Benchmark Tracing the Origin of Non - Hematopoietic Cells Using CD 45 PCR Restriction Fragment Length

The possibility of distinguishing the product of different CD45 alleles by immunocytochemistry has made this antigenic system a popular way of tracking the origin of hematopoietic cells in congenic models of transplantation, in which the animals diverge only in their CD45 alleles (15). The extracellular domains of the transmembrane phosphatases encoded by each allele differ in three amino acid residues, which generates distinct epitopes that are recognizable by specific antibodies (3,5,10). Yet, the fact that those antigens are expressed only in cells of hematopoietic origin precludes the use of the same approach to study nonhematopoietic organs. Several recent reports of transdifferentiation of hematopoietic cells into other tissues (1,2,4,6–8) highlight the relevance of having a simple way of assessing the occurrence of this phenomenon, by investigating the presence in various organs of non-hematopoietic cells that carry the donor CD45 allele. We present a method that exploits the existence of differences in restriction sites among CD45 alleles to allow tracking the origin of cells contributing to nonhematopoietic organs. The gene encoding the CD45 antigen [also known as leucocyte common antigen (LCA), protein tyrosine phosphatase receptor-type c polypeptide (Ptprc), or Ly5] maps to chromosome 1 (16) and comprises 34 exons (13,14). Exons 1a and 1b are untranslated and alternatively excluded. Exons 4 (also known as A), 5 (B), and 6 (C) can be alternatively spliced, giving rise to different cell type specific isoforms, which can also be recognized by distinct antibodies (CD45RA, RB, RC, and RO) that react across allelic variants (10–12). Hitherto, three murine CD45 alleles have been documented: CD45.1 (Ly5.1 or Ly5a), CD45.2 (Ly5.2 or Ly5b), and CD45.3 (Ly5.3 or Ly5c). Of note, the nomenclature for alleles 1 and 2 was reversed in 1987 (9), which is still a source of confusion. Most murine strains, such as BALB/c, C57Bl/6, CBA, and NZB/BLN, originally expressed the CD45.2 allele, while CD45.1 was present in SJL mice. However, inbred strains have been backcrossed to carry another CD45 allele (5,17), notably the C57Bl/6 line, commonly used for bone marrow transplant experiments involving CD45.1 and CD45.2 mismatch. In contrast to the extracellular portion, the cytoplasmic domain is conserved in both CD45.1 and CD45.2 proteins, but its coding sequence has two silent nucleotide changes that are responsible for two restriction site differences between alleles (Figure 1). The CD45.1 gene has a unique XhoI site at codon 738 (exon 23), and the CD45.2 gene has a unique KpnI site at codon 825 (exon 25) (17). To be able to generate DNA segments by PCR of sufficient size to be resolved in an agarose gel, we required some knowledge about the intronic sequences. Although all CD45 exons have been sequenced so far, there is no information about CD45 introns in public genomic databases. Therefore, we first sequenced the introns adjacent to the exons containing the relevant restriction sites. The sequences were read at Baylor College of Medicine core sequencing facility and deposited in GenBank® (accession nos. AY090071–AY090075 and AY096794–AY096795). After sequencing the introns of interest, primers were designed to anneal to sequences around the XhoI and the KpnI restriction sites (XhoI FWD: 5′TGTGATAATCCTTTTTGCAGTCTTCTT-3′—in intron 22—and XhoI REV: 5′-GTTTTAGTTCCTCACATTTTACATTCCTTA-3′—intron 23; KpnI FWD-3: 5′-TAGTATGGAGGAGAGCTTTATTGAG-3′—intron 24—and KpnI REV: 5′-TCCACTTGCACCATCAGACACC-3′—exon 25) (Figure 1). PCR was performed on template DNA samples (approximately 10 μg) obtained from mixing different proportions of CD45.1 and CD45.2 bone marrow cells (Figure 1) in a 100-μL solution containing 10 U Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM DRUG DISCOVERY AND GENOMIC TECHNOLOGIES