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Gene Knockout in MiceBY: Shikha Sharma | Category: DNA | Submitted: 2010-11-22 08:13:44
Article Summary: "There are the phenotypes of mice that lack a functional copy of a particular gene. As example, it was noted that mice lacking a functional copy of the p53 gene invariable develops malignant tumors. These animals are called as knockout mice..."
There are the phenotypes of mice that lack a functional copy of a particular gene. As example, it was noted that mice lacking a functional copy of the p53 gene invariable develops malignant tumors. These animals are called as knockout mice. The latter can provide a unique insight into the genetic basis of human disease as well as a mechanism for studying the various cellular activities in which the product of a particular gene might be engaged.
The technique used to generate knockout mice was developed in the late 1980s by Mario Capecchi at the University of Utah. The first step is to isolate an unusual type of cell that has virtually unlimited powers of differentiation. These cells, called embryonic stem cells, are found in the mammalian blastocyst, is composed of two distinct parts. The outer layer makes up the trophoectoderm, which giver rise to most of the extraembryonic membranes characteristic of a mammalian embryo. The inner surface of the trophoectoderm contacts a cluster of cells called as inner cell mass that projects into a spacious cavity called as blastocoel. The inner cell mass gives rise to the cells that make up the embryo. The inner cell mass contains the embryonic stem (ES) cells, which differentiate into all of the various tissues of which a mammal is composed.
ES cells can be isolated from blastocysts and cultured in vitro under conditions where the cells grow and proliferate. The ES cells are then transfected with a DNA fragment containing a nonfunctional, mutant allele of the gene to be knocked out as well as antibiotic resistant genes that can be used to select for cells that have been incorporated the altered DNA into their genome. Of those cells that take up the DNA, approximately one in 10 raised to the power 4 undergo a process of homologous recombination in which the transfecting DNA replaces the homologous DNA sequence. Through the use of this procedure, ES cells that are heterozygous for the gene in question are produced and then selected on the basis of their antibiotic resistance.
In the next step, a number of these donor ES cells are injected into the blastocoel of a recipient mouse embryo. The recipient embryo is obtained from an albino (non-pigmented) strain. The recipient embryo is then implanted into the oviduct of a female mouse that has been hormonally prepared to carry the embryo to term. As the embryo develops in its surrogate mother, the injected ES cells join the embryo's own inner cell mass and contribute to formation of embryonic tissues, including the germ cells of the gonads. These chimeric mice can be recognized because their coat contains characteristics of both the donor and recipient strains. To determine whether the germ cells also contain the knockout mutation, the offspring will be heterozygous for the gene in all of their cells. Heterozygotes can be distinguished by their coat coloration. These heterozygotes are then mated to one another to produce offspring that are homozygous for the mutant allele.
These are the knockout mice that lack a functional copy of the gene. In some cases, the deletion of a particular gene can lead to the absence of a particular function. Often, however, the absence of one gene product is compensated by the product of an entirely different gene, so that little effect on the phenotype is evident.
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