Optimisation of transfection conditions of CD34plus hematopoietic cells derived from human umbilical cord blood

• Autorzy: Oldak T. , Kruszewski M. , Machaj E.K. , Gajkowska A. , Pojda Z.
• Języki publikacji: EN

Abstrakty

EN

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34+ hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34+ hematopoietic cells were separated on immuno- magnetic MiniMACS columns. Pure population of CD34+ cells was incubated in a se­rum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of vari­ous pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 iF, 550 V/cm, and 10 ug of DNA per 500 ul. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capaci­tance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34+ hematopoietic cells derived from human umbilical cord blood.

Słowa kluczowe

EN

gene therapy; green fluorescent protein; cord blood; man; transfection condition; transfection; umbilical cord blood; hematopoietic cell

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2002
• Tom: 49
• Numer: 3
• Opis fizyczny: p.625-632,fig.

Twórcy

autor

Oldak T.

• Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, W.C.Roentgena 5, 02-781 Warsaw, Poland

autor

Kruszewski M.

autor

Machaj E.K.

autor

Gajkowska A.

autor

Pojda Z.

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