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.
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Acknowledgments
This work was supported by Grant No. 173008 from the Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors would like to thank Dr. Vera Ignjatovic for grammar corrections of the manuscript.
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Pruner, I., Djordjevic, V., Gvozdenov, M. et al. Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay. Biochem Genet 52, 159–165 (2014). https://doi.org/10.1007/s10528-013-9636-3
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DOI: https://doi.org/10.1007/s10528-013-9636-3