Transient transfection of primary cultured hepatocytes using CaPO4/DNA precipitation.
We present a detailed protocol for the transient transfection of non-proliferating primary cultured hepatocytes that is easily reproducible. Using a modification of the classical CaPO4/DNA precipitation method, this protocol is an inexpensive alternative to other methods that are often cumbersome, expensive, difficult to reproduce or harmful to primary hepatocytes. Because only 0.5 x 10(6) cells are needed for a single transfection experiment, several reporter genes can be introduced into hepatocytes of a single liver preparation. With our protocol, different plasmids can be introduced into one cell. In this way, cis-trans interactions can be examined and reporter gene expression can be normalized for transfection efficiency. Furthermore, we describe details of a transfection experiment with two different reporter gene vectors using a luciferase gene and a lacZ gene. The results presented may be helpful to other groups concerned with improved timing of transfection experiments.
Gaunitz
F
F
Universität Tübingen, Physiologisch-chemisches Institut, Germany. gaunitz@uni-tuebingen.de
Papke
M
M
Gebhardt
R
R
D000818:Animals; D002130:Calcium Phosphates / Q000737:chemistry*; D002478:Cells, Cultured; D004247:DNA / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D008099:Liver / Q000737:chemistry / Q000166:cytology*; D008156:Luciferases / Q000032:analysis; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D004247:DNA; C020243:calcium phosphate; D008156:Luciferases; D001616:beta-Galactosidase; C485829:calcium phosphate, dibasic, anhydrous