Comparison of hybridization methods and real-time PCR: their value in animal cell line characterization.
For biotechnological protein production, the International Conference of Harmonization (ICH) recommends appropriate testing of expression constructs and characterization of producer cell lines in order to assure uniform specifications, improve safety, and confirm stable productivity. Commonly, hybridization analyses are used for the evaluation of genetic stability, gene and transcript copy numbers as well as the integrity of the coding sequence. However, improvements in polymerase chain reaction (PCR) techniques have accelerated the analysis of genetic parameters and have nowadays become the method of choice for the evaluation of gene copy numbers and transcript levels. Nevertheless, Southern and Northern blot analyses are still valuable tools that deliver additional information to PCR results during cell clone characterization studies. In this study, we discuss advantages and drawbacks of hybridization and PCR methods in regard to their applicability and efficiency during the development of recombinant mammalian cell lines. A comparative review of the literature as well as an overview of findings from our own group will be given.
Böhm-Hofstätter
Helga
H
Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 11, 1190, Vienna, Austria.
Tschernutter
Marion
M
Kunert
Renate
R
D000818:Animals; D002460:Cell Line / Q000737:chemistry* / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*