Integrin alpha IIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells

摘要

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alpha llb beta 3, is caused by the presence of regulatory elements of the alpha llb promoter that direct high-Ievel, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted ex- pression of transgenes, we transduced human CD34+ periph- eral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alpha llb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the PI~a2 alloantigen form (Pross) of the integrin beta 3 subunit was subcloned into this construct (-889PI~a2 beta 3) and transduced into cells that endogenously synthesized beta 3 (Leu33) as a marker for detection of provirus-derived beta 3. The ability of this vector to target expression of PI~a2 beta 3 to megakaryocytes was first examined in cell lines. Immunoblot analysis wilh human anti-PI~a2 alloserum detected synthesis of PI~a2 beta 3 in trans- duced promegakaryocytic cells) however, PI~a2beta3 protein was not detected in transduced epithelial cells. Human hemato. poietic CD34 + cells were transduced with - 889PI~a2beta33 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphallbbeta3 complex was formed in progeny megakaryocytes where provirus-derived PIa2beta3 was detected associated with endogenous alpha llb subunit. Another alphallb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakar