[Construction of gene targeting vector for duplicating p35Nck5a gene and its application in the gene targeting of ES cells].
To duplicating the regulatory subunit p35Nck5a gene of mouse neuronal cdc2-like kinase in embryonic stem (ES) cells, about 12.2 kb of pGDTV vector for p35Nck5a gene duplication was constructed. The linearized pGDTV vectors were transfected into ES cells by electroporation. 245 drug-resistant cell clones were obtained in both G418 and GANC medium and the frequency of surviving cells was 6.22 x 10(-5). The ES cell clones were identified to have duplicated the p35Nck5a gene by use of both PCR and genomic Southern blotting, and the frequency of homologous recombination is 5.08 x 10(-7). The use of negative selection gene (HSV-tk) results in 7-fold increase at selection efficiency. This study lays the foundations of preparing mouse models of Alzheimer's disease.
Zhou
C W
CW
Department of Medical Genetics, Second Military Medical University, Shanghai, China.
Fu
J L
JL
Xue
H
H
D000544:Alzheimer Disease / Q000209:etiology; D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology*; D020440:Gene Duplication; D018390:Gene Targeting; D005822:Genetic Vectors; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics*; D013234:Stem Cells / Q000378:metabolism*
D009419:Nerve Tissue Proteins; C089495:neuronal Cdk5 activator (p25-p35)