Conditional targeting of p16(INK4a)exon 1a in mouse embryonic stem cells / 中华医学遗传学杂志

ABSTRACT

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>Twenty-four G418- and gancyclovir-resistant ES cell colonies were picked out and one of them was confirmed as positive by Southern hybridization.</p><p><b>CONCLUSION</b>Targeting vectors with 2 TK genes flanking the homologous arms are likely to produce good result of homologous recombination.</p>