title: Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR.
abstract: We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10(4) cells are required for the analysis.
authors.affiliation: Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: P
authors.initials: P
authors.lastname: Fu
authors.forename: P
authors.initials: P
authors.lastname: Senior
authors.forename: R T
authors.initials: RT
authors.lastname: Fernley
authors.forename: G W
authors.initials: GW
authors.lastname: Tregear
authors.forename: G P
authors.initials: GP
authors.lastname: Aldred
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018628:Gene Dosage; D051379:Mice; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis / Q000235:genetics; D044162:Parathyroid Hormone-Related Protein; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D051381:Rats; D014162:Transfection
chemical_list: D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D004247:DNA