EndoU is a novel regulator of AICD during peripheral B cell selection

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.; Tedder, Thomas F.

To disrupt the EndoU gene by homologous recombination, an 8 kb DNA fragment containing exons 1-7 was isolated from a B6 genomic BAC clone (RP24-169P4). Flanking BsmBI restriction sites were used to remove the distal end of exon 4, a portion of intron 4 and the splice donor site, which was replaced with a neomycin-resistance gene (neo r ) that introduced an inframe termination codon. A thymidine kinase (tk) gene was inserted at the 5 end of the EndoU targeting sequence. Appropriate homologous recombination in EF1 (B6:129 hybrid) embryonic stem cells (Duke Transgenic Mouse Facility) was confirmed by Southern blot analysis of HindIII digested genomic DNA using a probe outside of the targeting sequence. The targeted allele generated an expected 8.5 kb band, compared with the WT 7.5 kb fragment. Correct EndoU gene targeting in subsequent offspring was verified by PCR analysis using a common forward primer (PCR1) in combination with reverse primers either 3 of (PCR2) or within (PCR3) the inserted neo r gene.