Gene transfer techniques
Breeding with transgenic livestock
Gene transfer in various species
Application possibilities for gene transfer
Conclusions
Bibliography
G. Brem and H.-G. Wagner
Professor Gottfried Brem's address is Lehrstuhl für Molekulare Tierzucht, Ludwig-Maximilians-Universität, 8000 München 22, Germany. Dr H.-G. Wagner is Animal Production Officer (Artificial Insemination and Breeding Development), FAO.The objective of breeding and selection programmes is to improve the genetic potential of livestock and subsequently phenotypic production traits. The problems faced by conventional breeding programmes are, first, an accurate evaluation of genotype on which the phenotype is based and, second, the fact that although the entire genome of an animal is always assessed, only half of the genome of the selected animal is passed on to its offspring. These problems also apply to the modem technologies in animal breeding, including artificial insemination, embryo transfer, in vitro maturation and fertilization, and embryo microsurgery since the methods used in these technologies are also applied to the genome in haploid or diploid form as produced by natural processes of segregation and random mating. The possibility of making a specific gene or group of genes me direct subject of breeding work, thus excluding many other genes of little or no concern, is an option of great interest to animal breeders.
Since 1980, when the first successful gene-transfer experiment using DNA microinjection on a mouse was reported (Gordon et al., 1980), a method has been established that allows the transfer of a single isolated gene. By using molecular biology methods already established in the 1970s, genes that are of interest for breeding purposes can be isolated, sequenced, recombined with regulatory elements and tested in vivo and in vitro for their functional ability. The implementation of this basic research in animal breeding programmes is only just beginning and it is expected that drastic changes will emerge in the future. This article reviews the known techniques of gene transfer as well as the present state of the art of gene transfer in some domestic animals. The possibilities of application of gene transfer have been highlighted by Hammer et al. (1986); Kräusslich and Brem (1986); Brem (1986b); Smith, Meuwissen and Gibson (1987); and Church (1987).
Gene transfer is the transfer of in vitro recombined gene constructs into animals. When a gene construct is integrated into the genome of the animal it is described as a transgene. The coded protein produced by this transgene is the transgenic product. Animals that contain transgenes are transgenic and, if the transgene is passed on to the offspring, transgenic lines or populations will be created. The target of the technology is the trait that is to be influenced by the transgenic product.
1. Scheme of a gene transfer programme in pigs - Schéma d'une programme de transfert de gène chez le porc - Esquema de un programa de transferencia de genes en cerdos
For gene transfer in mammals, three different techniques are possible:
· microinjection of DNA into the pronuclei of zygotes;· DNA transfer using retroviral vectors;
· production of transgenic chimeras by injecting genetically transformed totipotent stem cells into embryos.
Gene transfer through direct DNA microinjection
Until now the microinjection of DNA into the pronuclei of zygotes is the only method used for gene transfer in domestic animals. The procedure consists of the following phases:
· cloning and recombination of a suitable gene construct;· preparation of donors;
· recovery of zygotes (fertilized oocytes);
· visualization of pronuclei (necessary in some domestic animal species);
· preparation of the DNA solution to be injected;
· microinjection of DNA solution into the pronuclei of the zygotes;
· transfer of injected zygotes into the oviducts of synchronized recipients;
· investigation of newly born animals to ascertain whether they have integrated the gene construct (Dot - Southern - Blot).
Figure 1 shows the procedure of a gene transfer programme with a pig as an example. The donor animals are superovulated by means of gonadotropin injections according to the programme established for each species and mated or inseminated twice. The recovery of the zygotes is performed 12-24 hours after fertilization through surgical flushing of the oviducts. Persisting cumulus cells are removed using hyaluronidase treatment. In almost all domestic species no nucleus structures are visible because of dark lipid-containing granule. Centrifuging is a useful method of making pronuclei visible.
Gene constructions needed for gene injection are recombined in plasmids or cosmids and then cloned. After splitting the recombined vectors with restriction endonuclease the gene construct is extracted, precipitated, washed and placed in an injection buffer. In order to avoid problems during injection, all solutions utilized during the preparation of the injection fluid must be sterile-filtered. The gene solution must be free of contamination and particles. The DNA solution is diluted so that one picolitre contains about 1 000 copies of the gene construct.
The equipment needed for microinjection (Figure 2) includes an invert microscope, two micromanipulators and injection equipment. Additional necessary items are an injection chamber and holding and injection pipettes. The injection pipette, with an outer diameter of 1-2 m m, is filled with DNA solution. During injection, the zygote is held with the holding pipette. The injection pipette is introduced into the pronucleus, passing through the zone pellucida, the cell membrane and the nucleus membrane. About one to two picolitres of the DNA solution are injected into the pronucleus, increasing its volume.
These injected zygotes are transferred, after brief in vitro culture, into the oviducts of synchronized recipient animals. Recipient animals are synchronized with the donors by means of hormonal treatment. After the birth of offspring from gene injection, high molecular DNA is isolated from tissue (blood or cells can be conveniently taken from the tail), to confirm successful integration. The integration of the injected DNA and the number of integrated copies can be determined after further processing with Southern Blot or Dot Blot hybridization. Integration sites in the chromosomes can be proved through hybridization of metaphase chromosomes, using the injected gene as a probe.
Transgenic animals are raised and mated. Offspring from these matings are tested to discover whether the transgene has been passed on. In mating hemizygous-transgenic F1 inter se attempts are made to produce homozygous transgenic animals.
Gene transfer with retroviral-vector assistance.
Retroviruses have an RNA genome, which is transcribed through the virus' own reverse transcriptase into DNA in infected cells and subsequently integrated into the genome of the cell. Integration occurs accidentally. However, only one copy can generally be found in the provirus. Based on this cycle retroviral vectors can be used as vehicles for gene transfer.
In 1974 it was shown for the first time that after injection of SV40 DNA into the blastocoel of mouse blastocysts, the DNA could then be found later in the cells of adult mice (Jaenisch, 1974; Jaenisch and Mintz, 1974). The Mo-Mulv-provirus DNA used in these experiments which followed was integrated into the genome and passed on to offspring, thus building up stable lines (Jaenisch, 1976; Stuhlmann, Jähner and Jaenisch, 1981).
For utilization as a vector, different parts of the viral genome are replaced by the gene construct which is to be transferred. Care has to be taken that the LTR and psi-region remain intact. The virus cover is provided by a second provirus, which lacks the coverage signal (psi-region) (Mann, Mulligan and Baltimore, 1983; Cone and Mulligan, 1984).
With the assistance of retrovirus vectors, the bacterial neo-gene was integrated into the germ line of mice and Stewart et al. (1987) were able to prove their expression under the control of the TK promoter. Transgenic mice, carrying a mutant DHFR gene, were produced by Van der Putten et al. (1985) by infecting zone-free mouse embryos with recombinant defective retroviruses without viral assistance. Rubenstein, Nicolas and Jacob (1986) were able to achieve integration of the provirus when coculturing denuded embryos with psi2-cells, transgenic mouse lines were produced by Soriano et al. (1986) through infection of pre-implantation embryos with recombined retroviruses containing the complete human beta-globingene including its own promoter. These mouse lines expressed the transgene in the haemopoetic system.
The successful utilization of retroviral vectors in domestic mammals has not yet been reported. In poultry, Salter et al. (1986) have injected the wild strain as well as the recombinant chicken-leucosis-virus into eggs before hatching. Some animals were found which showed integration.
Gene transfer through production of germ-line chimeras
The procedure to utilize totipotent transformed stem cells to transfer recombined gene constructs into the germ line is receiving increasing attention, especially in respect of the mouse. Totipotent stem cells are isolated from in vitro cultured blastocysts. Through aggregation with early embryonic division stages or through injection of these cells into blastocysts, chimeras can be produced. Up to 30 percent of the chimeras so produced are germ-line chimeras containing the genotype of the cell line. Mintz (1977) mentioned the possibility of using these stem cells as a vehicle for the introduction of new-genes. Figure 3 shows the model producing transgenic cattle through production of the chimeras with transformed stem cells.
Stewart, Vanek and Wagner (1985) and Robertson (1986) showed that gene constructs that have been transformed into embryonic stem cells were expressed in the somatic tissues of the mouse chimeras produced. The percentage of mice born expressing chimerism was very high and in some cases it could also be proved that some of the offspring had inherited the transgene. Because no suitable stem cells are so far available for use in-farm animals, this technique has not been applied for the latter. Important efforts are being made to produce these cell lines. First results in the pig have shown that it is possible to cultivate stem cell-like colonies from embryos (Piedrahita et al., 1988).
The advantage of such gene transfer through transformed stem cells would be that integration and eventually expression of the transgene could already be tested in the cell line. Perhaps of even more importance is the fact that the-transfer could possibly be done through manipulating morulae and blastocysts. Since embryos at that stage, particularly in the bovine, can be obtained non-surgically, this would mean that gene transfer programmes would be greatly facilitated.
Another method, which could also be a gene transfer technique through manipulation of blastocysts, is the microinjection of DNA packed into liposomes (Rottmann et al., 1985). Reed et al. (1988) have packed recombinant DNA into liposomes and have injected these into bovine blastocysts, but positive results of this experiment are not yet available.
In 1989, the use of sperm-mediated gene transfer for the gene transfer for the generation of transgenic mice and pigs was published (Lavitrano et al., 1989; Gandolfi et al., 1989). This method is quite simple and, as shown by the authors, also efficient. Other scientists have not been able to repeat these results (Brinster et al., 1989).
In the application of gene transfer in livestock production programmes, it is of key importance that the transferred gene be subsequently transferred to its progeny. This means that all, or at least some, of the germ cells of primary transgenic animals must contain the transgene Unfortunately little knowledge exists about the molecular biological processes that take place when the injected DNA is integrated in the genome. For example, the exact time of the integration is not known nor whether this is stable in all cells during the further development of the embryo. When transgenic animals have been checked after birth, particularly in trials to produce offspring, it has been shown that mosaics might occur. Mosaics are animals with different cell lines of different genotype that have, however, originated from one zygote. Transgenic mosaics contain both transgenic and non-transgenic cells and this sometimes can become a problem. It is obvious that the progeny cannot inherit the transgene from its transgenic parents if the transgene is not present in the gonads. From past experience, it appears that about 30 percent of the primary transgenic animals produced through microinjection are mosaics and therefore pass on the transgene to less than half or even to none of their progeny.
In animals that have integrated the injected gene construct in a stable way, inheritance follows that of a Mendel gene since, in general, integration takes place at one locus in the chromosome. These animals are called hemizygote transgenics. The term "heterozygous" is not appropriate since the allele corresponding to the transgene is missing in the homologous non-transgenic chromosome. If the gonads are mosaics of transgenic and non-transgenic cells, the percentage of transgenic progeny depends on the participation of these two cell lines in the production of gametes and can be between 0 and 50 percent. Mosaics normally only occur in the F0 generation. Offspring of transgenic F1 and the following generations contain, if they are positive, the gene construct in all somatic and gonadal cells. It has been observed, however, that a transgene has seldom not been transmitted reliably through several generations. The reason why a transgene does not remain stably integrated but can be lost from the genome has sometimes not been satisfactorily explained. Even more frequent than the instability of integration is the large variability in the expression of the transgene. In subsequent generations, all different forms of changes from an increase to a reduction or even to a complete absence of the expression have occurred. Even within full-sibs, a significant variability of transgenic expression has been observed. The reasons for this have not yet been clarified but one possibility could be the different grade of methylization of the DNA.
It is rare to be able to confirm several places of integration in a founder transgenic animal. If the integration sites in the chromosomes are far from each other, more transgenic progeny may accordingly occur. In a transgenic animal with two independent places of integration, it can be expected that 75 percent of the offspring will inherit the transgene, in which case 25 percent will inherit one of the two transgenes and 25 percent will have both integration sites in their genome. When mating hemizygote transgenic offspring inter se, normally 50 percent hemizygous and 25 percent homozygous transgenic animals, as well as 25 negative, should occur.
The uncontrolled integration in primary transgenic animals may result in the transgene being integrated in the area of an existing gene which is essential for the development of the foetus. This kind of integration mutation does not cause any problem in hemizygous animals as long as an intact allele exists on the homologous chromosome. By mating hemizygous animals showing insertional mutation, it is not possible to produce homozygous transgene progeny in cases where the gene that is essential for the development of the foetus is menaced by the insertional mutation. Wagner et al. (1983) made such observations in offspring of transgenic hGH mice. Even though no homozygote animals could be produced because of the observed prenatal mortality, it was possible to produce 50 percent normal transgenic offspring when mated to normal animals.
Before introducing transgenic animals into a population, it is essential to ensure that these animals are free from insertional mutations to avoid an increase in the frequency of lethal genes. These insertional mutations, however, can be of specific interest for basic research. Woychik et al. (1985) found that insertional mutation led to the known defect of extremity malformation. This would mean that insertional mutation can be used to isolate and characterize the respective endogenous gene.
Smith, Meuwissen and Gibson (1987) established a breeding plan for testing transgenic animals and for the foundation of homozygous lines.
As far as breeding and selection are concerned, the consequences of these problems of mosaics and insertional mutations are that one transgenic animal is insufficient for introduction of a well-defined gene construct into a population. Apart from the fact that an increasing number of inbreeding problems may occur, the chances of establishing a functional line based on one single transgenic animal are rather limited. For the successful utilization of a transgenic line in livestock production, the following prerequisites have to be fulfilled:
· stable transmission of the transgene to the offspring;· freedom from insertional mutation and the possibility of the production of homozygous transgenic animals;
· stable expression of the transgene with a positive biological influence on the target features.
At least five to ten primary transgenic animals are needed to allow the production of successful lines, in view of these uncertainties.
A review of the literature shows that to date the only successful method to produce transgenic rabbits, pigs, sheep, goats and cattle is DNA-microinjection technique into the pronucleus.
Rabbits. Rabbits are used as experimental models in gene transfer experiments. In 1985, the successful production of transgenic rabbits was reported, for the first time, and included the growth hormone construct MT-hGH (Hammer et al., 1985; Brem et al., 1985). The rate of degeneration of rabbit zygotes (see Figure 4) caused by injection was below 10 percent (Ross et al., 1988). The pre-implantation development capacity of injected zygotes is significantly lower compared with control embryos.
Pigs. Pig zygotes must be centrifuged to show the pronucleus (Wall et al., 1985). Fifty percent of the centrifuged non-injected zygotes develop in vivo up to the morula or blastocyst stage. After microinjection (see Figure 5), 10-20 percent development to various stages of embryonic development occurs (Hammer et al., 1986; Brem et al., 1988). Of the injected zygotes, 5.6 percent (Brem et al., 1985) to 11 percent (Hammer et al., 1985) developed and led to the birth of piglets. The integration rate in pigs is approximately 10 percent. Growth-hormone constructs used in initial experiments led to an expression rate of 50 percent.
The production of transgenic F1 offspring is possible. In the authors' own experiments, inheritance of the transgene could be proved in two out of five animals.
It is not necessary to centrifuge sheep embryos to make the pronucleus visible. According to "Nomarski optics", 80 percent of the pronuclei can be located if a microscope with interference contrast is available. The capacity for in vivo development of sheep zygotes with injection (26 percent) and without (10 percent) is half that of pig embryos after similar treatment (Hammer et al., 1986). Seven days after in vivo culture of non-treated and non-in vitro cultured sheep zygotes, Rexroad and Wall (1987) observed a development rate of 86 percent. An in vitro culture of five hours' duration reduced this development rate to 65 percent, and after the injection of a buffer solution a reduction to 42 percent was observed. Nineteen percent developed to the 32-cell stage after injection of DNA solution. In initial experiments, the integration rate was about 1 percent (Hammer et al., 1985; Ward, Murray and Nancarrow, 1986; Ward et al., 1986), while the survival rate of injected embryos up to birth was 7 percent (Hammer et al., 1985, 1986) and 6.2 percent (Nancarrow et al., 1987) respectively.
An attempt was also made to produce transgenic goats through DNA microinjection into centrifuged zygotes. No DNA integration has yet been proved in the relatively limited number of animals born (Armstrong et al., 1987; Fabricant et al., 1987).
Pronuclei of bovine zygotes also have to be revealed by centrifuging. Lohse, Robl and First (1985) injected a thymidine-kinase construct into fertilized cattle zygotes and showed that 24 hours after injection about 30 percent of the embryos had thymidine kinase of two standard deviations higher than the controls. Roschlau et al. (1988) injected 513 bovine zygotes with three different gene constructs of viral origin and was able to show the foreign DNA in 14 embryos. Fourteen pregnancies resulted from the transfer of 43 embryos that had shown development in vivo. Loskutoff et al. (1986) were able to obtain three pregnancies after injecting 72 zygotes and 17 eggs at the two-cell stage. McEvoy et al. (1987) have so far obtained four pregnancies after the transfer of 43 injected zygotes and eight two-cell stages to 17 recipients.
Church, McRae and McWhir (1986) injected 852 bovine zygotes with an alphafetoprotein construct and found four with gene-integration in the 111 embryos which developed further. Following injection of bovine zygotes, the calf-birth rate was 19.9 percent; the respective figure for untreated controls was 42.8 percent. Seven out of 126 calves (5.6 percent) had integrated the DNA (Church, 1987). Biery, Bondioli and de Mayo (1988) obtained an integration rate from 0.22 percent up to 1.67 percent, based on injected zygotes.
In recent years, several application possibilities for gene transfer in domestic animals have been discussed. Until now it has been possible to influence only traits that are based on a single gene or on a limited number of genes. There is only a very limited number of traits of interest to breeders, which are based on a single gene. The difference between qualitative and quantitative traits is not always fully clear, as demonstrated in the trials of Palmiter et al. (1982, 1983).
In these authors' well-known experiments, growth, one of the classical quantitative traits in animal breeding, was changed to become a quasi-qualitative trait through the transfer of a single growth hormone gene which was related to a feedback independent regulation mechanism. Similar consequences can be expected by application of somatotropins in dairy cattle.
Although our knowledge is still rather limited, the results of these experiments can be interpreted in the form of a working hypothesis. This is because of the high variability through which these single genes contribute to performance in the form of an additive gene effect. It can only be hoped that further research will contribute to a better understanding of performance based on genetics.
As far as gene transfer in cattle is concerned, there are realistic prospects that it will be possible to influence positively different production traits. Traditional selection programmes using conventional breeding techniques have achieved important results and will continue to do so. However, it seems preferable to concentrate the very expensive and complex technique of gene transfer to fields which until now could only be improved with limited success through conventional breeding programmes, such as breeding for increased disease resistance.
At present, research in the field of transgenic livestock is concentrated on the following fields.
Growth. Attempts are being made to increase growth and body composition of animals through the transfer of these genes which are responsible for growth-hormone regulation. Experiments with the respective proteohormones in growing animals have shown that such effects can be achieved. These gene transfer experiments have been undertaken particularly in pigs (Hammer et al., 1985, Brem et al., 1985; Vize et al., 1987) and in sheep (Ward, Murray and Nancarrow, 1986; Ward et al., 1986).
Disease resistance. Only a very limited number of genes is known that are able to influence the resistance of domestic animals to diseases. A model for such work is the influenza resistance caused by the Mx-gene (Staeheli et al., 1986). The authors have made attempts to produce influenza-resistant pigs through the transfer of three Mx-gene constructs.
Quality of animal products. The improvement of the quality or composition of animal products through the transfer of respective gene constructs could provide new prospects for animal production. A model proposed by Mercier (1987) has the reduction of the lactose content of milk as its objective. In the milk of transgenic sheep and cattle carrying a lactose gene, combined with an udder specific promoter, lactose is split into galactose and glucose. Such milk could then also be consumed by the large percentage of the world population suffering from lactose intolerance. Through gene transfer, researchers have tried to promote synthesis of cysteine in the mammal organism and to influence positively wool growth (Ward, Murray and Nancarrow, 1986; Ward et al., 1986).
Gene farming. A suitable combination of tissue-specific promoters and the transfer of these genes into domestic animals may lead to efficient and biologically reliable production of proteins (Lathe et al., 1987). In particular, efforts have been made to use animals as bioconversion systems (Clark et al., 1987). Gordon et al. (1987) and Simons, McClenaghan and Clark (1987) have also achieved the expression of human T-PA and sheep beta-lactoglobulin respectively in the milk of transgenic mice.
For future research activities in the field of gene transfer, the following three subjects should be given particular attention.
Investigations to improve the effectiveness of gene transfer methods. While trying to optimize the technique of gene transfer through microinjection, attempts should also be made to establish and to optimize the other techniques in gene transfer.
Isolation and characterization of genes of interest for breeding purposes. The results of basic research in molecular biology are an excellent foundation for practical work on the genome of domestic animals.
Isolation and characterization of suitable regulation elements. The provision of suitable promoters and enhancers is even more difficult than the search for structure genes. For convenient use in breeding programmes, it is of the utmost importance to produce gene constructs that allow the transferred constructs to be expressed in the right tissue at the right time, as well as in the right amount.
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