Details

Autor(en) / Beteiligte

• Ryan, Claire E.
• Salvetti, Thomas R.
• Baum, Ilana R.
• Figueroa, Brandon A.
• LeBere, Brittany E.
• Alberti, Michael O.

Titel

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

Ist Teil von

• Molecular and cellular probes, 2024-06, Vol.75, p.101962-101962, Article 101962

Ort / Verlag

England: Elsevier Ltd

Erscheinungsjahr

2024

Quelle

Elsevier ScienceDirect Journals

Beschreibungen/Notizen

• 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 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca 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-PtprcaPepcb/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 Ptprca and Ptprcb 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. •JAXBoy mice differ from C57BL/6J mice by two adjacent SNPs in the Ptprc gene.•Current methods employ probe-based real-time PCR to genotype Ptprc alleles.•We developed a single-tube PCR assay to discriminate Ptprc alleles on agarose gels.•Adding a long oligo 5′-tail to one of the Ptprc amplicons allows for gel separation.•This method can be adapted to discriminate other nucleotide polymorphisms.