Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose
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Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose. / Li, Zhixiong; Schwieger, Maike; Lange, Claudia; Kraunus, Janine; Sun, Hanying; van den Akker, Eric; Modlich, Ute; Serinsöz, Ebru; Will, Elke; von Laer, Dorothee; Stocking, Carol; Fehse, Boris; Schiedlmeier, Bernd; Baum, Christopher.
in: Experimental hematology, Jahrgang 31, Nr. 12, 01.12.2003, S. 1206-14.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose
AU - Li, Zhixiong
AU - Schwieger, Maike
AU - Lange, Claudia
AU - Kraunus, Janine
AU - Sun, Hanying
AU - van den Akker, Eric
AU - Modlich, Ute
AU - Serinsöz, Ebru
AU - Will, Elke
AU - von Laer, Dorothee
AU - Stocking, Carol
AU - Fehse, Boris
AU - Schiedlmeier, Bernd
AU - Baum, Christopher
PY - 2003/12/1
Y1 - 2003/12/1
N2 - 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.
AB - 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.
KW - Animals
KW - Bone Marrow Cells
KW - Gene Dosage
KW - Gene Transfer Techniques
KW - Genetic Vectors
KW - Hematopoietic Stem Cells
KW - Immunomagnetic Separation
KW - Mice
KW - Mice, Inbred Strains
KW - Retroviridae
KW - Transduction, Genetic
KW - Transgenes
M3 - SCORING: Journal article
C2 - 14662326
VL - 31
SP - 1206
EP - 1214
JO - EXP HEMATOL
JF - EXP HEMATOL
SN - 0301-472X
IS - 12
ER -