Reed, S. E., Staley, E. M., Mayginnes, J. P., Pintel, D. J. & Tullis, G. E. Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J. Virol. Methods 138, 85-98

Department of Ophthalmology, Boston University, Boston, Massachusetts, United States
Journal of Virological Methods (Impact Factor: 1.78). 01/2007; 138(1-2):85-98. DOI: 10.1016/j.jviromet.2006.07.024
Source: PubMed


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.

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    • "THP-1 macrophages were differentiated with 200 nM PMA for 24 h. Transfection was performed using Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA) according to manufacturer's protocol or PEI MAX (Polysciences, Warrington, PA, USA) as described previously [21] "
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    • "The Daudi cells were harvested by centrifugation and washed once. Transfections of HeLa cells with either OAS1 cDNAs or OAS2 p69 cDNA under the control of the CMV promotor and control plasmids were performed with either TurboFect Transfection Reagent (Fermentas) following the manufactures protocol or using the polyethylenimine (PEI) method [40] for the immunofluoresence cytochemistry studies. In brief, linear PEI (25,000 Da) was used and complexes of DNA:PEI at ratios 1:8 (w/w) were allowed to form and added to the cells. "
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    • "The helper plasmids used (pDP1, pDP2) [54] generated combined serotype of 1 and 2, which efficiently transduces neurons, and can be purified on a heparin column [55]. Briefly, HEK293T/17 cells (ATCC) were plated in T225 flasks and transfected by linear polyethylenimine (PEI) [56], [57] with equal molar amount of either responder GADD34 cont, GADD34 CA or activator, and pDP1 and pDP2. The cells were collected 72 hours after transfection by scraping. "
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