Creating a loss-of-function CFTR

Creating a loss-of-function CFTR (cystic fibrosis transmembrane conductance regulator) allele in mice through homologous recombination. Mutations in the human ortholog are the cause of cystic fibrosisCreating a loss-of-function CFTR (cystic fibrosis transmembrane conductance regulator) allele in mice through homologous recombination. Mutations in the human ortholog are the cause of cystic fibrosis

(a) Create knockout allele by homologous recombination (b) Generate knockout mouse from ES cells. in embryonic stem cells. Homology with CFTR gene Linearized targeting Neomycin Thymidine vector injected into resistance kinase ES cells CFTR'ICFTR‘ \ 55.23% 29.2322: be \ eeeeeeeeeueeee containing the mouse k k \ cells into host blastocysts, CFTR gene and rePlace mar er mar er \ creating blastocysts that the central region with are genetic chimeras a posrtive selectable g , / l \ N containing both Wild- -type marker gene, disruptlng Three possible fates i , e _ e cells and heterozygous ‘ the CFTR 9909- of injected DNA. 1 When coat-color mutant cells. 3 ’ ‘ ' mutants are used, \ J l chimeric offspring are V ’ . ‘ ‘ readily identified: B— = z ‘ 01 Homologous 0 Illegitimate 9- No ,_ brown; bb = black. / , e ; recombination recombination recombination A ' l ‘ Inject blastocysts into ; uterus Neomycin resistant, Neomycin resistant, Neomycin sensitive, ganciclovir resistant ganciclovir sensitive ganciclovir resistant Progeny mice are mosaic. ‘ After transformation, and application of both positive , and negative selection, only those cells that have acquired the positive marker (neo’i) but not the / Mate mosaic mice \ negativermarker (HSV thymidine kinase) will survrve. j Generate with wild type, If A, , _ r r _ _ e _ , _. ,4 homozygous transgenels present w u _ x knockout in germ cells, it will mouse be heritable. Select ES cells that are Some progeny will both neomycin and be heterozygous for ganciclovir resistant. the knockout alleles. V . other mice will be CFTRb /CFTR‘ CFle‘lcftr CFTR/d‘t wild type l Meshetewwee- l memm CFTR‘ICFTR‘ CFTR‘lcftr cftr/cftr Homozygous knockout mouse Homologous recombinants will be heterozygous.