P16～(INK4a)EXON 1a KNOCKOUT IN MOUSE EMBRYONIC STEM CELLS

INK4a/ARF locus distinguishes itself by its unusual structure and function. It contains two overlapping genes with exons 1α, 2 and 3 encode p16^INK4aand exons 1β , 2 and 3 encode p19^ARF. Mice with their exons 2 and 3 of the INK4a/ARF knocked out are viabl and fertile but develop spontaneous tumors at an early age and highly sensitive to carcinogenic treatment. Resent studies, however, reveal that mice with their exon 1β of INK4a/ARF knocked out, without interference the expression of p16^INK4a, show almost the same phenotype as those with their exons 2 and 3 knocked out. If p19^ARFbut not p16^INK4afunctions as a tumor suppressor, it will be difficult to explain the fact that more than 30% of the human cancers are related to p16^INK4a inactivation but mutations in exon 1β are extremely rare. Knocking out the exon 1 α might be a reseanalbe way to investigate the phenotype of exon 1α-null mouse and clarify the function of mouse p16^INK4a. In this study, we have made the following efforts to achieve this goal. According to the exonl a and its upstream sequence published elsewhere, a pair of PCR primers was designed by PCGEN. The PCR product, using R1 ES cell genomic DNA as template, was collected as probe to screen the mouse genomic library. A 14.5Kb Sal Ⅰ/Sal Ⅰ genomic DNA fragment was obtained and then sequenced. Bioinformatics analysis suggests that the structure of this DNA fragment may be responsible for the p16^INK4a inactivation found in some tumors. A common targeting vector with 1.5kb Sal Ⅰ/Acc Ⅱ fragment as short arm and 5.9kb Xba Ⅰ/Xho Ⅰ fragment as long arm was built based on the plasmid pSSC-9. The conditional targeting vector contains 2.1 kb EcoR Ⅰ/Xba Ⅰ fragment as short arm and 5.9 kb Spe Ⅰ/Not Ⅰ fragment as long arm. One loxP was inserted at 240bp upstream of the initiate code of INK4a/ARF exon 1α and the second loxP inserted at 1633bp downstream of the initiate code of INK4a/ARF exon 1α. Both exon 1α and the selection marker Neo will be deleted in targeted cells when mediated by Cre. Linearized and purified conditional targeting vector was used to electroporate ES cells. After G418 and Gancyclovir selection, 24 ES drug-resistant colonies were picked out and one of them was confirmed as positive by Southern hybridization.