To elucidate molecular mechanisms governing functional differences between human CD34+ and CD34- cells, we set up a molecular screen to identify genes that are preferentially expressed in CD34+ cells. We recently reported that in a subtracted cDNA library (cord blood CD34+ vs. CD34- cells), genes expressed preferentially in CD34+ cells were highly enriched (20% and 10% of the cloned gene fragments were from the c-kit and CD34 gene, respectively). Among the 73 initially sequenced gene fragments, 27 (37%) were novel, or homologous only to entries in EST databases (Genomics, 65-283). One of these novel/EST clones (C17) was found 4 times (5.5%). The C17 cDNA is 1 kb long, and encodes a protein of 136 amino acids with a putative signal peptide at its N-terminus. Transient expression in transfected human cells demonstrated that the C17 protein is indeed a secreted molecule. To date we have not found any existing molecule that shows significant homology to the amino acid sequence of the Cl 7 gene. A secondary structure analysis predicts that the C17 peptide contains 4 alpha-helices, a characteristic of hematopoietic cytokines and interleukins. In order to determine functions and regulation of Cl 7 gene, we first examined its expression profile in over 50 human tissues and many types of primary and cultured human cells. C17 expression is largely restricted to tissues associated with hematopoiesis and the blood/ lymph circulation. At cellular level, C17 expression is highly restricted to CD34+ cells. By RT-PCR and Northern blot analyses, C17 gene expression was detected in human CD34+ cells from cord blood, adult bone marrow (ABM) and G-CSF mobilized peripheral blood (mPB), but not in bulk CD34- cells, PBL or marrow stromal cells. C17 gene is also expressed in sorted Thy+CD34+Lin- cells from ABM and mPB. Second, we mapped the location of C17 gene in the human genome. It is uniquely mapped to chromosome 4pl5-16 where the AC 133 and CD38 gene reside. Third, we obtained an upstream 129 kb genomic fragment overlapping with the C17 cDNA sequence. The proximal 1.5 kb fragment flanking sequence was first tested for its ability as a promoter to drive GFP or luciferase reporter expression. We found that the 1.5 kb fragment is functional to direct either transgene expression in transfected human cells. Since lentiviral vectors with self-inactivating (SIN) modification allow transgene expression in stably transduced cells from a non-LTR promoter, we are constructing SIN lentiviral vectors containing the C17 promoter. These vectors may allow specific expression of a transgene (C17 or MDR) in cultured and primary human CD34+ cells.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology