Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprc^b/CD45.2/Ly5.2) and congenic B6.SJL-Ptprc^aPepc^b/Boy (Ptprc^a/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprc^a allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-Ptprc^aPepc^b/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5′-tail to allow for simultaneous identification of the Ptprc^a and Ptprc^b alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.