Adenovirus-mediated transfection of cultured cells.
We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles. The procedure allows effective delivery of DNA into the cytoplasm and, therefore, results in a higher fraction of cells expressing exogenous proteins. Using this method, we routinely obtain 60%-90% of COS cells or Chinese hamster ovary cells expressing beta-galactosidase, as determined by in situ staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). We have also obtained much improved levels of expression in cells that are difficult or impossible to use in transient expression assays, such as rat-1 fibroblasts or primary osteoblast cultures. We successfully used the method to express heteromeric proteins that require subunit assembly for proper function. The method also proved effective to express functions in which the exogenous protein needs to couple to the endogenous cellular machinery. Thus, this transient transfection method should prove valuable for many functional studies in a broad variety of cell lines and primary cultures.
Forsayeth
J R
JR
University of California at San Francisco.
Garcia
P D
PD
D000109:Acetylcholine / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005697:Galactosides / Q000032:analysis; D007211:Indoles / Q000032:analysis; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics; D014162:Transfection / Q000379:methods*
D005697:Galactosides; D007211:Indoles; D011978:Receptors, Nicotinic; D000109:Acetylcholine; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside