The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products
| Autor: | Alvin W. Nienow, H. Patel, Julian A. J. Hanak, Anthony Gordon Crewe Hitchcock, Saethawat Chamsart |
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| Rok vydání: | 2001 |
| Předmět: |
DNA
Bacterial Time Factors Lysis Bioengineering Biology medicine.disease_cause Applied Microbiology and Biotechnology Bioreactors Plasmid Escherichia coli medicine Bioreactor Electrophoresis Agar Gel Plasmid preparation Chromatography Gene Transfer Techniques Genetic Therapy Hydrogen-Ion Concentration Molecular biology Solutions Electrophoresis Agarose gel electrophoresis Stress Mechanical Alkaline lysis Plasmids Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 75:387-392 |
| ISSN: | 1097-0290 0006-3592 |
| DOI: | 10.1002/bit.10046 |
| Popis: | Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy. Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied. Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer. Comparison has been made with unstressed material subjected to similar holding times. These tests essentially show that under all these conditions |
| Databáze: | OpenAIRE |
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