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      Calcium-Mediated In Vitro Transfection Technique of Oligonucleotides with Broad Chemical Modification Compatibility.

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          Abstract

          Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca2+-enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.

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          Author and article information

          Journal
          Methods Mol Biol
          Methods in molecular biology (Clifton, N.J.)
          Springer Science and Business Media LLC
          1940-6029
          1064-3745
          2020
          : 2176
          Affiliations
          [1 ] Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
          [2 ] Department of Molecular Innovation in Lipidology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan.
          [3 ] Medicinal Chemistry Research Laboratory for Medium Molecular Drug Discovery, Shionogi & Co., Ltd., Osaka, Japan.
          [4 ] Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. tsuyoshi.yamamoto@nagasaki-u.ac.jp.
          [5 ] Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan. tsuyoshi.yamamoto@nagasaki-u.ac.jp.
          Article
          10.1007/978-1-0716-0771-8_10
          32865788
          98fe8efd-0fac-4fe7-b1a3-09d22c4feb56
          History

          siRNA,2′4′-BNA/LNA gapmers,LNA,Naked oligonucleotides,Nanoparticles,Oligonucleotide drugs,Phosphorodiamidate morpholino oligonucleotide,Transfection

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