Isolation of CERT-deficient (Certgt/gt) mice. (A) The genomic organization of Cert is shown, and the location of the gene trap vector in the ES cell line RRF047 is depicted in the middle panel. This insertion results in truncation of the product within the pleckstrin homology (PH) domain of the CERT protein and results in a null or severely hypomorphic mutant. The insertion site was confirmed by sequence analysis of PCR products. The third intron of Cert is 26,177 bp long. The gene trap was inserted 18,394 nucleotides downstream of the 5′ end of this intron. START, steroidogenic acute regulatory protein related. (B–D) The genotype of the Cert+/gt heterozygous intercross was confirmed by genomic PCR across the insertion point (B), RT-PCR across exons 3 and 4 (C), and Western analysis (D) as described in Materials and methods. +/+, wild type; +/−, heterozygous mutant; −/−, homozygous mutant. (E) Certgt/gt embryos have growth defects. The mutant embryos are smaller in size compared with wild type. These changes are obvious even in E7.5 embryos.
