Genetically engineered fluorescent cell marker for labeling CD34+ hematopoietic stem cells

Abstract

To address the challenge of labeling and tracking stem cells in vivo, we have engineered a fluorescent cell marker CD34EGFP by utilizing the mechanism of the cell-specific activity of CD34 promoter in CD34(+) stem cells. A retroviral vector derived from a murine stem cell virus was constructed to integrate the CD34EGFP gene into the genome of the cells for labeling. Our experiment demonstrates that the 454 bp segment upstream of the murine CD34 cDNA sequence has full function of promoter activity and can serve as a cell-specific promoter for driving the expression of EGFP in CD34(+) hematopoietic stem cells (HSC), providing a living color for labeling stem cells. The CD34EGFP marker was tested in various types of cells, including terminally differentiated cells, CD34(+) mouse myeloid leukemia progenitor cells, CD34(-) hematopoietic cells, and CD34(+) HSCs. We show that the engineered CD34EGFP cell marker is expressed in the CD34(+) stem or progenitor cells but not in CD34(-) or terminally differentiated cells. RT-PCR assay indicates that the transcription level of the CD34EGFP gene from CD34 promoter is almost the same as that from CMV promoter in CD34(+) progenitor cells. The approach we present here offers a framework for genetic engineering of fluorescent cell markers for labeling and tracking stem cells in vivo. We anticipate that a variety of cell markers could be generated by coupling variants of fluorescent proteins with various cell-specific promoters.