Preparation of adenovirus-polylysine-DNA complexes.
This unit describes preparation of adenovirus-polylysine-DNA complexes, which is useful for transfection of DNA into a variety of cell types. A DNA complex is prepared with biotinylated adenovirus and streptavidin-polylysine, coupled to transferrin, and used to transfect cells. Several support protocols describe methods for adenovirus growth and purification, biotinylation, inactivation with psoralen, and quantitation of the adenovirus particles. Additional support protocols describes preparation of streptavidin-polylysine and transferrin-polylysine, necessary for the basic procedure. The DNA used for transfection must be free of lipopolysaccharide (LPS), and two methods for removing LPS are described. A more direct polylysine-virus linkage that is simple and requires no exotic reagents can be used for transfection. This protocol requires polylysine-modified adenovirus, prepared as described. An alternate protocol describes transfecting cells with free virus and DNA condensed with a polycation.
Cotten
M
M
Institute for Molecular Pathology, Vienna, Austria.
Baker
A
A
Birnstiel
M L
ML
Zatloukal
K
K
Wagner
E
E
D000256:Adenoviridae / Q000235:genetics* / Q000302:isolation & purification*; D000818:Animals; D002460:Cell Line; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification*; D005821:Genetic Techniques; D005826:Genetics, Medical; D006801:Humans; D046911:Macromolecular Substances / Q000302:isolation & purification; D011107:Polylysine / Q000302:isolation & purification*; D014162:Transfection
D004274:DNA, Recombinant; D046911:Macromolecular Substances; D011107:Polylysine