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 μg of DNA per 106 cells; transfection with 24 μg 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.
Bibliographical noteFunding Information:
This work was supported by grant AI20385 from the NIH. I thank Eric J. Berman (recipient of a March of Dimes Summer Research Scholarship) for preparation of labelled mosquito DNA, Dr A. M. Johnston and M. R. Swerdel for critically reading the manuscript and Eleanor Kells for typing the manuscript. Plasmid hsp-CAT was the generous gift of Dr I. B. Dawid.