Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of the Dlk1-Dio3 region in mice

doi:10.4252/wjsc.v4.i8.87

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World Journal of Stem Cells

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1948-0210

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

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World J Stem Cells. Aug 26, 2012; 4(8): 87-93
Published online Aug 26, 2012. doi: 10.4252/wjsc.v4.i8.87

Reprogramming somatic cells by fusion with embryonic stem cells does not cause silencing of the Dlk1-Dio3 region in mice

Nariman R Battulin, Anna A Khabarova, Ul’yana A Boyarskikh, Aleksey G Menzorov, Maxim L Filipenko, Oleg L Serov

Nariman R Battulin, Anna A Khabarova, Aleksey G Menzorov, Oleg L Serov, Institute of Cytology and Genetics SD RAS, Lavrentyeva 10, Novosibirsk 630090, Russian

Ul’yana A Boyarskikh, Maxim L Filipenko, Institute of Chemical Biology and Basic Medicine SD RAS, Lavrentyeva 8, Novosibirsk 630090, Russian

Author contributions: Battulin NR, Khabarova AA, Menzorov AG and Boyarsikh UA performed the majority of experiments; Boyarskikh UA and Filipenko ML provided vital reagents and analytical tools; Battulin NR, Boyarsikh UA and Serov OL analyzed the data; Battulin NR, Menzorov AG and Serov OL designed the study and wrote the manuscript.

Supported by Grant from the Russian Academy of Sciences, Siberian Branch N 48

Correspondence to: Nariman R Battulin, PhD, Institute of Cytology and Genetics SD RAS, Lavrentyeva 10, Novosibirsk 630090, Russian. battulin@bionet.nsc.ru

Telephone: +7-383-3634915 Fax: +7-383-3331278

Received: September 21, 2011
Revised: March 12, 2012
Accepted: March 18, 2012
Published online: August 26, 2012

Abstract

AIM: To examine the imprinted Dlk1-Dio3 locus in pluripotent embryonic stem (ES) cell/fibroblast hybrid cells.

METHODS: Gtl2, Rian, and Mirg mRNA expression in mouse pluripotent ES cell/fibroblast hybrid cells was examined by real-time reverse transcription-polymerase chain reaction. Pyrosequencing and bisulfate sequencing were used to determine the DNA methylation level of the Dlk1-Dio3 locus imprinting control region.

RESULTS: The selected hybrid clones had a near-tetraploid karyotype and were highly pluripotent judging from their capacity to generate chimeric embryos and adult chimeras. Our data clearly demonstrate that Gtl2, Rian, and Mirg, which are imprinted genes within the Dlk1-Dio3 locus, are active in all examined ES cell/fibroblast hybrid clones. In spite of interclonal variability, the expression of the imprinted genes is comparable to that of ES cells and fibroblasts. Quantitative analysis of the DNA methylation status of the intergenic differentially methylated region (IG DMR) within the Dlk1-Dio3 locus by pyrosequencing and bisulfite sequencing clearly showed that the DNA methylation status of the imprinted region in the tested hybrid clones was comparable to that of both ES cells and fibroblasts.

CONCLUSION: Reprogramming process in a hybrid cell system is achieved without marked alteration of the imprinted Dlk1-Dio3 locus.

Keywords: Cell fusion; Embryonic stem cells; Reprogramming; Pluripotency; Imprinted Dlk1-Dio3 region; DNA methylation