Optimization of transfection for CFTE29o- cell culture to develop editing of F508del mutation in CFTR gene
https://doi.org/10.1234/XXXX-XXXX-2016-8-36-39
Abstract
Genome editing technologies, including the newly appeared CRISPR/Cas9, are the most promising for development of etiotropic treatment of cystic fibrosis. Traditional transfection methods are commonly used to deliver components of CRISPR/Cas9 system. We aimed at optimizing non-viral delivery of plasmid pEGFP-N1 into human tracheal epithelial cells CFTE29o- with homozygous F508del mutation. Efficiency of lipofection with various reagents, Metafectene, Metafectene Pro, Unifectine-56 and Maxifectine-56, was very low and poorly reproducible. Electroporation, even in «soft» conditions, led to high cell mortality. Calcium phosphate transfection turned out to be suitable for CFTE29o- showing efficiency of 46.3-48.0%. High efficacy of this method allows to transfect CRISPR/Cas9 plasmids for their further comparative characterization in order to optimize genome editing of F508del mutation in CFTR gene.
About the Authors
S. A. Smirnikhina
Research Centre for Medical Genetics
Russian Federation
Russian Federation
A. V. Bannikov
Research Centre for Medical Genetics
Russian Federation
Russian Federation
A. V. Lavrov
Research Centre for Medical Genetics; The Russian National Research Medical University named after N.I. Pirogov
Russian Federation
Russian Federation
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Review
For citations:
Smirnikhina S.A., Bannikov A.V., Lavrov A.V. Optimization of transfection for CFTE29o- cell culture to develop editing of F508del mutation in CFTR gene. Medical Genetics. 2016;15(8):36-39. (In Russ.) https://doi.org/10.1234/XXXX-XXXX-2016-8-36-39
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