CANINE CD34 - CLONING OF THE CDNA AND EVALUATION OF AN ANTISERUM TO RECOMBINANT PROTEIN

Increasingly, enriched populations of hematopoietic progenitors are us ed in experimental and clinical transplantation studies. The separatio n of progenitors is based an the expression of CD34, a marker preferen tially expressed on progenitor cells. The dog model has been important for preclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To iden tify and isolate canine hematopoietic progenitors, we have cloned a cD NA encoding a CD34 homologue from a canine myelomonocytic leukemia cel l line, ML2. The CD34 homologue cDNA predicts an amino acid sequence t hat is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C-terminal end of the extracellular domain, but shows considerable divergence from these sequences at the amino-terminal end of the protein. In Western blotting studies, canine CD34 homologue (caCD34) appears to be a heavily and variably glycosyl ated protein with a molecular weight of approximately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the exp ression of caCD34 protein, the extracellular domain of caCD34 was expr essed as an lg fusion protein and used as an immunogen to generate a r abbit polyclonal antiserum. The antiserum reacted against the fusion p rotein, against vascular endothelium, and with three leukemic cell lin es, Approximately 1% of canine bone marrow cells stained brightly with antibodies to caCD34 and this population was 25- to 50-fold enriched for colony-forming units-granulocyte-macrophage as compared to unfract ionated marrow mononuclear cells. These findings suggest that the cani ne CD34 homologue is expressed on bone marrow progenitor cells and, th us, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoi esis and stem cell transplantation. (C) 1996 by The American Society o f Hematology.