medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.1234/XXXX-XXXX-2016-8-36-39medgen-163Research ArticleСтатьиОптимизация условий трансфекции клеточной культуры CFTE29o- для разработки редактирования мутации F508del в гене CFTROptimization of transfection for CFTE29o- cell culture to develop editing of F508del mutation in CFTR geneСмирнихинаС. А.SmirnikhinaS. A.smirnikhinas@gmail.comБанниковА. В.BannikovA. V.noemail@neicon.ruЛавровА. В.LavrovA. V.noemail@neicon.ruФедеральное государственное бюджетное научное учреждение «Медико-генетический научный центр»РоссияResearch Centre for Medical GeneticsRussian FederationФедеральное государственное бюджетное научное учреждение «Медико-генетический научный центр»; Российский национальный исследовательский медицинский университет им. Н.И. ПироговаРоссияResearch Centre for Medical Genetics; The Russian National Research Medical University named after N.I. PirogovRussian Federation2016071020161583639Copyright © Смирнихина С.А., Банников А.В., Лавров А.В., 20162016Смирнихина С.А., Банников А.В., Лавров А.В.Smirnikhina S.A., Bannikov A.V., Lavrov A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.medgen-journal.ru/jour/article/view/163

Технологии геномного редактирования, включая недавно появившийся метод CRISPR/Cas9, являются наиболее перспективными для разработки этиотропного лечения муковисцидоза. Для доставки компонентов системы CRISPR/Cas9 в клетки обычно используют традиционные методы трансфекции. Целью данной работы была оптимизация невирусной доставки плазмиды pEGFP-N1 в клетки трахеального эпителия человека CFTE29o- с гомозиготной мутацией F508del. Эффективность липофекции различными реагентами, Metafectene, Metafectene Pro, Unifectine-56 и Maxifectine-56, была низкой и плохо воспроизводимой. Электропорация, даже при «мягких» условиях, приводила к высокой смертности клеток. Подходящим методом для CFTE29o- оказалась кальций-фосфатная трансфекция, эффективность которой составила 46,3-48,0%. Высокая эффективность метода позволяет трансфицировать плазмиды CRISPR/Cas9 для их дальнейшей сравнительной характеристики в целях оптимизации геномного редактирования F508del в гене CFTR .

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.

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The authors declare that there are no conflicts of interest present.