| Production of replication-defective retrovirus by transient transfection of 293T cells. | |
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MedLine Citation:
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PMID: 18989403 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model. |
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Authors:
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L Cristina Gavrilescu; Richard A Van Etten |
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Publication Detail:
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Type: Video-Audio Media Date: 2007-12-04 |
Journal Detail:
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Title: Journal of visualized experiments : JoVE Volume: - ISSN: 1940-087X ISO Abbreviation: J Vis Exp Publication Date: 2007 |
Date Detail:
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Created Date: 2008-11-07 Completed Date: 2009-02-10 Revised Date: 2010-12-02 |
Medline Journal Info:
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Nlm Unique ID: 101313252 Medline TA: J Vis Exp Country: United States |
Other Details:
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Languages: eng Pagination: 550 Citation Subset: IM |
Affiliation:
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Molecular Oncology Research Institute, Tufts-NEMC, USA. cgavrilescu@tufts-nemc.org |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Adenovirus E1A Proteins
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genetics,
metabolism Animals Bone Marrow Transplantation Cell Line, Transformed Genetic Vectors Humans Mice Oncogenes Retroviridae / genetics*, growth & development* Retroviridae Infections / genetics Transfection / methods* Virus Replication / genetics* |
| Chemical | |
Reg. No./Substance:
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0/Adenovirus E1A Proteins |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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