scholarly journals Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production and Clinical Application: Improved Safety and High Titer

2000 ◽  
Vol 2 (3) ◽  
pp. 262-275 ◽  
Author(s):  
Philip L. Sheridan ◽  
Mordechai Bodner ◽  
Andrea Lynn ◽  
Trung K. Phuong ◽  
Nicholas J. DePolo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Clinical Application ◽  
Large Scale ◽  
High Titer ◽  
Producer Cell

scholarly journals High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish

2002 ◽  
Vol 76 (5) ◽  
pp. 2192-2198 ◽  
Author(s):  
Wenbiao Chen ◽  
Shawn Burgess ◽  
Greg Golling ◽  
Adam Amsterdam ◽  
Nancy Hopkins
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Insertional Mutagenesis ◽  
Germ Line ◽  
High Titer ◽  
Retroviral Vectors ◽  
Embryonic Cells ◽  
Zebra Fish ◽  
Glycoprotein G ◽  
Producer Cell

ABSTRACT Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate

scholarly journals Packaging Cells Based on Inducible Gene Amplification for the Production of Adeno-Associated Virus Vectors

1998 ◽  
Vol 72 (9) ◽  
pp. 7024-7031 ◽  
Author(s):  
Naoki Inoue ◽  
David W. Russell
Keyword(s):  
Cell Lines ◽  
Gene Amplification ◽  
Large Scale ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Scale Production ◽  
Adeno Associated Virus ◽  
Tetracycline Transactivator ◽  
Producer Cell

ABSTRACT Although vectors based on adeno-associated virus (AAV) offer several unique advantages, their usage has been hampered by the difficulties encountered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replication origin. The packaging and producer cell lines developed express SV40 T antigen under the control of the reverse tetracycline transactivator system, which allows inducible amplification of chromosomal loci linked to the SV40 origin. Culturing these cells in the presence of doxycycline followed by adenovirus infection resulted in helper and vector gene amplification as well as higher vector titers. Clonal producer cell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 104vector particles produced per cell. These stocks were free of detectable replication-competent virus. The lack of a transfection step combined with the reproducibility of stable producer lines makes this packaging method ideally suited for the large-scale production of vector stocks for human gene therapy.

Reliable Generation of Stable High Titer Producer Cell Lines for Gene Therapy

Intervirology ◽  
2007 ◽  
Vol 50 (3) ◽  
pp. 197-203 ◽  
Author(s):  
Ina Rattmann ◽  
Veronika Kleff ◽  
Anja Feldmann ◽  
Carsten Ludwig ◽  
Ursula Regina Sorg ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cell Lines ◽  
High Titer ◽  
Producer Cell

scholarly journals Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5104-5110 ◽  
Author(s):  
Robert E. Throm ◽  
Annastasia A. Ouma ◽  
Sheng Zhou ◽  
Anantharaman Chandrasekaran ◽  
Timothy Lockey ◽  
...  
Keyword(s):  
Cell Lines ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Cell Clone ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Retroviral Vectors ◽  
Scale Production ◽  
Producer Cell

AbstractRetroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

scholarly journals Development of high-titer retroviral producer cell lines by using Cre-mediated recombination.

1997 ◽  
Vol 71 (10) ◽  
pp. 7820-7826 ◽  
Author(s):  
E F Vanin ◽  
L Cerruti ◽  
N Tran ◽  
G Grosveld ◽  
J M Cunningham ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Titer ◽  
Producer Cell
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