Somatic cell gene targeting
The generation of HOIP-deficient cells was accomplished using a homologous recombination approach described previously [7], [8]. Segments of Rnf31 gene sequence used in the targeting construct (Fig. 1A) were amplified by PCR from A20.2J genomic DNA. The oligonucleotide primers used to generate the 5′ flank (1224 bp) were 5′-ttttctagagcggtggcttaagtgaccc-3′ and 5′-tattctagatgcagcatctgagaaagcaagc-3′. The 3′ flank (6032 bp) primers were 5′-aaaaccggtgtatgcttctttacgggagaaaaatattag-3′ and 5′-tataccggtatgaagccaaaggaacactgagag-3′. Restriction endonuclease sites in the oligonucleotide primers allowed insertion of the PCR products into the targeting vector. A20.2J cells were transfected (by electroporation [7]) with the targeting construct and subcloned in medium containing 600 µg/ml G418 sulfate. Homologous recombination in G418-resistant clones was detected by PCR of genomic DNA, as described [7]. Oligonucleotide primers used for screening were 5′-cttcctgatctcagctttaccgtcac-3′ (homologous to genomic sequence; approximate position noted in Fig. 1) and 5′-caatccatcttgttcagccat-3′ (homologous to sequence in NeoR). Clones in which one copy of Rnf31 had been disrupted were transiently transfected with an expression plasmid encoding Cre, in order to remove NeoR. G418-sensitive subclones were subjected to a second round of targeting to disrupt the remaining copy of Rnf31. G418-resistant clones were tested for homologous recombination by PCR and HOIP protein expression by Western blot.