student research day - Case Western Reserve University School of ...
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Jacob M. McGrath<br />
Genotyping bradykinin B2 receptor/apolipoprotein E dual knockout mice by the polymerase chain reaction<br />
Jacob M. McGrath, Marvin T. Nieman and Alvin H. Schmaier<br />
Division <strong>of</strong> Hematology/Oncology, Department <strong>of</strong> Medicine<br />
<strong>Case</strong> <strong>Western</strong> <strong>Reserve</strong> <strong>University</strong><br />
It has previously been established that bradykinin B2 receptor (BKB2R) knockout(KO) mice are protected from<br />
thrombosis, while apolipoprotein E (APOE) knockout mice are at increased risk for thrombosis. To determine<br />
whether a double knockout will eliminate thrombosis protection seen in the BKB2R, the two strains must be cross-<br />
bred. Since both genes were disrupted by the introduction <strong>of</strong> the neomycin (neo) resistance gene, assaying<br />
progeny for the presence <strong>of</strong> this gene is insufficient to determine whether dual-knockouts have been produced. The<br />
5’ flanking sequence to the neo gene in the BKB2R knockout mice was determined, and 5’ primers suitable for the<br />
polymerase chain reaction (PCR) in this region, as well as 3’ primers in the neo gene sequence, were<br />
identified. Multiple PCRs were carried out using DNA from both BKB2R KO mice and wild type mice, and one primer<br />
pair was found to produce a distinct band unique to BKB2R KO DNA. The fidelity <strong>of</strong> this band as part <strong>of</strong> the BKB2R<br />
was determined by DNA sequence analysis, and found to correspond to the known sequence <strong>of</strong> the mouse genome<br />
at the junction <strong>of</strong> the interrupted BKB2R gene and the 5’ end <strong>of</strong> the neo gene. Together with a similar method that<br />
has been developed for genotyping APOE knockout mice, using a 5’ primer hybridizing within the coding region <strong>of</strong><br />
the neo gene and a 3’ primer hybridizing to the 3’ flanking region <strong>of</strong> the APOE gene, this reaction can be used to<br />
assay for the presence <strong>of</strong> both KO genes in a single animal. These studies produced an incontrovertible screening<br />
method for the double BKB2R and APOE KO mouse. This assay facilitates investigations on the influence <strong>of</strong><br />
competing thrombosis risks.<br />
Supported by NIH T35 Training Grant HL082544<br />
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