HGM2002 Poster Abstracts: 8. Disease Mechanisms
POSTER NO: 476
Establishment of Glucokinase Conditional Gene Knockout Embryonic Stem Cells based on the Cre-LoxP System
Yali Zhang, Huanran Tan
Pancreatic beta cells and hepatocytes are the two major cell types in maintaining glucose homeostasis, in which there is specific glucokinase playing an essential role. In humans, glucokinase gene mutations can cause maturity onset diabetes of the young, type 2 (MODY-2). Therefore, we considered generating an MODY animal model and assessing the effects of disrupted glucokinase function in both beta cells and hepatocytes based on the Cre-LoxP system Objective: In order to create tissue-specific knockout mouse models and to establish the technique system of conditional gene knockout, two directly repeated LoxP sequences were inserted into to flank the exon9 and exon10 of glucokinase gene in embryonic stem (ES) cells. Methods: Linearizd replacement targeting vector was electroporated into ES cells. The cells were grown and selected by both G418 and Gancyclovir. G418 and Gancyclovir-double-resistant clones were screened for homologous recombination by PCR and further confirmed by Southern blotting analysis. Results: 161 ES cell clones to resist either G418 or Gancyclovir were selected. Among these resistant clones, 41 clones were positive to PCR and only 8 clones were positive to Southern blot. Conclusion: 8 ES cells clones that had undergone the desired recombination event were obtained, meanwhile, we established the useful and convenient methods to screen the ES cells, that is, combining PCR and Southern blot with two different restriction enzymes.
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