Design of a novel bicistronic expression vector with demonstration of a p16INK4-induced G(1)-S block(1).
Traditional eukaryotic gene expression systems have many shortcomings that make them unsuitable for the analysis of cytotoxic and growth-arrest genes. An intestinal alkaline phosphatase bicistronic expression vector was specifically designed to facilitate such studies. Intestinal alkaline phosphatase serves as a marker of cells that have been transfected and, therefore, must also be co-expressing the gene under study. Using flow cytometry, a trivariate analysis was performed on p16INK4-transfected U87 glioblastoma cells. An average G1-S block of 77.5% was demonstrated compared to controls despite a 1% transfection efficiency. This vector has universal applications, including: (a) analysis of cytotoxic and growth-inhibitory genes in transient assays; (b) 100% enrichement in gene expression studies, especially in low transfection efficiency experiments; and (c) facilitation of the study of cell cycle kinetics.
Poulos
N E
NE
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717, USA.
Farmer
A A
AA
Chan
K W
KW
Stanbridge
E J
EJ
D000469:Alkaline Phosphatase / Q000096:biosynthesis*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002453:Cell Cycle; D002460:Cell Line; D019941:Cyclin-Dependent Kinase Inhibitor p16; D003587:Cytomegalovirus; D018274:Electroporation; D004742:Enhancer Elements, Genetic; D004791:Enzyme Inhibitors / Q000378:metabolism*; D016193:G1 Phase; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D005909:Glioblastoma; D006801:Humans; D007700:Kinetics; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D016196:S Phase; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase