Document Detail


Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors.
MedLine Citation:
PMID:  16950522     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). Our linear PEI protocol is as effective as commercial reagents in the transfection of HeLa cells and XDC293 cells, a derivative of HEK293 cells, but at a fraction of the cost. Greater than 90% of XDC293 cells and 98% of HeLa cells transfected using our method were positive for EGFP expression as determined by flow cytometery. Our protocol should be useful for many different applications such as large-scale production of recombinant protein and viruses, which requires transient transfection of mammalian cells in large batches. We have used this protocol to produce recombinant adeno-associated virus (AAV) in XDC293 cells and in HeLa cells. This requires transient expression of three adenovirus gene-products (E2A, E4orf6, and VA RNAs) as well as the AAV replication (Rep78, Rep68, Rep52, and Rep40) and capsid (VP1, VP2, and VP3) proteins. Production of a recombinant AAV that expresses green fluorescent protein was assessed by quantitative PCR and by transduction of HeLa cells. Linear PEI is a better transfection reagent than calcium phosphate for the production of recombinant AAV in both HEK293 and HeLa cells. In addition, when both HeLa and XDC293 cells were by our method, HeLa cells in the absence of E1A generated three-fold more recombinant AAV than XDC293 cells, which constitutively express E1A.
Authors:
Sharon E Reed; Elizabeth M Staley; John P Mayginnes; David J Pintel; Gregory E Tullis
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2006-09-06
Journal Detail:
Title:  Journal of virological methods     Volume:  138     ISSN:  0166-0934     ISO Abbreviation:  J. Virol. Methods     Publication Date:  2006 Dec 
Date Detail:
Created Date:  2006-10-30     Completed Date:  2007-02-05     Revised Date:  2007-12-03    
Medline Journal Info:
Nlm Unique ID:  8005839     Medline TA:  J Virol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  85-98     Citation Subset:  IM    
Affiliation:
Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, United States.
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MeSH Terms
Descriptor/Qualifier:
Cell Line
DNA, Viral / analysis
Dependovirus / genetics*
Flow Cytometry
Genetic Vectors*
Green Fluorescent Proteins / biosynthesis,  genetics
Humans
Polyethyleneimine*
Polymerase Chain Reaction
Recombination, Genetic*
Transfection / methods*
Grant Support
ID/Acronym/Agency:
NS444494/NS/NINDS NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Viral; 0/enhanced green fluorescent protein; 147336-22-9/Green Fluorescent Proteins; 9002-98-6/Polyethyleneimine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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