Calcium Phosphate Transfection of 3T3 Fibroblasts

2010 ◽  
Vol 2010 (2) ◽  
pp. pdb.prot5372-pdb.prot5372 ◽  
Author(s):  
S. T. Smale
Keyword(s):  
Calcium Phosphate ◽  
3T3 Fibroblasts ◽  
Calcium Phosphate Transfection

scholarly journals The uptake and stability of simian-virus-40 DNA after calcium phosphate transfection of CV-1 cells

1984 ◽  
Vol 218 (2) ◽  
pp. 475-482 ◽  
Author(s):  
A J Strain ◽  
A H Wyllie
Keyword(s):  
Calcium Phosphate ◽  
Transfection Efficiency ◽  
Plaque Assay ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Fraction ◽  
Optimum Conditions ◽  
Viral Plaque ◽  
Sv40 Dna ◽  
Calcium Phosphate Transfection

The uptake and fate of purified SV40 (Simian virus 40) DNA, transfected into permissive CV-1 cells by calcium phosphate precipitates, was examined. By using a viral plaque assay, optimum conditions for transfection were established and transfection efficiencies of up to 10(6) plaque-forming units/micrograms of SV40 DNA were obtained. After a 2h exposure to 3H-labelled SV40 DNA-calcium phosphate co-precipitates under basal conditions, up to 7% of the input DNA became cell-associated, with approx. 4% reaching the nuclear fraction. This value was never exceeded, even under conditions known to enhance significantly the ultimate transfection efficiency, such as increased exposure time, addition of carrier DNA or treatment with DMSO (dimethyl sulphoxide) or glycerol. Substantial degradation of this SV40 DNA occurred within a further 4h, apparently in both nucleus and cytoplasm. Degradation of form-II and form-III SV40 DNA, which have lower transfection efficiencies than form-I DNA, was no more rapid than degradation of form-I DNA. The results indicate that less than 0.5% of the transfected DNA which reached the nucleus is protected from nuclease attack. The mechanism of action of agents such as glycerol, DMSO or carrier DNA remains obscure, but they may be involved in conferring greater stability to the intracellular SV40 DNA rather than merely affecting its rate of entry into the cell.

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