Human hematopoietic cell culture, transduction, and analyses.

Current Protocols in Human Genetics
Jesper BondeJ A Nolta

Abstract

This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-term cultures. Support protocols describe methods for maintenance of vector-producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long-term cultures. Other protocols provide PCR-based methods to analyze individual colonies for transduction, methods to analyze cells harvested from long-term cultures, and procedures for freezing and thawing of hematopoietic cells.

References

Nov 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·D M BodineA W Nienhuis
Mar 19, 1996·Proceedings of the National Academy of Sciences of the United States of America·J A NoltaD B Kohn
Mar 19, 1999·Methods : a Companion to Methods in Enzymology·T E ThomasC J Eaves
Oct 30, 2001·Leukemia·M Bhatia
Jul 13, 2002·The Journal of Pathology·Dominique Bonnet
Dec 17, 2003·British Journal of Haematology·Peter LangJohann Greil
Dec 20, 2003·BioTechniques·Todd E MeyerroseJan A Nolta
Apr 30, 2004·Clinica Chimica Acta; International Journal of Clinical Chemistry·Katharina RuzickaMathias M Mueller
Nov 19, 2004·Current Opinion in Hematology·Jesper BondeJan A Nolta

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