Expression of CD34 mRNA and protein in cattle.

CD34 is a transmembrane glycoprotein expressed by hematopoietic progenitors and endothelial cells. It is used widely in the clinic for purification of human hematopoietic stem cells transplants, and as an endothelial marker for several species. The aim of this study was to produce an anti-bovine CD34 antibody and to characterize the expression of CD34 mRNA and protein in cattle tissues. The bovine CD34 cDNA was cloned by RT-PCR, and the expression of bovine CD34 mRNA investigated by RT-PCR and in situ hybridization. Polyclonal antibodies were raised against CD34 polypeptide fragments expressed in Escherichia coli, and affinity purified. Alternative splicing of bovine CD34 mRNA was observed. Both splice variants were readily observed in endothelium, while the variant encoding a truncated cytoplasmic domain was mostly undetectable in bone marrow mononuclear cells. A polyclonal antibody against an extracellular fragment of the CD34 polypeptide was characterized using Western blots, cytocentrifuge preparates, and paraffin sections. CD34 immunoreactivity was enriched in lineage-depleted bone marrow cells. The antibody labelled most blood vessel endothelia in fetal and adult cattle, with highest intensity in capillaries. Newly forming capillaries in granulation tissue were also stained. Lymphatic vessels and the endothelium of liver sinusoids were negative.