Efficient expression of foreign genes in human CD34+ hematopoietic precursor cells using electroporation
| Autor: | Michael H. Wu, Stephanie F. Williams, D N Liebowitz, M E Dolan, SL Smith |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Reporter gene
Cell Survival Genome Human Electroporation Genetic enhancement Cell Culture Techniques Gene Transfer Techniques CD34 Antigens CD34 Stem cell factor Biology CD38 Hematopoietic Stem Cells Transfection Molecular biology Colony-Forming Units Assay Haematopoiesis Genes Reporter Genetics Humans Molecular Medicine Stem cell Molecular Biology |
| Zdroj: | Gene Therapy. 8:384-390 |
| ISSN: | 1476-5462 0969-7128 |
| Popis: | Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cells from chemotherapeutic damage. Electroporation is a non-chemical, nonviral, highly reproducible means to introduce foreign genes into mammalian cells that has been used primarily for rapidly dividing cells. CD34(+) cells isolated from mobilized peripheral blood of patients were cultured for 48 h in serum-free culture medium supplemented with Flt-3 ligand, stem cell factor and thrombopoietin. Cell cycle analysis showed an increase in % S-phase from 2% on day 0 to 28% on day 2 without significant loss of mean fluorescence intensity (MFI). Optimal electroporation conditions for CD34(+) cells were 550 V/cm, 38 ms, 30 microg DNA/500 microl at cell densities between 0.2 x 10(6) and 10 x 10(6) cells/ml resulting in transient EGFP gene expression in 21% (+/- 1%) of CD34(+) precursor cells, as determined by flow cytometry 48 h after electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjugated monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) and 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The reporter gene was found to be integrated into the LTC-IC genomic DNA as determined by inverse PCR and DNA sequencing. These results suggest that electroporation has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells. |
| Databáze: | OpenAIRE |
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