prospect of completely new breeding strategies and other novel applications. However, despite a decade of gene transfer experiments in farm animals, only a few applications have achieved fruition (reviewed in Wall et al. 1992). This is mainly because of fundamental experimental difficulties with these species. In comparison with mice, farm animals have very long generation intervals and the time scale of a transgene project is thus extremely prolonged (Brem 1988, 1989). Naturally this problem is also experienced in conventional breeding programmes, but the major advantage of gene transfer is that significant advances can be made in one generation, whereas conventional breeding techniques require several.

Among the theoretically possible techniques of transferring genes, the only one successfully applied to farm animals so far is microinjection of DNA. Researchers have been rather reluctant to perform gene transfer experiments using retroviral vectors due to the slight risk of recombination with wild-type viruses.

Introduction

The use of transgenic rodents, in particular transgenic mice, to address a number of diverse biological problems has been so vast that this review chapter cannot cover every facet of transgenesis. We hope that by explaining the technology associated with the generation of transgenic mice and by focusing on a few current areas of research where transgenic animals are proving invaluable, we will help the reader understand the use of transgenic animals in research, as well as the power and limitations of this approach.

In this review a transgenic animal is defined as an animal that carries a foreign piece of DNA stably integrated into the genome of every cell in that animal. Thus, all somatic cells and the germ-cells of a transgenic animal carry the same DNA fragment at the same chromosomal location, and this DNA fragment can be transmitted in a Mendelian fashion to offspring. This DNA element is termed a transgene.

Generation of transgenic mice

There are essentially three ways of generating animals with the capacity to transmit a genetic element through the germ-line to their offspring. These are (a) retroviral integration into an early-developing embryo, (b) injection of DNA into the pronucleus of a newly fertilized egg or (c) genetic manipulation of embryonic stem cells (Fig. 5.1). All three routes have been employed to generate transgenic mice, while a few transgenic rat strains have been established chiefly through the pronuclear injection route.

Retroviral infection

If preimplantation embryos are exposed to retrovirus, a proportion of the embryonic cells will stably integrate proviral sequences into their genome, usually as one copy per cell.

Introduction

Birds are virtually unique among domesticated species in being bred for the nutrient value of their ova, and few organisms have been subjected to as much genetic selection as poultry. These two features encapsulate both the attractions and the frustrations of producing transgenic birds.

Current breeding practices in the poultry industry depend on natural genetic variation that is exploited by selective reproduction. New variants are introduced from mutations or translocations, but there is a progressive loss of ‘wild-type’ characteristics. By inserting foreign DNA into organisms it is possible to bypass reproductive barriers and induce gene flow between vastly different organisms from quite distinct populations. Thus, the potential benefits of genetic engineering are extremely attractive, for they could dramatically increase both the genetic repertoire and the rate of change of a particular breed. Unfortunately, one of the characteristics of birds that makes them so commercially attractive – their large eggs – is also one of the features that makes it necessary to devise new approaches to their genetic engineering. The most common way of introducing foreign DNA into mammals is microinjection into the male pronucleus (Palmiter & Brinster 1986). The large size of birds' eggs makes it virtually impossible to see these structures and, to make matters worse, polyspermy is common. There may be up to 20 male pronuclei in a fertilized fowl egg with no indication as to which will eventually fuse with the female pronucleus.

Introduction

What are transgenic animals?

Since the earliest days of animal domestication, people have sought to improve their flocks and herds and companion animals by selective breeding. Although the results of such breeding are impressive, the procedure is by nature slow and to some extent imprecise. So the dream existed that perhaps one could take a more direct hand in stock improvement by recovering the genetic factors involved and adding these selectively to individual animals. The first attempts to do this took place some thirty years ago, but since the methods of DNA purification were suboptimal and methods had not been developed for gene isolation and cloning, complete genomic DNA preparations were used. These were simply mixed with animal eggs or embryos in the hope that transfer might occur, and then the resulting adults were screened for phenotypic features previously present only in the donors. Many of us can remember such experiments being undertaken with DNA from pigmented Ambystoma (the Mexican axolotl), such DNA being added to or injected into albino embryos of this species in the hope that the pigment gene would be transferred. Since that gene was likely to be swamped by the DNA for at least 1 million alternative sequences, one can see with the benefit of hindsight that the odds against success were enormous.

The developments which have now tipped the balance in our favour are chiefly threefold. The first, along with improvements in methods of DNA extraction and purification, was the discovery of restriction enzymes, which made it possible to dissect DNA into manageable fragments of precise length and sequence.

The range of transgenic animals

As seen in previous chapters, animal species of many kinds have been used for experiments in transgenic induction. Some offer particular benefits to the experimentalist; others have proved difficult or even impossible to use. In this book we have attempted to review all of those in which transgenic induction has been successful. The present range is as follows: protozoans such as Paramecium and Trypanosomid species, a few species of sea urchins, the nematode Caenorhabditis elegans, a few insects such as the silk moth Bombyx, the mosquito Anopheles and the fruit fly Drosophila, the predatory mite Metaseiulus, fish of many species, the amphibian Xenopus laevis, the domestic chicken and both laboratory mammals such as rats, mice and rabbits and large mammals such as cattle, sheep and pigs.

The failures and successes of applying transgenic technology to all of these experimental systems makes fascinating reading, yet clearly we are only at the threshold of this interesting study area. As well as the major transgenic systems that we have addressed here, there are a number of interesting minor ones, which will be briefly reviewed in this chapter. Of course, since this book is about transgenic animals, we have not discussed the extensive work on transgenic plants, fungi such as yeast and Aspergillus or bacteria, in which transgenism is a way of life, facilitated as it is by the extensive range of natural plasmids.

Phenotypic changes that occur in an organism after the attainment of adulthood may be traced to the changes in specific proteins/enzymes that are coded by specific genes. For example, changes in the cell membrane that alter its permeability may be due to changes in its lipid components, which are synthesized by specific enzymes. The turnover of collagen decreases with age because the level of collagenase declines. This increases cross-linking between collagen fibrils and in turn the tensile strength of collagen. Wrinkling of the skin in mammals is the result of these changes. During aging, a decrease in the enzyme tyrosinase leads to greying of the hair in mammals. The level of free radicals that damage macromolecules increases with age because superoxide dismutase (SOD) which is coded by a gene decreases. Thus, the functional changes that occur as an organism ages, whether at the organ or cellular or molecular level, are due to specific enzymes that are coded by specific genes. Environmental and intrinsic factors, however, influence the rate and the degree of such changes. Nevertheless, the fact that all individuals of a species have a specific life-span pattern indicates that the primary reason for a functional change is due to one or more genes. Therefore, an understanding of the changes in gene expression during aging may throw light on the basic cause of this process.

The science of aging, though young in comparison to the science of development, has advanced considerably during the past three decades. Up until 1980, most of the research that had been done was on the physiological and biochemical aspects of aging. The information obtained from this research was covered in my book, Biochemistry of Ageing, published by Academic Press in 1980. Since the development in the early 1980s of genetic engineering technology, the research on aging has concentrated on the role of the gene and the changes that occur at the genetic level during the aging process. Much of this research is being focused on the genes that are expressed in either a greater or lesser degree in old age, the changes that occur in the promoters of genes, the trans-acting factors that alter with age, and the genes that vary in expression during aging under stress. However, with all the research that has been aimed at elucidating the changes in genes, we still do not know how many genes are involved in the aging process, nor do we know the specific genes responsible for aging. Also, whether the same set of genes undergoes alterations in expression in all organs during aging or whether there are tissue-specific alterations in the expression of genes remain to be answered.

In Genes and Aging I have attempted to assemble the information on age-related changes in genes that has appeared primarily in the last ten years, since it is during this period that biochemists and molecular biologists have concentrated on this problem.