PMC:3148254 / 28570-30091 JSONTXT

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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. 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