Transgenic Animals

The term "transgenics" refers to the science of inserting a foreign gene into an organism's genome . Scientists do this, creating a "transgenic" organism, to study the function of the introduced gene and to identify genetic elements that determine which tissue and at what stage of an organism's development a gene is normally turned on. Transgenic animals have also been created to produce large quantities of useful proteins and to model human disease.

In the early 1980s Frank Ruddle and his colleagues created the first transgenic animal, a transgenic mouse. Researchers making transgenic mice use a very fine glass needle to inject pieces of DNA into a fertilized mouse egg. They inject the DNA into one of the egg's two pronuclei , before the pronuclei fuse to become the nucleus of the developing embryo's first cell. After the DNA is injected, multiple copies, usually joined end-to-end, insert randomly into the host organism's nuclear DNA.

Multiple injected embryos are then transferred to a surrogate mother mouse to develop to term. Only a small percentage of the embryos survive the injection, and even of those that survive, not all have successfully incorporated the foreign DNA into their genome. Once the mice are born, researchers must identify which mice have the foreign gene in their genome. The animals that contain the added foreign DNA, or transgene are referred to as transgenics.

Targeted Gene Replacement and "Knockouts"

A gene that is injected into a fertilized egg is generally integrated randomly into the host genome. This means that scientists originally had no control over where in the host genome the foreign gene would land, nor could they control the number of copies of the gene that would be integrated. Where and how many copies of a gene are inserted can profoundly affect its function, so scientists looked for ways to make more precise insertions.

In the late 1980s Mario Capecchi and colleagues pioneered a method to target the inserted gene to a desired position in the genome. These researchers took advantage of an observation that, on rare occasions, an injected, mutated copy of a gene lines up precisely with the original form of the gene in the mouse genome. By a process called homologous recombination, the aligned DNA segments are cut and rejoined to each other. The result is a precise stitching of the introduced DNA into the targeted gene in the mouse genome. This means that scientists found they could make minor modifications to a gene before injecting it and, by homologous recombination, or "gene targeting," replace the natural gene with this transgenic version. For commercial applications, the transgene is often a gene coding for a functional human protein, which is then mass-produced in the host organism and isolated. For research purposes, it is often more useful to insert a mutated, nonfunctional version of the gene, to see what happens when the normal, functional version is missing. Creating such "knockout" organisms is a key tool used for studying genes that control development.

Selection of Gene Targeted Cells

Homologous recombination is a very rare event, and scientists using it to modify or "knock out" mouse genes must identify the cells in which it has occurred. In addition to injecting the gene they are trying to incorporate, scientists also inject "selectable" genes whose products permit cells to live or cause them to die in the presence of a particular drug. The two most common selectable genes used in gene targeting are the neomycin resistance (neo ^r) gene, which allows cells to survive in the presence of the antibiotic neomycin, G418, and the thymidine kinase (TK ) gene from the herpes virus. Cells with this gene die in the presence of the antiviral agent gancyclovir. The neo ^r and TK genes are generally used together for maximum selection.

The first step in gene targeting is to clone the gene that is to be replaced from the mouse genome. The cloned gene is placed into a targeting vector along with a selectable gene such as the neo ^r gene. (The targeting vector is a larger piece of carrier DNA.) When the targeting vector lines up with the native mouse gene and homologous recombination stitches the genes in the targeting vector into the genome, the neo ^r gene will be included. By adding G418 to the cell growth media, only those cells that have incorporated into their genome the transgenes, including the neo ^r gene, will survive. This is referred to as positive selection, selecting for cells that contain the desired integration product.

The TK gene is placed in the same targeting vector, but it is placed outside of the cloned mouse gene pieces. If homologous recombination occurs such that the added DNA lines up precisely with the native gene, the TK gene will be excluded. However, if the targeting vector integrates randomly into the genome the entire vector is inserted and the TK gene will be included. The addition of gancyclovir kills all cells that have the TK gene, thereby killing those where the insertion was random. The combination of positive neo ^r selection and negative TK selection results in the survival of only those cells containing targeted gene replacements.

From Transgene to Transgenic Organism

To get the targeted gene into the mouse genome, Capecchi used a very specialized embryonic cell previously isolated by Matthew Kaufman and Martin Evans. These embryonic stem (ES) cells were isolated from an early stage of embryonic development (the blastocyst ). When grown under controlled conditions in culture dishes, the ES cells have the remarkable capacity to become cells belonging to any tissue type. They can become muscle, cartilage, blood vessel or nerve cells, for example. Even more astonishing, when ES cells are injected back into a blastocyst, they mix with the cells of the recipient embryo and contribute some cells to every tissue in the body. Thus, if researchers place a transgene into the ES cell's genome and inject those ES cells into a blastocyst, the transgene could end up in every tissue type of the mouse.

Typically, the targeting vector is placed into ES cells derived from a mouse that has a brown coat. ES cells with the targeted gene replacement are identified through positive and negative selection. They are then injected into blastocysts from mating mice with black coats. These blastocysts are then placed into a surrogate mother and allowed to develop to term. The offspring that have incorporated transgene-containing ES cells into some of their tissues are identified by having patches of brown coat color.

To allow the targeted gene replacement to be passed to subsequent generations, the ES cells must also contribute to the developing embryo's eggs or sperm. To determine if the targeted gene has been incorporated into a mouse's eggs or sperm, the mice with brown patches are mated to black-coated individuals. The brown coat color is a dominant trait, so any off-spring with brown coats can be assumed to have arisen from germ cells that derived from the manipulated ES cells and thus contain the targeted gene replacement. These mice are mated to brown-coated siblings to produce homozygous transgenics, which are identified by determining if the offspring contain two copies of the transgene replacing both normal copies of the gene in the genome. These homozygous mutants are studied to look for phenotypic changes due to the transgene.

Applications

Gene targeting has been used to identify the function of hundreds of mouse genes. One dramatic example was the deletion of the Lim-1 gene by Richard Behringer and colleagues. The mice carrying this deletion died during embryonic development because of a complete lack of brain and head structure development. This demonstrated that the Lim-1 gene was critical for head development.

The gene responsible for sex determination in mice was also identified thanks to the use of transgenic animals. When a gene called Sry (sex-determining region of the Y chromosome) was microinjected into mouse embryos, the resulting transgenic mice were all male. Indeed, even in the mice that had two X chromosomes and were thus genetically female, the presence of the Sry gene was sufficient to cause them to develop testes and led to complete sex reversal. This clearly demonstrated that the Sry gene alone was responsible for sex determination.

Gene targeting is also being exploited by scientists to create models of human disease. For instance, mutations have been made in the mouse version of the cystic fibrosis transmembrane conductance regulator gene. Although mice with the mutated gene do not develop the devastating symptoms of cystic fibrosis in their lungs, they do develop the intestinal and pancreatic duct defects associated with the disease and thereby provide a model to study at least part of the disease. Transgenic mice overexpressing the amyloid precursor protein form deposits in the brain that resemble the amyloid plaques found in Alzheimer's patients. Mouse models such as these can potentially be used to test drug therapies and to learn more about the progression of the disease.

One important application of transgenic technology is the generation of transgenic livestock as "bioreactors." Key human genes have been introduced into sheep, cows, goats, and pigs so that the human protein is secreted into the milk of the transgenic animal. In theory, large quantities of the human protein can be produced in the animal's milk and subsequently purified for use in medical therapies. An early example of this technology by John Clark and colleagues was the production of transgenic sheep expressing the human blood-clotting factor IX needed by many patients with hemophilia. These researchers placed the human factor IX gene under the control of a piece of sheep DNA that normally turns on the beta-lactoglobulin gene in the mammary tissue. Though the sheep secreted factor IX into their milk, the levels of the protein were very small. With advances in the efficiency of creating and expressing genes in transgenic farm animals, therapeutic proteins can now be isolated.

see also Agricultural Biotechnology; Alzheimer's Disease; Biotechnology; Cloning Genes; Cloning Organisms; Cystic Fibrosis; Embryonic Stem Cells; Gene Targeting; Hemophilia; Recombinant DNA; Rodent Models; Sex Determination; Transgenic Microorganisms; Transgenic Organisms: Ethical Issues; Transgenic Plants; Y Chromosome.

Suzanne Bradshaw