Fig. 1. Targeted disruption of the Abcc10 gene in mice.
A), Schematic of the targeting strategy. Shown are a portion of the Abcc10 gene in which exons encoding part of nucleotide binding domain-1 reside, the targeting construct, the predicted targeted locus and endonuclease restriction sites, predicted EcoR1 fragments for the wild-type and targeted locus (top and bottom, respectively) and location of the 3’ probe used for Southern blot analysis. The targeting construct is designed to delete exons 6 – 8 (amino acids 204–332), which include the coding sequence of the entire Walker B motif required for ATPase activity, and to cause a frameshift which results in a prematurely terminated Abcc10 protein. Proper recombination replaces 2.3 kb of Abcc10 sequence with the 1.8 kb neomycin cassette and introduces an EcoR1 site. Exons are shown as black rectangles. B), Southern blot analysis of EcoR1-digested DNA prepared from representative ES cell clones. Wild-type restriction fragments (12 kb) are seen in all lanes, and the 7.5–kb fragment (indicated by *) resulting from proper recombination are found in the 2 lanes labeled +/−. C), Relative expression levels of Abcc10 mRNA in mouse tissues as assessed by real-time Taqman PCR assay. Values are averages and SD’s of two reactions with different amounts of RNA. Data are presented using an arbitrary scale with mRNA levels in testes set to 100. For each sample, the values are average and standard deviation of data from two PCR reactions performed with two amounts of total RNA (50 ng and 12.5 ng) in the RT reaction. D) Immunoblot detection of Abcc10 in primary kidney cell lines and spleen. Fifty micrograms of kidney cellular lysate and 20 µg of spleen membranes were analyzed. Abcc10 is indicated by the bracket and a molecular weight market is shown to the right