QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 1. Targeted disruption of the CAR gene in mice. (A) Targeting strategy. A map of the relevant genomic region containing the first ATG-containing exon of CAR (top), the targeting vector (middle) and the mutated locus after recombination (bottom) are shown. Dor25, GS3 and Neo2L represent oligonucleotides used for PCR screening. The positions of probes used for Southern screening are depicted as bars #1 and #2. E=EcoRV, X=XhoI. (B) Detection of wild-type and targeted alleles by Southern blot analysis. Genomic DNA of mice of the F1-generation was digested with EcoRV and XhoI and hybridized with the combination of probes #1 and #2. DNA of wild type (+/+) mice gave an 8 kb signal (EcoRV fragment), +/– heterozygotes gave an additional signal for the 6.4 kb EcoRV/XhoI fragment. (C) PCR analysis of genomic DNA with the oligonucleotides Dor25, GS3 and Neo2L described in A. A 1kb product is generated from the wild-type allele and a 1.5 kb product from the targeted allele. (D) Western blot of SDS-PAGE extracts of mouse embryos (E11.5) of the three different genotypes with antibodies against mouse CAR. Note that heterozygotes expressed less CAR protein than homozygous wild-type embryos. (E) Activity and specificity of affinity purified rabbit anti-mouse CAR antibodies illustrated by western blot analysis of mock transfected CHO cells and CAR-transfected CHO cells (as indicated). Molecular mass markers are indicated on the left. (F) Specificity control for affinity purified anti-CAR antibodies (VE15) used for indirect fluorescence staining of the heart region of E11.5 CAR^+/+ and CAR^-/- embryos (as indicated).

Return to article