Conditional Pten Inactivation in Mouse

A 129/Sv mouse genomic library (Stratagene, La Jolla, California, United States) was screened with a mouse Pten probe containing exons 4–6. To generate the targeting construct, a 4.1 Kb KpnI–BamHI fragment containing 5′ Pten genomic DNA and a 2.0 Kb XbaI fragment containing 3′ genomic DNA were cloned into pPNT. The targeting construct was linearized with NotI and electroporated into CJ7 ES cells. Transfectants were selected in G418 (350 μg/ml) and gancyclovir (2 μM) and expanded for Southern blot analysis using a 3′ probe. Chimeric mice were produced by microinjection of two independently generated targeted ES cell clones with normal karyotypes into E3.5 C57BL6/J blastocysts, then transferred to pseudopregnant foster mothers. Chimeric males were mated with C57BL6/J females (Jackson Laboratory, Bar Harbor, Maine, United States), and germline transmission of the mutant allele was verified by Southern blot analysis of tail DNA from agouti coat-colored F1 offspring. Next, Pten^loxP-neo/+ mice were mated with EIIA-Cre transgenic mice (Lakso et al. 1996 (link)), and tail DNA from offspring was subjected to Southern blot analysis using probe 6.1. Through these crosses, mosaic mice harboring a Pten wild-type allele, a Pten targeted allele (Pten^loxP-neo), and a floxed allele (Pten^loxp) in their germline were generated. These mosaic mutants were mated with wild-type mice and tail DNA from offspring subjected to Southern blot analysis using probe 6.1 and to PCR analysis using primer 1 (5′-AAAAGTTCCCCTGCTGATGATTTGT-3′) and primer 2 (5′-TGTTTTTGACCAATTAAAGTAGGCTGTG-3′). PCR conditions were 35 cycles (30 sec at 95°C, 1 min at 55°C, and 1 min at 72°C) using HotStarTaq Master Mix (Qiagen, Valencia, California, United States), primer (0.25 μM), and DNA (50 ng). To detect the deleted allele, primer 3 (5′-CCCCCAAGTCAATTGTTAGGTCTGT-3′) was used. Pten^loxP/loxP mice were next mated with PB-Cre transgenic mice (Maddison et al. 2000 (link)) or male PB-Cre4 transgenic mice (Wu et al. 2001 (link)) for conditional prostate-specific Pten inactivation.