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12-‐‑10-‐‑16 BIL 250 -‐‑ Knockout Mouse Knocking out a Gene Step One: isolating a "knocked out" gene in cell culture A cloned gene of interest is taken from a library It is inactivated via insertion of a known gene (the one used in the example here confer resistance to neomycin, which is lethal to cells not carrying the neor gene) The vector also is given another marker, this one called "tk", which makes any cell carrying it lethally sensitive to the drug ganciclovir. (The "tk" gene has a restriction site that excludes it if the neomycin resistance-inactivated vector is inserted into the desired location, replacing the wild type gene of interest) Embryonic Stem Cells (ES cells) from a wild type agouti mouse are cultured in vitro Vectors are added to the mouse cell culture and the scientist crosses his/her fingers www.bio.miami.edu/dana/dox/knockout.html 1/4 12-‐‑10-‐‑16 BIL 250 -‐‑ Knockout Mouse Three things could happen: Vector will insert into the desired target gene (in this case, it's the Mystery Gene "M", whose function is unknown), replacing it with the mutant (inactivated) version (with the neor insertion) Vector will insert ectopically (in a random location) Vector won't insert at all The desired result, of course, is replacement of the wild type gene (M) with the artificially knocked out version (m). To isolate cells in which replacement has successfully taken place: Putatively transfected cells from your suspension are grown in vitro. The agar medium contains both neomycin and gangclovir. Hence, neomycin will kill the cells that don't have a neor insertion (the knocked out "m" allele) gangclovir will kill the cells that have ectopic insertions, as those will have retained the "tk" gene. This leaves only the cells that have the insertion of the mutant, inactivated form replacing the wild type gene of interest. These successfully transfected cells can be cloned and used for further study. Step Two: Creating a Chimera Mouse (carrying the knocked out gene) www.bio.miami.edu/dana/dox/knockout.html 2/4 12-‐‑10-‐‑16 BIL 250 -‐‑ Knockout Mouse Your cell culture of diploid cells now contains one normal chromosome and one with a knocked out version of the wild type. These modified Embryonic Stem (ES) cells from the wild type agouti mouse are injected into a blastocyst from a strain of black mice (black fur is a recessive version of the A gene, coding for agouti fur). The modified embryos are inserted into a surrogate mother mouse, who gives birth to them. The baby mice who have patches of both black and agouti fur are the chimeras who have grown from embryos that successfully incorporated the modified Embryonic Stem Cells from the agouti mice. Choose a male chimera for greatest reproductive output. Step Three: Breeding Knockout Mice www.bio.miami.edu/dana/dox/knockout.html 3/4 12-‐‑10-‐‑16 BIL 250 -‐‑ Knockout Mouse If you mate the male chimera to a black female, 50% of the offspring should carry the knocked out gene from the father mouse. By taking tissue samples, isolating DNA and examining it directly for evidence of the insert, the individuals carrying the knocked out gene can be identified. These Mm heterozygotes can then be mated together. According to Mendel's Law, 25% of the resulting offspring will be mm. The knocked out gene may not have an obvious phenotypic expression, in which case the knockout mouse DNA can be examined directly for evidence of homozygous recessive condition. In the example above, however, the knocked out gene resulted in a curly tail in the homozygous knockout mice: The M gene must have something to do with normal development of the caudal vertebrae in wild type mice. And so it goes. www.bio.miami.edu/dana/dox/knockout.html 4/4