OBJECTIVE: Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods. MATERIALS AND METHODS: HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice. RESULTS: After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7%+/-21.7%, n=23) and 30 to 95% (69.8%+/-20.4%, n=17), respectively. Even after one round of infection, gene transfer was efficient (31.2%+/-15.1%, n=12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells. CONCLUSION: The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.
OBJECTIVE: Current protocols of retroviral gene transfer into murine hematopoietic stem cells (HSC) result in variable gene transfer efficiency and involve various procedures that are not clinically applicable. We developed and evaluated a reliable transduction protocol that is more related to clinical methods. MATERIALS AND METHODS: HSC were enriched from steady-state bone marrow by magnetic cell sorting (lineage depletion) and cultured in defined serum-free medium containing an improved growth factor cocktail (Flt3-ligand, stem cell factor, interleukin-3, interleukin-11). Cell-free ecotropic retroviral vector particles, generated by transient transfection of human 293T-based packaging cells, were preloaded at defined titers on CH296-coated tissue culture plates, thus largely avoiding serum contamination. These conditions were evaluated in 17 experiments involving 29 transduction cultures and 185 recipient mice. RESULTS: After two rounds of infection, the gene marking rates in cultured mononuclear cells and stem/progenitor cells (Lin(-)c-Kit(+)) were 15 to 85% (53.7%+/-21.7%, n=23) and 30 to 95% (69.8%+/-20.4%, n=17), respectively. Even after one round of infection, gene transfer was efficient (31.2%+/-15.1%, n=12). Using identical conditions, gene transfer rates were highly reproducible. Average transgene expression in reconstituted animals correlated well with pretransplant data. Using a moderate multiplicity of infection, the majority of transduced cells carried less than three transgene copies. In addition, coinfection was possible to establish two different vectors in single cells. CONCLUSION: The protocol described here achieves efficient retroviral transduction of murine bone marrow repopulating cells with a defined gene dosage, largely avoiding procedures that decrease stem cell output and repopulating capacity. This protocol may help to improve the predictive value of preclinical efficiency/toxicity studies for gene therapeutic interventions and basic research.
Authors: Samantha L Ginn; Sophia H Y Liao; Allison P Dane; Min Hu; Jessica Hyman; John W Finnie; Maolin Zheng; Marina Cavazzana-Calvo; Stephen I Alexander; Adrian J Thrasher; Ian E Alexander Journal: Mol Ther Date: 2010-03-30 Impact factor: 11.454
Authors: Daniel C Wicke; Johann Meyer; Guntram Buesche; Dirk Heckl; Hans Kreipe; Zhixiong Li; Karl H Welte; Matthias Ballmaier; Christopher Baum; Ute Modlich Journal: Mol Ther Date: 2009-10-20 Impact factor: 11.454
Authors: Maria Carolina Florian; Kalpana J Nattamai; Karin Dörr; Gina Marka; Bettina Uberle; Virag Vas; Christina Eckl; Immanuel Andrä; Matthias Schiemann; Robert A J Oostendorp; Karin Scharffetter-Kochanek; Hans Armin Kestler; Yi Zheng; Hartmut Geiger Journal: Nature Date: 2013-10-20 Impact factor: 49.962
Authors: Brandon K Wyss; Justin L Meyers; Anthony L Sinn; Shanbao Cai; Karen E Pollok; W Scott Goebel Journal: Exp Hematol Date: 2008-02-04 Impact factor: 3.084
Authors: Brandon K Wyss; Abigail F W Donnelly; Dan Zhou; Anthony L Sinn; Karen E Pollok; W Scott Goebel Journal: Exp Hematol Date: 2009-07 Impact factor: 3.084