title: Determination of transgene copy number in stably transfected mammalian cells by PCR-capillary electrophoresis assay.
abstract: The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.
authors.affiliation: Laboratory for Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P.O. Box 23, 11010, Belgrade, Serbia, iva.pruner@gmail.com.
authors.forename: Iva
authors.initials: I
authors.lastname: Pruner
authors.forename: Valentina
authors.initials: V
authors.lastname: Djordjevic
authors.forename: Maja
authors.initials: M
authors.lastname: Gvozdenov
authors.forename: Branko
authors.initials: B
authors.lastname: Tomic
authors.forename: Dragica
authors.initials: D
authors.lastname: Radojkovic
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019075:Electrophoresis, Capillary; D018628:Gene Dosage; D006801:Humans; D016133:Polymerase Chain Reaction; D011516:Prothrombin / Q000235:genetics; D014162:Transfection; D019076:Transgenes
chemical_list: D011516:Prothrombin