Research ExplorerPublicationsSimplified generation of high-titer retrovirus producer cell...

Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene

Standard

Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene. / Loew, Rainer; Selevsek, Nathalie; Fehse, Boris; von Laer, Dorothee; Baum, Christopher; Fauser, Axel; Kuehlcke, Klaus.

In: MOL THER, Vol. 9, No. 5, 05.2004, p. 738-46.

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

Harvard

Loew, R, Selevsek, N, Fehse, B, von Laer, D, Baum, C, Fauser, A & Kuehlcke, K 2004, 'Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene', MOL THER, vol. 9, no. 5, pp. 738-46. https://doi.org/10.1016/j.ymthe.2004.02.010

APA

Loew, R., Selevsek, N., Fehse, B., von Laer, D., Baum, C., Fauser, A., & Kuehlcke, K. (2004). Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene. MOL THER, 9(5), 738-46. https://doi.org/10.1016/j.ymthe.2004.02.010

Vancouver

Loew R, Selevsek N, Fehse B, von Laer D, Baum C, Fauser A et al. Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene. MOL THER. 2004 May;9(5):738-46. https://doi.org/10.1016/j.ymthe.2004.02.010

Bibtex

@article{351faf50207845429911c62cf6c5fbcc,
title = "Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene",
abstract = "Retroviral producer cells are generated by the introduction of a viral genome into {"}helper{"} cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.",
keywords = "Animals, Antigens, CD/genetics, Antigens, CD34/genetics, Cell Culture Techniques, Gene Expression, Gene Rearrangement/genetics, Genetic Markers, Genetic Vectors/genetics, Humans, Integrases/genetics, Membrane Cofactor Protein, Membrane Glycoproteins/genetics, Mice, Proviruses/genetics, Recombination, Genetic/genetics, Retroviridae/genetics, Terminal Repeat Sequences/genetics, Viral Proteins/genetics",
author = "Rainer Loew and Nathalie Selevsek and Boris Fehse and {von Laer}, Dorothee and Christopher Baum and Axel Fauser and Klaus Kuehlcke",
year = "2004",
month = may,
doi = "10.1016/j.ymthe.2004.02.010",
language = "English",
volume = "9",
pages = "738--46",
journal = "MOL THER",
issn = "1525-0016",
publisher = "NATURE PUBLISHING GROUP",
number = "5",

}

RIS

TY - JOUR

T1 - Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene

AU - Loew, Rainer

AU - Selevsek, Nathalie

AU - Fehse, Boris

AU - von Laer, Dorothee

AU - Baum, Christopher

AU - Fauser, Axel

AU - Kuehlcke, Klaus

PY - 2004/5

Y1 - 2004/5

N2 - Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.

AB - Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.

KW - Animals

KW - Antigens, CD/genetics

KW - Antigens, CD34/genetics

KW - Cell Culture Techniques

KW - Gene Expression

KW - Gene Rearrangement/genetics

KW - Genetic Markers

KW - Genetic Vectors/genetics

KW - Humans

KW - Integrases/genetics

KW - Membrane Cofactor Protein

KW - Membrane Glycoproteins/genetics

KW - Mice

KW - Proviruses/genetics

KW - Recombination, Genetic/genetics

KW - Retroviridae/genetics

KW - Terminal Repeat Sequences/genetics

KW - Viral Proteins/genetics

U2 - 10.1016/j.ymthe.2004.02.010

DO - 10.1016/j.ymthe.2004.02.010

M3 - SCORING: Journal article

C2 - 15120335

VL - 9

SP - 738

EP - 746

JO - MOL THER

JF - MOL THER

SN - 1525-0016

IS - 5

ER -