Human hematopoietic cell culture, transduction, and analyses

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.