- Tytuł:
- Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood
- Autorzy:
-
Ołdak, Tomasz
Kruszewski, Marcin
Machaj, Eugeniusz
Gajkowska, Agnieszka
Pojda, Zygmunt - Powiązania:
- https://bibliotekanauki.pl/articles/1043723.pdf
- Data publikacji:
- 2002
- Wydawca:
- Polskie Towarzystwo Biochemiczne
- Tematy:
-
CD34+ cells
umbilical cord blood
green fluorescent protein
transfection - Opis:
- 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 immunomagnetic 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 μF, 550 V/cm, and 10 μg of DNA per 500 μl. 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.
- Źródło:
-
Acta Biochimica Polonica; 2002, 49, 3; 625-632
0001-527X - Pojawia się w:
- Acta Biochimica Polonica
- Dostawca treści:
- Biblioteka Nauki
Artykuł