Gene-targeting the <i>Ptp4a3</i> locus.

<p>A) The <i>Ptp4a3</i> floxed allele was created by inserting loxP sites (red arrowheads) around exon 2. This mutation resulted in the disruption of a HindIII sequence that increases the size of the DNA fragment produced by restriction digest from 5.6 to 10.2 kb. Following expression of Cre recombinase, the sequence between the loxP sites is removed leaving a single loxP site and decreasing the size of the restriction fragment to 6.4 kb. This mutation results in deletion of the initiation codon causing a frameshift mutation producing a nonfunctional protein product. B) Southern blot analysis of genomic DNA was used to monitor these genetic changes and genotype mice containing the wildtype, floxed, or knockout alleles. Each lane is from an individual mouse. C) Quantitative RT-PCR analysis on total mRNA from fetal heart tissue revealed no change in <i>Ptp4a1</i> and <i>Ptp4a2</i> mRNA levels while <i>Ptp4a3</i> was reduced and not detectable in heterozygous and homozygous-null tissue, respectively. (* = p<0.05; n = 4/genotype). D) The PTP4A3 protein product was detectable by western blot in whole protein lysates from wildtype and heterozygous fetal heart tissue, but not in homozygous <i>Ptp4a3-</i>null samples.