Gene targeting strategy for generation of MrgE KO mice. (A) The targeting vector was assembled on the pGT-N28 backbone (NEB, Ipswich, MA) and so designed such that a total of 776 base pairs from within the 930 base pair coding region located in exon 2 of the 2 exon MrgE gene (first exon is un-translated) was deleted and replaced with a 7,076 base pair IRES-lacZ reporter and neomycin resistance cassette. Solid Line: intron 1; boxes colored red: exons 1 and 2; box colored blue: coding sequence located in exon 2; uncoloured box: IRES-LacZ-NEO cassette replacing part of the coding sequence located in exon 2; black triangle: FRT sequence. (B) Genotyping assay was designed to differentiate among wild type (+/+), heterozygote (+/-) or homozygote knockout (-/-) MrgE mice and performed using a multiplex PCR reaction, with a shared 5' forward primer (5'-GCA GAC ATC AGC CAT GAC GT-3') and a 3' reverse primer unique to the targeted allele (#2416, 5'-ATC AGC TTA CCA TGG CCA AGA TCC C-3') or to the MrgE locus (5'-ATC TAT CTC TTG GAT GTG GCC TG-3'). The WT amplicon is 201 and KO allele 922 bps.