Transgenic organisms

The use of recombinant DNA techniques to introduce new characters (ie. genes) into organisms (including humans) that were not present previously.

The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome, in contrast to spontaneous mutation. Foreign DNA is introduced into the animal, using recombinant DNA technology, and then must be transmitted through the germ line so that every cell, including germ cells, of the animal contain the same modified genetic material.

If the germ cell line is altered, characters will be passed on to succeeding generations in normal reproduction.

If the somatic cell line alone is altered, only the organism itself will be affected, not its offspring.

Transgenesis may involve whole organisms, rather than individual cells, and there may be in vivo alteration of body function.

One use of transgenesis is gene therapy which is the alteration of the genetic make-up of of an individual organism in an attempt to correct an inborn error of metabolism, ie. cure inherited diseases. But this is generally only carried out with somatic cells and, therefore, will only affect one generation.

Do not confuse transgenesis with cloning which is the production of identical copies of molecules, cells or whole organisms. Cloning does not necessarily involve gene manipulation.

Historical background

During the 1970s, the first chimeric mice were produced (Brinster, 1974). The cells of two different embryos of different strains were combined together at an early stage of development (eight cells) to form a single embryo that subsequently developed into a chimeric adult, exhibiting characteristics of each strain. Subsequently this was done with other animals, eg. sheep and goat ----> "geep" (1982).

Transgenesis was first described in 1981 (Gordon and Ruddle, 1981) using DNA microinjection of mice ova. Various other species such as rats, rabbits, sheep, pigs, birds, and fish soon followed.

It has been gaining application among biotechnologists since the development of transgenic "super mice" in 1982 and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987.

Two other main techniques have been developed: those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986).

Procedure for transgenesis

Conventional recombinant DNA techniques are used to construct the transgene so that the desired gene product will be expressed in the desired location. Typical transgenes contain nucleotide sequences that correspond to the gene of interest, with all the components necessary for efficient expression of the gene, including a transcription-initiation site, the 5' untranslated region, a translation-initiation codon, the coding region, a stop codon, the 3' untranslated region, a polyadenylation site and a promoter. Different promoters can be used to cause gene expression in all tissues of the body (non-specific) or only in specific tissues:

PROMOTER	GENE EXPRESSION IN
(beta)-actin promoter	many tissues of the transgenic animal
simian virus 40 T antigen promoter	many tissues of the transgenic animal
adipocyte P2 promoter	fat cells
myosin light-chain promoter	muscle
amylase promoter	acinar pancreas
insulin promoter	islets of Langerhans beta cells
beta-lactoglobin promoter	mammary glands

In pharming expression in the mammary glands is usually desired as this leads to the appearance of the product in the milk of the animal - very convenient.

The three principal methods used for the creation of transgenic animals are DNA microinjection, embryonic stem cell-mediated gene transfer and retrovirus-mediated gene transfer.

1. DNA microinjection

This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981) which are the most difficult of all cells to genetically manipulate. The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The DNA construct (usually about 100 to 200 copies in 2 pl of buffer) is introduced by microinjection through a fine glass needle into the male pronucleus - the nucleus provided by the sperm before fusion with the nucleus of the egg. The diameter of the egg is 70 痠 and that of the glass needle is 0.75 痠; the experimenter performs the manipulations with a binocular microscope at a magnification of 200 x. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male.

Microinjection is the commonest method at present and is generally more successful with laboratory animals than farm animals.

Figures in parentheses are percent efficiency compared to original number of ova injected.

2. Embryonic stem cell-mediated gene transfer

This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.

This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.

3. Retrovirus-mediated gene transfer

To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.

For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation.

There is also fusion of host cells with membranous vesicles (eg. liposomes) containing DNA.

(Plant cells can be modified using, eg. tobacco mosaic virus or the Ti plasmid of Agrobacterium tumefaciens.)

Some examples of the use of transgenic organisms

Transgenic animals have been used for simulating diseases and testing new therapies, eg. cardiovascular and neurodegenerative diseases. Animal models provide an opportunity to test methods for the prevention or delay of disease in humans. Some examples:

Genetic alteration	Method of alteration	Human disease equivalent
Introduction of mutant collagen gene into wildtype mice	Nuclear microinjection of inducible minigene	Osteogenesis imperfecta
Inactivation of mouse gene encoding hypoxanthine-guanine phosphoribosyl transferase (HPRT)	Insertion of retrovirus into HPRT locus in embryonic stem cells	HPRT deficiency
Mutation at locus for X-linked muscular dystrophy	Male mutagenesis followed by identification of female carriers	X-linked muscular dystrophy
Introduction of activated human ras and c-myc oncogenes	Nuclear microinjection of inducible minigene	Induction of malignancy
Introduction of mutant (Z)allele of human alpha -1-antitrypsin gene	Microinjection of DNA fragment bearing mutant allele	Neonatal hepatitis
Introduction of HIV tat gene	Microinjection of DNA fragment	Kaposi's sarcoma
Introduction of beta-globin sickle gene	Microinjection	Sickle-cell anaemia
Introduction of mouse renin gene	Microinjection	Hypertension
Introduction of (beta)-amyloid protein precursor (APP gene)	Microinjection	Alzheimer's disease

Improving plants

Transgenic methods have now been developed for a number of important crop plants such as rice, cotton, soybean, oilseed rape and a variety of vegetable crops like tomato, potato, cabbage and lettuce. New plant varieties have been produced using bacterial or viral genes that confer tolerance to insect or disease pests and allow plants to tolerate herbicides, making the herbicide more selective in its action against weeds and allowing farmers to use less herbicide.

A new variety of cotton, for example, has been developed that uses a gene from the bacterium Bacillus thuringiensis to produce a protein that is specifically toxic to certain insect pests including bollworm, but not to animals or humans. (This protein has been used as a pesticide spray for many years.) These transgenic plants should help reduce the use of chemical pesticides in cotton production, as well as in the production of many other crops which could be engineered to contain the Bacillus thuringiensis gene. In another case, a gene from the potato leaf-roll virus has been introduced into a potato plant, giving the plant resistance to this serious potato disease.

Transgenic technologies are now being used to modify other important characteristics of plants such as the nutritional value of pasture crops or the oil quality of oilseed plants like linseed or sunflower.

Improving livestock

The main aim in using transgenic technology in animal agriculture is to improve livestock by altering their biochemistry, their hormonal balance or their important protein products. Scientists hope to produce animals that are larger and leaner, grow faster and are more efficient at using feed, more productive, or more resistant to disease. Examples of transgenic breeding programs include:

The welfare of the animals, including any changes in metabolism that may cause health problems, is an important consideration in these programmes. Both researchers and regulatory bodies also examine closely any changes in the composition of meat or other products that will ultimately be eaten.

Advantages of transgenesis over selective breeding for animals and plants

Transgenic technology is an extension of agricultural practices that have been used for centuries: selective breeding and special feeding or fertilization programmes. It may reduce or even replace the large-scale use of pesticides and long-lasting herbicides. When fully developed, it would offer a number of advantages over traditional methods.

Pharming

Many valuable pharmaceutical products can now be made using transgenic animals such as mice, rabbits, sheep, goats, pigs and cows. Often the product conveniently appears in the milk of the animal. Transgenic plants can also be used to make pharmaceuticals.

References

Brinster, R. (1974). The effect of cells transferred into mouse blastocyst on subsequent development. J. Exp. Med.:1049-1056.

Donnelly, S., McCarthy, C.R. and Singleton, R. Jr. (1994). The Brave new World of Animal Biotechnology, Special Supplement, Hastings Center Report.

Federation of European Laboratory Animal Science Associations (FELASA) September 1992, revised February 1995. Transgenic Animals - Derivation, Welfare, Use and Protection.

Gordon, J.W. and Ruddle, F.H. (1981). Integration and stable germ line transformation of genes injected into mouse pronuclei. Science 214:1244-1246

Gossler, A. et al. (1986). Transgenesis by means of blastocyst-derived embryonic stem cell line. Proc. Natl. Acad. Sci. 83:9065-9069.

Jaenisch, R. (1976). Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. 73:1260-1264.

Moore, C.J. and Mepham, T.B. (1995). Transgenesis and animal welfare. ATLA 23:380-397.

US Congress, Office of Technology Assessment (1989). New Developments in Biotechnology: Patenting Life. Special Report OTA-BA-370. 3pp. Washington DC: US Printing Office.

For Further Reading

"See How They (Don't) Grow." Successful Farming. March 1991, p. 33.

"Transgenic Animals in the Production of Therapeutic Proteins." Biotechnology International. Century Press, 1992, p. 317.

"Whole Animals for Wholesale Protein Production." Bio/Technology. August 1992, p. 863

Animal species	Number of ova injected	Number of offspring	Number of transgenic offspring
rabbit	1907	218 (11.4%)	28 (1.5%)
sheep	1032	73 (7.1%)	1 (0.1%)
pig	2035	192 (9.4%)	20 (1.0%)

TRANSGENESIS

Transgenesis