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Design of a Novel Bicistronic Expression Vector with Demonstration of a p16^INK4-induced G₁-S Block¹

Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92717

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 p16^INK4-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% enrichment in gene expression studies, especially in low transfection efficiency experiments; and (c) facilitation of the study of cell cycle kinetics.

¹ This work was supported by the American Brain Tumor Association/Cele Konigsberg Fellowship (to N. E. P.), Optical Biology Shared Resource of the Cancer Center Support Grant CA-62203, and National Cancer Institute Grant CA 19401.

² Present address: Department of Neurosurgery, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195.

³ Present address: Center For Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Drive, San Antonio, TX 78245.

⁴ To whom requests for reprints should be addressed. Phone: (714) 824-7042; Fax: (714) 824-8598.

Received 1/ 4/96. Accepted 3/ 1/96.

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