Transgenic Animals

Louis-Marie Houdebine

• Publisher: CRC Press
• Year: 2022
• Print ISBN: 9789057020698
• eBook ISBN: 9781000448436
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

During the past 20 years, transgenesis has become a popular technique and a crucial tool for molecular geneticists and biologists. Transgene expression is now better-controlled and even specifically inducible by exogenous factors. While these techniques have quite significantly transformed the experimental approaches taken by biologists, the applications are more limited than expected and concerns have arisen regarding biosafety as well as physiological, social, and philosophical issues. Transgenic Animals: Generation and Use contains articles on the techniques used to generate transgenic animals and a section on the preparation of vectors for the optimally controlled expression of transgenes. It also examines the use of transgenic animals in the study of gene function and human diseases, the preparation of recombinant proteins and organs for pharmaceutical and medical use, and the improvement of genetic characteristics of farm animals. Finally, it discusses more recent problems generated by transgenic animals including conservation of transgenic lines, specific database patenting, biosafety, and bioethics.Drawn from both academia and industry, the contributors to this monograph present in one concise volume all the relevant information on the different aspects of transgenesis. This book can be used as both a reference book and a textbook for specialized university courses and will be of interest to everyone involved in basic research in animal biology, molecular genetics, animal biotechnology, pharmaceutical science, and medicine.

PART I

1. Overview

Louis Marie Houdebine

Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-josas Cedex, France

About fifteen years after the pioneer experiments which showed that a stable gene transfer into animals, an expression and a phenotypic effect of the transgenes were possible (Gordon et al., 1980 and Palmiter et al., 1982), it is interesting to consider the state of art in animal transgenesis. This technique has already provided an invaluable amount of information for understanding the mechanisms which govern the life of animals. However, the situation is somewhat contrasted. The applications of transgenesis in agronomy for animal production remain very limited, in comparison to the numerous successes in plant production. This is obviously due to the technical problems which persist in generating transgenic farm animals at a reasonable cost. The real success of animal transgenesis lies undoubtedly in the field of basic research. Many laboratories throughout the world routinely use transgenic mice, Drosophila and Caenorhabditis elegans, to study gene and biological functions. Relevant biological models are also created each year by gene transfer to study human diseases and new pharmaceuticals. Transgenic farm animals are ready to be used as the source of recombinant proteins for pharmaceutical use. Recently, very encouraging experiments have shown that some human genes transferred into pigs can protect their grafted heart from hyperacute rejection caused by the activation of the primate complement. The introduction of new genetic traits in domestic animals by transgenesis is still in its infancy. The success is limited not only by the techniques for generating transgenic animals but also by the lack of valid identified genes.

The mapping of domestic animal genomes will certainly provide researchers with genes of interest. One may imagine that the first success in this field will be the protection of animals against diseases and the modification of milk composition. The improvement of the essential biological functions for breeding (growth, prolificity, meat quality, etc…) seems much less accessible due to the great complexity of the biological mechanisms involved. The generation of transgenic animals has raised totally new problems such as the conservation and the patenting of the animals, the biosafety of the gene transfer experiments for researchers and for the environment, the quality of the new products originating from the animals and also the ethics of animal genome manipulation.

In the present book, all these points are considered. For this purpose, many authors have been solicited, in order to have the point of view of many different people. The authors were asked to write relatively short chapters providing the most relevant information in their field. For these reasons, the chapters have no standard structure and are of variable length.

The Generation of Transgenic Animals

The first group of chapters describes the methods used to generate different transgenic mammals such as rat (J.J. Mullins), rabbit (C. Viglietta et al.), pig (V.G. Pursel), goat (W.G. Gavin), sheep (Y. Gibson) and cow (M. Gagné et al., and A.M. Deloos) are described. The possibility of enhancing the integration rate of the foreign gene by adding cow repeated DNA sequences to the vector is considered by M. Gagné et al. The efficiency of polycation-DNA complex injected into embryo cytoplasm rather than pronuclei is evaluated by W.H. Velander. A critical evaluation of gene transfer into cow through spermatozoa is given by K. Schellander et al.

In practice, the success of transgenesis, specially for farm animals, is highly dependent on the techniques of reproduction. The possibility of using one-cell embryos generated after in vitro oocyte maturation and in vitro fertilization in sheep, goat and cow rather than by conventional superovulation is depicted by N. Crozet and A.M. Deloos. The use of one-cell embryos kept frozen before microinjection is shown by N. Tada. The cloning of embryos harbouring interesting transgenes by nuclear transfer into the cytoplasm of oocytes is expected to accelerate diffusion of the new genetic traits (N. Strelchenko).

Gene transfer into bird embryos is still not a fully resolved problem. Several complementary approaches are studied: the direct gene microinjection and in vitro development of the embryos (T. Ono and N. Naito), the use of retroviral vectors (C. Ronfort et al.) of biolistics (K. Simkiss) and liposomes (E.J. Squires) to transfer a foreign genes into embryos or spermatozoa. The possibility of culturing chicken embryonic cells and generating chimaerae offers quite interesting additional possibilities (R.J. Etches).

Gene transfer into lower vertebrates is possible after microinjection into early embryo cytoplasm (D.H. Delvin and P. Collas et al.). In fish (K. Inoue), as in chicken (E.J. Squires et al.) and mammals (K. Schellander et al.), gene transfer through spermatozoa appears possible but with a rather low yield and often with rearrangement or inactivation of the transgenes.

In invertebrates, direct microinjection into early embryos is also the technique routinely used. In Drosophila, the use of a transposon as a vector greatly facilitates the integration of the foreign DNA (Kaiser et al.). Gene transfer is carried out in the nematode C. elegans (D. Thierry-Mieg et al.) in marine invertebrates (E. Mialhe) and in arthropods (Presnail and Hoy, 1992).

The Use of Cellular Vectors for Transgenesis

The use of cellular vectors to carry the foreign DNA into the embryo is an attractive possibility. Cultured spermatozoa precursors, ES cells and EG cells can theoretically be used for this purpose. Spermatozoa fully matured or not can be injected into oocytes and give rise to fertilization and embryo development with an acceptable yield. Alternatively, cultured spermatogonial cells can be reintroduced into a recipient testis and give rise to the birth of offspring by normal fertilization (Brinster et al., 1994). This pioneer experiment is very encouraging. Longer periods of spermatogonial cell culture are necessary however to allow gene integration and homologous recombination. The number of cultured cells to be reintroduced into the testis to colonize this organ to a significant degree is probably so high for farm animals that this approach may turn out to be applicable only to laboratory animals. ES and EG cells can be fused to developing embryos to generate chimaerae. The success remains limited to mouse. Attempts to establish stable ES or EG cell lines from other species have failed so far. This may be due to the lack of basic knowledge on what are really totipotent cells. This point is discussed by J.E. Fléchon whereas the possible use of totipotent cells is described by N. Strelchenko, P.J. Donovan and A. Nagy. The possibility to generate lambs after transfer of nuclei from cultured sheep embryonic cells into cytoplasm of oocyte have opened new avenues for transgenesis (Campbell et al., 1996).

The Direct Gene Transfer into Somatic Cells

The direct gene transfer into somatic cells is the technical basis for gene therapy. It can be used in some cases as a relatively simple substitute to transgenesis to study gene function in vivo. It is also an attractive tool for vaccination using injections of pure DNA (Krishnan et al., 1995) for in vivo transfection to study gene activity (P. Couble) and for the evaluation of mammary cells to perform post-translational modifications of recombinant proteins (Archer et al., 1994). Retroviral vectors (C. Ronfort et al.), adenoviral vectors (J.F. Dedieu et al. and Nakayima et al., 1995) adeno-associated viral vectors (R.J. Salmulski) or retrosposons (C.P. Hogson) can be used for this purpose. Interestingly, viral vectors can be used in adults to express transiently and locally the Cre recombinase to induce a local gene recombination in transgenic animals harbouring the integrated lox recombination sequence (see Chapter 48 by S. Viville). Somewhat unexpectedly, DNA injected intravenously into pregnant mice is spontaneously transferred to fetuses (T. Subamoto et al., 1995). If this observation is confirmed, this simple technique might be useful to study gene expression and action in mouse foetus without prior transgenesis.

The Fate of Transgenes

The fate of the foreign DNA microinjected into embryos is not well known. In mammals, tandem arrays of foreign genes are integrated whereas in lower vertebrates a random association of individual copies of the foreign gene occurs (R.H. Delvin). The mechanism of persistence and integration has been studied by R.J. Wall et al., J.O. Bishop, Chen et al., 1995 and Pawlik et al., 1995. Transgenic founder animals are more frequently mosaic than originally imagined in mammals (Y. Echelard) and also in fish (R.H. Devlin) in the first generation. Animals are no more mosaic in offspring as was expected.

Detection of transgenes in preimplantation embryos would be very helpful for farm animals to reduce the number of recipient females. A systematic study carried out by G.T. O’Neil revealed that no reliable method is presently available. The use of the Vargula luciferase gene as a reporter co-transgene is a potentially interesting tool (Thompson et al., 1995). The tyrosinase gene which has a phenotype effect on pigmentation may be used to study the presence of a coinjected gene of interest. This test can be used reliably but only in newborns and adults (Umland et al. and Methot et al., 1995).

The transgenes may be methylated (K.J. Snibson) and subjected to genomic imprinting (J.R. Chaillet) which lead to their inactivation. These phenomena are interesting models to study the mechanisms of gene expression and they may limit the long term use of transgenic animals.

The Vectors for Transgenesis

Transgenesis has pointed out somewhat unexpectedly several aspects of the control mechanism of gene expression: introns are required for transgenes and generally not for genes transfected into cultured cells (Palmiter et al., 1991 and Attal et al., 1995). The role of intron seems quite complex. Interestingly, they seem to participate in some cases in nucleosome phasing (Lin et al., 1995).

Episomal vectors would be of great help to generate transgenic animals. Attempts have had limited success so far (J. Attal et al.). This kind of vectors have the potential advantage of generating transgenic animals with a high yield and of being independent of host chromatin. Only a few transgenes are independent of their site of integration in chromatin and have a copy number-dependent expression. A few gene insulators have been partially identified and used successfully when added to the gene constructs (A. Sippel et al.).

The coinjection of a gene known to be functional as a transgene with the gene construct of interest may significantly enhance the expression of the latter (A.J. Clark). Relatively long DNA fragments can be faithfully reconstituted from overlapping fragments with good yield before integration into the animal chromatin (Pieper et al., 1992). Long genomic DNA fragments cloned in Pi phages (Sternberg, 1992 and S.P.A. McCormick et al.) in BAC vectors (Birren) or in YAC vectors (T. Umland et al.) are expected to bring most, if not all of the regulatory elements leading to a more predictable and confident expression of transgenes. Interestingly, the YAC vectors allow a spontaneous homologous recombination with the host genome with a relatively high frequency (T. Umland et al.). A combination of homologous recombination with YAC vectors in yeast to introduce new sequences in the vector and a standard microinjection into pronuclei can potentially lead to gene replacement. If this fact is confirmed, the use of ES cells for gene targeting may no longer be compulsory.

Contiguous YAC and P₁ vectors covering up to 2 Mb of a chromosome can also be used to generate transpolygenic mice which constitute a partial in vivo library. These long DNA fragments may be stably integrated without any rearrangement and the genes they contain are expressed. This elegant approach has been used to identify and to study the gene responsible for Down’s syndrome located in chromosome 21 in human and in chromosome 16 in mouse (Smith et al., 1995). No more than 8 lines of mice harbouring a DNA fragment in a YAC or a P₁ were sufficient to cover the 2 Mb.

Fragments of chromosomes can be microinjected and integrated into an animal genome (Richa et al.) bringing simultaneously several loci to the host.

The control of the transgene by an external stimulus not acting on host genes is highly desirable. Several systems and one using tetracycline as an inducer are available...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Table of Contents
• Preface
• Contributors
• Part I Overview
• Part II The Techniques for Gene Transfer
• Part III The Fate of Microinjected DNA into Mammal Embryos
• Part IV The Use of Transgenic Animals
• Part V The New Problems Generated by Transgenic Animals
• Index