Factors affecting polybrene-mediated transfection of cultured Aedes albopictus (mosquito) cells.
The polycation 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) is superior to calcium phosphate for the introduction of purified DNA into cultured Aedes albopictus (mosquito) cells. Adsorption of the polybrene-DNA complex to mosquito cells was essentially linear for 6 h. However, the rate of adsorption of DNA increased when the DNA-polybrene mixture was preincubated for several hours prior to addition to cells. A recombinant plasmid carrying an inducible chloramphenicol acetyltransferase gene under the control of a Drosophila heat shock protein (hsp) promoter was used to show that expression of transfected DNA was highest when cells were treated with a freshly prepared polybrene-DNA mixture. Optimal expression was observed in cells transfected with 4-13 micrograms of DNA per 10(6) cells; transfection with 24 micrograms of DNA resulted in reduced CAT expression. Variation in the polybrene-DNA ratio improved transfection with high levels of DNA. In mosquito cells, CAT expression was independent of DNA methylation.
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D000123:Acetyltransferases / Q000235:genetics; D000327:Adsorption; D000330:Aedes / Q000235:genetics*; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase; D002999:Clone Cells; D004247:DNA / Q000378:metabolism; D004274:DNA, Recombinant; D006360:Heat-Shock Proteins / Q000235:genetics; D006583:Hexadimethrine Bromide / Q000494:pharmacology*; D008745:Methylation; D011073:Polyamines / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D006360:Heat-Shock Proteins; D011073:Polyamines; D006583:Hexadimethrine Bromide; D004247:DNA; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase