Ligand Activated Site Specific Recombination Of Gene And The Generation Of Transgenic Mice

938 WordsMar 23, 20164 Pages
Ligand activated site specific recombination in mice Inactivation of definite genes by homologous recombination in the mouse has proved to be revolutionary for the control of mammalian development and the determination of its physiology. Some factors like the selection marker, deficit gene product and intra-utero lethality due to the inactivation of the gene limited the gene targeting and elucidation of the knockout phenotypes. Time and the tissue specific inactivation of the target gene by engineered DNA recombinase was successful in elimination of the limiting factors. However the somatic mutations could not be introduced using the recent mouse gene targeting technologies, pristine version of Cre/Lox system has been used to achieve conditional site-specific recombination in the mice. In this paper repeated lox P recognition sites were used to flank DNA by using bacteriophage P1 Cre recombinase. The experiment started with the construction of the plasmids and the generation of transgenic mice. PCMVCre-ERT was made by the cloning of E CORI fragment from pCre-ERT into the E CORI of the expression vector. pCre-ERT was then formed by replacing the HINDIII-BglII fragment of pCre-ER with the fragment secluded from the expression vector. Glycine was then mutated to arginine by mutagenesis. The restriction fragment of the IE gene of the CMV virus was cloned into the BSM+. The simian virus promoter was then replaced with the CMV promoter by

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