How does trangenic technology work?
high throughput screening
Definition
Transgenic technology refers to the genetic engineering of an organism so that its genomic DNA is altered to over- or underexpress certain genes. All progeny of a transgenic organism will share the same genotype as the parents (assuming both parents are homozygous). A knockout refers to a transgenic organism in which a gene has been replaced or disrupted with an artificial piece of DNA. The most common type of transgenic organism used in research is the knockout mouse, though knockout rats and knockout rabbits have also been developed. It has become routine to develop knockout mice with disruptions in specific genes. These knockout mice allow scientists to view the effect of this gene disruption on the resulting phenotype. Often the phenotype is a direct result of the gene knockout and can provide clues as to the biological role of the gene, but occasionally the phenotype can also be the result of compensatory or indirect effects of the gene knockout. Sometimes the procedure to create a transgenic organism can result in a phenotype completely unrelated to the disrupted gene, but is an effect of the artificial DNA used to create the disruption. In addition, some gene knockouts create a lethal phenotype where the organism fails to develop in utero, making in vivo studies exceedingly difficult.
Creation of a knockout mouse
The production of a knockout mouse begins by the harvesting of embyronic stem cells (ES cells) from early-stage mouse embryos 4 days after fertilization. ES cells are used because they can differentiate into any cell type and a gene knocked out of these cells will be absent in all tissue. ES cells can be used to generate knockout mice as long as 10 years after they are harvested. Researchers use 1 of 2 ways to introduce artificial DNA into the chromosomes of the ES cells. Both methods are carried out in cultured cells.
Homologous recombination
Homologous recombination or gene targeting involves manipulation of a gene in the ES cell nucleus. This is accomplished by introducing artificial DNA sharing a homologous sequence to the gene of interest. The homologous sequence is chosen such that it flanks the target gene's DNA sequence both upstream and downstream on the chromosome. The cell's own machinery recognizes the homologous sequences and swaps the target gene for the artificial DNA. The artificial DNA is inactive, containing only a reporter gene used to track successful recombination. This method is useful because the target gene can be precisely knocked out at a high rate of efficiency.
Gene trapping
In gene trapping, investigators also manipulate genes in the ES cell, but instead of a homologous sequence, a random sequence is used containing a reporter gene. The artifical DNA is designed to insert randomly into any gene, preventing the RNA splicing mechanism from working properly, thus knocking out the gene's function. The advantage of gene trapping is that the investigators do not need to know the DNA sequence of specific genes, but rather a single vector can be used in a high throughput manner to generate many mice in which a variety of genes have been disrupted. This method is not useful to target a specific gene, and is not as efficient as homologous recombination.
Production and refinement
In both techniques, a modified viral vector or linear bacterial DNA is used to carry the artificial DNA into the ES cells. Following successful insertion of the artificial DNA, the ES cells are grown in culture for several days and are then injected into early-stage mouse embryos which are then implanted into the uterus of a female mouse for further development. The resulting mouse pups (if they survive to birth) have tissue derived from the engineered ES cells and also normal tissue from the non-altered embryos into which the ES cells were injected. They are heterozygous knockouts. These heterozygotes are crossbred to produce homozygous knockout mice. Homozygous knockouts can be identified using genomic PCR for the gene of interest.