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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, Vol. 31, No. 12, 01.12.2003, p. 1206-14.

Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review

Harvard

Li, Z, Schwieger, M, Lange, C, Kraunus, J, Sun, H, van den Akker, E, Modlich, U, Serinsöz, E, Will, E, von Laer, D, Stocking, C, Fehse, B, Schiedlmeier, B & Baum, C 2003, 'Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose', Experimental hematology, vol. 31, no. 12, pp. 1206-14.

APA

Li, Z., Schwieger, M., Lange, C., Kraunus, J., Sun, H., van den Akker, E., Modlich, U., Serinsöz, E., Will, E., von Laer, D., Stocking, C., Fehse, B., Schiedlmeier, B., & Baum, C. (2003). Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose. Experimental hematology, 31(12), 1206-14.

Vancouver

Li Z, Schwieger M, Lange C, Kraunus J, Sun H, van den Akker E et al. Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose. Experimental hematology. 2003 Dec 1;31(12):1206-14.

Bibtex

@article{6de89f93e1e24caabbcb54017546c690,
title = "Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose",
abstract = "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.",
keywords = "Animals, Bone Marrow Cells, Gene Dosage, Gene Transfer Techniques, Genetic Vectors, Hematopoietic Stem Cells, Immunomagnetic Separation, Mice, Mice, Inbred Strains, Retroviridae, Transduction, Genetic, Transgenes",
author = "Zhixiong Li and Maike Schwieger and Claudia Lange and Janine Kraunus and Hanying Sun and {van den Akker}, Eric and Ute Modlich and Ebru Serins{\"o}z and Elke Will and {von Laer}, Dorothee and Carol Stocking and Boris Fehse and Bernd Schiedlmeier and Christopher Baum",
year = "2003",
month = dec,
day = "1",
language = "English",
volume = "31",
pages = "1206--14",
journal = "EXP HEMATOL",
issn = "0301-472X",
publisher = "Elsevier Inc.",
number = "12",

}

RIS

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 -