Optimization of calcium phosphate transfection for bovine chromaffin cells: relationship to calcium phosphate precipitate formation.
Optimal conditions for formation of calcium phosphate-DNA precipitates and for chromaffin cell transfection by the calcium phosphate method were examined. A relationship was observed between turbidity of calcium phosphate solutions and the ability of calcium phosphate-DNA mixtures to give efficient transfection of bovine chromaffin cells. Under optimal conditions up to 35% of chromaffin cells in cultures transfected with plasmid DNA encoding human proenkephalin or Escherichia coli beta-galactosidase expressed the respective proteins. Important factors for transfection were the pH (6.95) and buffer employed for calcium phosphate-DNA precipitate formation, the amount and type of DNA, and the absence of serum in the cultures. Additionally, phosphate and calcium concentrations in the culture medium during incubation of cells with DNA are critical. Optimal conditions for transfection of chromaffin cells were also useful for transfection of clonal BSC-40 cells, an African green monkey kidney cell line. These results suggest that the optimal conditions described here for chromaffin cells may have broad applicability to other cell types. In addition, the results suggest that it is possible to optimize the solutions used for transfection conditions by monitoring calcium phosphate formation.
Wilson
S P
SP
Department of Pharmacology, University of South Carolina School of Medicine, Columbia 29208, USA.
Liu
F
F
Wilson
R E
RE
Housley
P R
PR
D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D002122:Calcium Chloride; D002130:Calcium Phosphates / Q000737:chemistry*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D011232:Chemical Precipitation; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D010710:Phosphates; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000235:genetics / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D002021:Buffers; D002130:Calcium Phosphates; D008081:Liposomes; D010710:Phosphates; D004247:DNA; D001616:beta-Galactosidase; D002122:Calcium Chloride; D005990:Glycerol; C018279:sodium phosphate