A safe packaging line for gene transfer: separating viral genes on two different plasmids.
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TL;DR: Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus and results suggest that gag, pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer.
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Abstract: A retrovirus packaging cell line was constructed by using portions of the Moloney murine leukemia virus in which the gag, pol, and env genes of the helper virus were separated onto two different plasmids and in which the psi packaging signal and 3' long terminal repeat were removed. The plasmid containing the gag and pol genes and the plasmid containing the env gene were cotransfected into NIH 3T3 cells. Clones that produced high levels of reverse transcriptase and env protein were tested for their ability to package the replication-defective retrovirus vectors delta neo and N2. One of the gag-pol and env clones (GP+E-86) was able to transfer G418 resistance to recipient cells at a titer of as high as 1.7 X 10(5) when it was used to package delta neo and as high as 4 X 10(6) when it was used to package N2. Supernatants of clones transfected with the intact parent gag-pol-env plasmid 3P0 had comparable titers (as high as 6.5 X 10(4) with delta neo; as high as 1.7 X 10(5) with N2). Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus. These results suggest that gag, pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer.
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Citations
DEVELOPMENT OF VIRAL VECTORS FOR HUMAN GENE THERAPY: RETROVIRUS AND ADENOVIRUS (pART I)
Bruce C. Trapnell,Michael N. Pensiero +1 more
- 01 Jan 1997
TL;DR: It is remarkable to note that the gene-enzyme basis of disease was first hypothesized by Garrod only 86 years ago, and more than 600 individuals have received transferred genes.
2
Retroviral mediated gene transfer in megakaryocytic cell lines.
TL;DR: The results demonstrate the feasibility and provide a method for infecting cultured megakaryocytic cell lines with retroviral of vectors such that a molecular analysis ofmegakaryocyte differentiation can be accomplished.
2
Progress Towards the Clinical Application of Somatic Cell Gene Therapy
C. Bordignon
- 01 Jan 1993
TL;DR: Successful gene transfer has been reported in vitro and in vivo in the mouse, and with lower efficiency in hematopoietic progenitors derived from dogs and primates, including humans.
2
The Use of Retroviral Vectors in Human Disorders
M. Scarpa,C. T. Caskey +1 more
- 01 Jan 1989
TL;DR: With the application of recombinant DNA technology to medical practice, geneticists have gained powerful tools for the diagnosis and therapy of genetic disease at the molecular level.
2
Construção e caracterização in vitro de um vetor retroviral bicistrônico codificando endostatina e interleucina-2 para utilização em terapia gênica
Fernanda Bernardes Calvo
- 09 Dec 2009
TL;DR: The objective of this project was to construct and characterize “in vitro” an IRES-based bicistronic retroviral vector encoding endostatin and intereukin-2, which provides a powerful tool for studies of cell biology of cancer and new therapeutic strategies.
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