[Conditional targeting of p16(INK4a)exon 1a in mouse embryonic stem cells].
OBJECTIVE
To study the relationship between targeting vector structure and homologous recombination rate and investigate whether the mouse p16(INK4a) plays a role in tumor suppression.


METHODS
A conditional  targeting vector with 2.0 kb EcoR I/Xba I fragment as short arm and 5.9 kb SpeI/NotI fragment as long arm was built. Of the 2 direct locus crossing-   over(loxPs) in the vector, one was inserted at 240 bp upstream of the initiate code of p16(INK4a)  exon 1a and the other at 1633 bp downstream of the initiate code. Both exon 1a and the selection marker Neo will be deleted in targeted cells when mediated by Cre. After linearlization and purification, t he targeting vector was introduced into ES cells through electroporation.


RESULTS
Twenty-four G418-   and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization.


CONCLUSIONS
Targeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.
Gong
Zhenming
Z
Medical Genetics Department, Second Military Medical University, Shanghai, 200433 P.R.China. jlfu@guomai.sh.cn
Zheng
Jingmin
J
Fu
Jiliang
J
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005091:Exons / Q000235:genetics*; D015774:Ganciclovir / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D005839:Gentamicins / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000900:Anti-Bacterial Agents; D000998:Antiviral Agents; D019941:Cyclin-Dependent Kinase Inhibitor p16; D005839:Gentamicins; C010680:antibiotic G 418; D013937:Thymidine Kinase; D015774:Ganciclovir