Determination of Transgene Copy Number and Expression Level Using Denaturing Gradient Gel Electrophoresis

• Published in: BioTechniques Vol. 24; no. 1; pp. 126 - 131
• Main Authors: Ringel, M.D, Schwindinger, W.F, Saji, M, Zeiger, M.A, Levine, M.A
• Format: Journal Article
• Language: English
• Published: Natick, MA Future Science Ltd 01.01.1998; Eaton; Taylor & Francis Group
• Subjects: Animals; Biological and medical sciences; Biotechnology; Blotting, Southern; Diverse techniques; Electrophoresis; Fundamental and applied biological sciences. Psychology; Gene Dosage; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Mice; Mice, Transgenic; Miscellaneous; Molecular and cellular biology; Polymerase Chain Reaction; RNA, Messenger - analysis; Synthetic digonucleotides and genes. Sequencing; Transgenes; Genetic transformation; Gel electrophoresis; Copy number; Denaturation; Transgenic animal; Reverse transcription; Gene expression; Polymerase chain reaction; Cell line; Messenger RNA; Gene; Measurement method; Quantitative analysis; Concentration gradient
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/98241st06

Transgenic mice and cell lines are frequently developed to study human disease. Accurate determination of transgene copy number and levels of mRNA are necessary to understand the phenotypic changes observed in these models. Currently, transgene copy number and expression are estimated by Southern blot analysis of genomic DNA and Northern blot analysis of mRNA. We report a novel PCR-based method for determining transgene copy number and levels of transgene expression using competitive PCR between endogenous genomic genes and mutant transgenes followed by denaturing gradient gel electrophoresis (DGGE). We are able to accurately quantify a range of 1-10 copies of transgene incorporated per diploid genome. After reverse-transcribing RNA to cDNA, we are able to quantify levels of transgene mRNA that correlate with biochemical and histological evidence of transgene activity. In conclusion, resolving PCR and reverse transcription-PCR products by DGGE is a rapid and reproducible method that allows for accurate determination of transgene copy number and expression. This technique provides a more complete understanding of transgene effects.