Supporting Figure

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Fig. 7. Targeting of the DGKd gene. (A) Structural organization of DGKd protein. The region deleted in the mutant allele, which encodes the second zinc finger motif and part of the catalytic domain, is shown. The position of the epitope of the anti-DGKd antibodies is also indicated. (B) Strategy for generating a targeted mutation of the DGKd gene. The DGKd gene, the deletion targeting vector, and the recombinant mutant allele are shown. Exons 6-8 (numbered boxes) were replaced with a neo cassette for positive selection by homologous recombination. Thymidine kinase gene sequences (TK1 and TK2) were used for negative selection. The shaded box below the recombinant mutant allele indicates the location of the probe used in Southern genotyping analysis. Arrowheads above the DGKd gene show the position of oligonucleotide sequences used for RT-PCR analysis. Lines with double arrowheads show predicted EcoRI fragment sizes for WT and disrupted DGKd alleles. (C) Expression of the DGKd and b-actin mRNA in primary embryo fibroblasts from WT (+/+), heterozygous (+/-), and DGKd knockout (-/-) mice. (D) Western blot showing DGKd expression in primary keratinocytes and primary dermal fibroblasts. The band migrating above DGKd is nonspecific. (E) RT-PCR analysis of newborn mouse brain showing mRNA expression levels of all known murine DGK isotypes. (F) RT-PCR analysis of DGKd and b-actin expression in whole mouse embryos (embryonic day 7-17) and specific tissues from adult mice. The primers used to detect DGKd were common to both DGKd1 and DGKd2.