Generation of Myogenic MELAS-iPSCs

We introduced a myogenic differentiation system³⁴ (link) into MELAS-iPSCs using a piggyBac vector, PB200_hMyoD,³⁵ (link) including (Tet-On)-MYOD-IRES-EGFP cDNA and a neomycin resistance gene. A01 MELAS-iPSCs were transfected with a transposase-expressing plasmid, pHL-EF1a-hcPBase³⁴ (link) (1.5 μg), and PB200-hMyoD (1.5 μg) using Nucleofector 2b (Lonza) and an Amaxa human stem cell Nucleofector kit 2 (Lonza), according to the manufacturer’s protocol, and were then plated on feeder cells. Forty-eight hours after the transfection, a G418 disulfate aqueous solution (50 μg/mL; Nacalai Tesque) was added to select appropriate MyoD-iPSC clones with high EGFP expression.

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Yahata N., Boda H, & Hata R. (2020). Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs. Molecular Therapy. Methods & Clinical Development, 20, 54-68.

Publication 2020

Nucleofector 2b Amaxa human stem cell Nucleofector kit 2 G418 disulfate aqueous solution

Cdna Clones Feeder cells G418 Gene Human Ipsc Ires Melas Myogenic Neomycin Plasmid Stem cell Transfection Transposase Vector

Corresponding Organization : Fujita Health University

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Variable analysis

independent variables

Transfection of MELAS-iPSCs with a transposase-expressing plasmid, pHL-EF1a-hcPBase, and PB200-hMyoD vector

dependent variables

Selection of appropriate MyoD-iPSC clones with high EGFP expression

control variables

Nucleofector 2b (Lonza) and an Amaxa human stem cell Nucleofector kit 2 (Lonza) used for transfection according to the manufacturer's protocol
Feeder cells used for plating the transfected MELAS-iPSCs
G418 disulfate aqueous solution (50 μg/mL; Nacalai Tesque) used for selection of MyoD-iPSC clones

controls

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

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