Efficient transfection and expression of heterologous genes in PC12 cells.
The PC12 pheochromocytoma cell line has been a favorite model system for cell and neurobiologists, but has proven relatively refractory to standard DNA transfection methods. We have found that the cationic lipid "lipofectin" provides a simple, gentle, and nontoxic procedure that vastly improves transfection efficiencies in PC12 cells. Transient expression of chloramphenicol acetyl transferase (CAT) driven by a Rous sarcoma virus long terminal repeat (LTR) is much more efficient using lipofectin when compared with calcium phosphate as a transfection procedure. Additionally, transient transfection of nerve growth factor (NGF)-differentiated PC12 cells proceeds with equal efficiency relative to naive, uninduced cells. Using the lipofectin procedure, the frequency of stable transfection is 100-fold higher than that reported with standard calcium phosphate precipitation protocols. To examine the effectiveness of different promoters for efficient expression of heterologous DNA in PC12 cells, three different promoter-bearing constructs were utilized. Each construct contains a different promoter sequence upstream from a chicken calsequestrin cDNA. A human cytomegalovirus (CMV) immediate early promoter construct produced the highest level of expression, followed by a human beta-actin promoter construct. Expression from a mouse Moloney sarcoma virus LTR construct could not be detected. These results overcome the previous transfection problems of low efficiency and low viability that have plagued many PC12 cell investigations.
Department of Biological Science, University of California, Santa Barbara 93106.
S R
SR
Muller
P D
PD
Sullivan
D O
DO
Clegg
S C
SC
Feinstein
D000818:Animals; D002130:Calcium Phosphates; D002454:Cell Differentiation / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase; D004247:DNA; D005821:Genetic Techniques; D005822:Genetic Vectors; D007700:Kinetics; D008081:Liposomes; D009414:Nerve Growth Factors / Q000502:physiology; D010673:Pheochromocytoma / Q000235:genetics; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D002130:Calcium Phosphates; D008081:Liposomes; D009414:Nerve Growth Factors; D000644:Quaternary Ammonium Compounds; C055197:calcium phosphate-DNA precipitate; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase