Journal: PLoS Genetics
Article Title: Senataxin Plays an Essential Role with DNA Damage Response Proteins in Meiotic Recombination and Gene Silencing
Figure Lengend Snippet: Targeted disruption of the mouse Setx gene. A. Diagram of the Setx wild type allele (WT), targeting vector, and mutant alleles (neo+ and KO). Primers used for PCR genotyping (In3F, In3R, LoxPR) and the length of the PCR fragments obtained for WT (In3F and In3R yielding a 600 bp product) and KO (In3F and LoxpR yielding a 339 bp product) are indicated. E, exon; I, intron. NeoR represents the neomycin cassette, and triangles the loxP sites. B. Representative image of PCR genotyping using In3F, In3R and LoxPR primers. Wild type (+/+), heterozygotes (+/−) and knockout (−/−) alleles generate PCR products of 600 bp, 600 bp and 339 bp, and 339 bp, respectively. A negative control for the PCR reaction (−ve) is also shown. M, 100 bp marker. C. RT-PCR of 35 day-old mice testes samples using primers specific to Setx cDNA indicates the absence of Setx expression in KO testes. GAPDH was used as an internal standard. D. Immunoprecipitation of senataxin using anti-human senataxin antibodies (Ab-1/Ab-3) from 35 day-old mouse testes extracts confirmed the absence of the protein in the Setx −/− . Immunoprecipitation of senataxin from human lymphoblastoid cell extracts from normal (C3ABR) and an AOA2 patient (SETX2RM) confirmed the similar size of senataxin in both species. A species-matched non-specific serum (NSIg) was used as a negative control in for the IP experiments. As expected, no senataxin protein was brought down from Setx +/+ testes following the IP with the non-specific serum (NSIg).
Article Snippet: Reactions (25 µl) contained 14.5 µl of sterile water, 50 ng of cDNA template, 1× PCR Buffer II (Roche, Switzerland), 2.5 mM MgCl2 (Roche, Switzerland), 20 µM dNTPs, 1 µM of each primer, and 5 µl of AmpliTaq Gold DNA Polymerase (Roche, Switzerland).
Techniques: Plasmid Preparation, Mutagenesis, Polymerase Chain Reaction, Knock-Out, Negative Control, Marker, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Expressing, Immunoprecipitation