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Titolo:
Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation
Autore:
Wu, MH; Liebowitz, DN; Smith, SL; Williams, SF; Dolan, ME;
Indirizzi:
Univ Chicago, Hematol Oncol Sect, Chicago, IL 60637 USA Univ Chicago Chicago IL USA 60637 matol Oncol Sect, Chicago, IL 60637 USA Univ Chicago, Dept Med, Ctr Canc Res, Chicago, IL 60637 USA Univ Chicago Chicago IL USA 60637 ed, Ctr Canc Res, Chicago, IL 60637 USA
Titolo Testata:
GENE THERAPY
fascicolo: 5, volume: 8, anno: 2001,
pagine: 384 - 390
SICI:
0969-7128(200103)8:5<384:EEOFGI>2.0.ZU;2-5
Fonte:
ISI
Lingua:
ENG
Soggetto:
EX-VIVO EXPANSION; GREEN FLUORESCENT PROTEIN; MEDIATED DNA TRANSFER; PROGENITOR CELLS; STEM-CELLS; LYMPHOID-CELLS; FLT3 LIGAND; TRANSDUCTION; CULTURE; GROWTH;
Keywords:
CD34; electroporation; gene delivery; EGFP; integration;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
29
Recensione:
Indirizzi per estratti:
Indirizzo: Dolan, ME Univ Chicago, Hematol Oncol Sect, 5841 S Maryland Ave,Box MC2115, Chicago,IL 60637 USA Univ Chicago 5841 S Maryland Ave,Box MC2115 Chicago IL USA 60637
Citazione:
M.H. Wu et al., "Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation", GENE THER, 8(5), 2001, pp. 384-390

Abstract

Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cellsfrom 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 fluorescenceintensity (MFI). Optimal electroporation conditions for CD34(+) cells were550 V/cm, 38 ms, 30 mug DNA/500 mul 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 genesinto human hematopoietic precursor cells.

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Documento generato il 01/04/20 alle ore 09:04:26