Generation of Genome Integration-free Induced Pluripotent Stem Cells from Fibroblasts of C57BL/6 Mice without c-Myc Transduction

Yuko Jincho,Ryoko Araki,Yuko Hoki,Chihiro Tamura,Miki Nakamura,Shunsuke Ando,Yasuji Kasama,Masumi Abe

doi:10.1074/jbc.m110.115915

Yuko Jincho, Ryoko Araki + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m110.115915

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Abstract

Although the induction of genome integration-free induced pluripotent stem cells (iPSCs) has been reported, c-Myc was still required for the efficient generation of these cells. Herein, we report mouse strain-dependent differences in the c-Myc dependence for iPSC generation and the successful generation of genome integration-free iPSCs without c-Myc transduction using C57BL/6 mouse embryonic fibroblasts. We performed 49 independent experiments and obtained a total of 24 iPSC clones, including 18 genome integration-free iPSC clones. These iPSCs were indistinguishable from embryonic stem cells and from iPSCs generated using other methods. Furthermore, the generation of three-factor iPSCs free of virus vectors revealed the contribution of c-Myc to the genomic integration of external genes. C57BL/6 is an inbred mouse strain with substantial advantages for use in genetic and molecular biological studies due to its use in the whole mouse genome sequencing project. Thus, the present series of C57BL/6 iPSCs generated by various procedures will serve as a valuable resource for future genetic studies of iPSC generation.

Highlights

(hereafter referred to as 3F) could generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs), the exclusion of c-Myc yielded less than 10% of the number of iPSCs when compared with 4F infection and took twice as long [4, 5]
We report for the first time the successful generation of iPSCs from MEFs using a 2A plasmid vector that contains only three factors and excludes the c-Myc gene [12]
IPSC Generation from C57BL/6 Embryonic Fibroblasts, a High Yield in the Absence of c-Myc Transduction—It is well known that many biological phenomena, tumorigenesis, occur with variable frequency in laboratory mouse strains (16 –18)

Summary

EXPERIMENTAL PROCEDURES

Mouse Strains and MEFs—The following mouse strains were used: C57BL/6, C.B-17/lcr-ϩ/ϩ Jcl (Crea Japan, Shizuoka, Japan); C3H, 129, C57BL/6-Tg(CAG-EGFP) (Japan SLC, Hamamatsu, Japan); and Tg mice homozygous for NanogGFP (RBRC02290; RIKEN BioResource Center). MEFs were prepared from embryonic day 13.5 embryos in each case and used within three cell passages. Moloney-based retroviral vectors (pMXs) containing murine cDNA inserts for Oct3/4, Sox, c-Myc, and Klf were obtained from Addgene (Cambridge, MA). These plasmids were transfected into PlatE cells using FuGENE 6 transfection reagent (Roche Applied Science, Basel, Switzerland). The medium was collected at 48 h after transfection, filtered, supplemented with 4 ␮g/ml polybrene and added to MEFs that had been plated the previous day at 1.25 ϫ 105 cells/3.5-cm diameter dish.

Differences in iPSC Generation between Mouse Strains

RESULTS

DISCUSSION