Establishing Stable Cell Lines

Conjugating cells were subjected to transfection by electroporation using a Gene Pulser II (Bio-Rad, Hercules, CA) as described previously (Iwamoto et al., 2014 (link), 2015 (link)). The resulting cell suspension was cultivated for 18 h and then treated with paromomycin sulfate (Sigma-Aldrich, St Louis, MO) at 120 µg/ml when using pIGF1, pIGF1C, pEGFP-neo4 and p2xmNeon_6xmyc_Neo4 vectors, or puromycin dihydrochloride (Fermentek, Jerusalem, Israel) at 200 µg/ml when using a pmCherry-pur4 vector. Cadmium chloride was also added at 0.5 µg/ml to induce the expression of drug-resistant genes for pEGFP-neo4, p2xmNeon_6xmyc_Neo4, and pmCherry-pur4 vectors. Resistant cells usually appeared within a few days after the drug was added. We checked that at least five independent clones (i.e. grown in five different wells) exhibited the same intracellular localization of each GFP–Nup.

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Iwamoto M., Osakada H., Mori C., Fukuda Y., Nagao K., Obuse C., Hiraoka Y, & Haraguchi T. (2017). Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in the ciliate Tetrahymena. Journal of Cell Science, 130(10), 1822-1834.

Publication 2017

Gene Pulser II paromomycin sulfate

Cadmium chloride Cells Clones Drug Electroporation Expression genes Gene Intracellular Paromomycin sulfate Puromycin dihydrochloride Transfection Vectors

Corresponding Organization :

Other organizations : National Institute of Information and Communications Technology, Tohoku University, Hokkaido University, Osaka University

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Variable analysis

independent variables

Transfection method: Electroporation using a Gene Pulser II
Plasmid vectors: pIGF1, pIGF1C, pEGFP-neo4, p2xmNeon_6xmyc_Neo4, pmCherry-pur4
Selection drugs: Paromomycin sulfate, Puromycin dihydrochloride
Induction agent: Cadmium chloride

dependent variables

Intracellular localization of GFP–Nup

control variables

Cultivation time: 18 h

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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