Journal: Molecular and Cellular Biology
Article Title: Mice Devoid of Fer Protein-Tyrosine Kinase Activity Are Viable and Fertile but Display Reduced Cortactin Phosphorylation
Figure Lengend Snippet: Domain structure of Fer and targeting strategy of fer locus. (A) Fer consists of three CC motifs, a central SH2 domain, and a kinase domain. The catalytic subdomains are shown, with subdomain IX encoded by exon 19 contained within genomic clone 3B. The targeting vector contains a 6-kbp long arm of homology, followed by the PGK-neo cassette, a 0.9-kbp short arm of homology, and a PGK-tk cassette. The D743R mutation was generated within exon 19, which also introduces an Nru I (N) site. A schematic of the targeted allele following homologous recombination is shown, with positions of PCR primers and Southern blot probe indicated. Positions of Xba I (X), Xho I (Xh), Nhe I (Nh), Eco RI (E), and Sac I (S) sites are shown. Sites in parentheses were destroyed. (B) Southern blot analysis of Sac I-digested genomic DNA from animals that were wild type (+/+), heterozygous (+/−), or homozygous (−/−) for fer D743R . Hybridization with a probe located 3′ to the sequence used for the short arm of homology reveals bands of 4.0 kbp for the wild-type allele and 5.8 kbp for the fer D743R allele. (C) Genotyping PCR analysis of genomic DNA from +/+, +/−, or −/− animals. The resulting 871-bp fragment is resistant to Nru I digestion in wild-type samples, yields additional 605- and 266-bp bands in heterozygous samples, and yields only 605- and 266-bp fragments in homozygous samples.
Article Snippet: The PCR-generated 871-bp fragment was subsequently digested with Nru I (NEB), yielding bands of 605 and 266 bp in the presence of the D743R mutation.
Techniques: Plasmid Preparation, Mutagenesis, Generated, Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Hybridization, Sequencing