Reprogramming of mesenchymal stem cells and adult fibroblasts following nuclear transfer in rabbits

Hao, Ru (2009): Reprogramming of mesenchymal stem cells and adult fibroblasts following nuclear transfer in rabbits. Dissertation, LMU München: Tierärztliche Fakultät

Abstract

The main aim of this thesis was to find out which donor cells would be most suitable for production of cloned rabbits with a targeted modification of their genome, and their corresponding patterns of reprogramming hetero- and euchromatic histone modifications (H3K4m2/3 and H3K27m3). For these purposes, we carried out NT with rabbit mesenchymal stem cells (MSCs) and adult fibroblasts (RAFs). As totally 13 cell lines of MSCs have been tested, they didn’t show stably higher development potential than RAFs, even though the blastocyst rate of embryos cloned from MSC A/B reached to 76%. Finally two cloned rabbits were produced from meschnymal stem cells. H3K27m3 was undetectable in all stages of nuclear transfer embryos except for one cell stage and blastocysts. It seems that H3K27m3 is faithfully reprogrammed in transferred nuclei of all donor cell types, with minor differences in zygotes and blastocysts. Strong signals for H3K4m2/3 were detected at the one to two-cell stages of in vivo embryos with a slight decrease at the 4-cell stage, followed by a more drastic decrease at the 8-cell stage, where the signal minimum was reached. In 16-cell embryos signals slightly increased and then reached in morulae and blastocysts the levels observed in one-two cell embryos. In all types of nuclear transfer embryos fluorescence intensity ratios differed from that of in vivo embryos. The minimum was not reached at the 8-cell stage but at the 4-cell stage. Reprogramming of H3K4m2/3 modification occurred quite differently with either type of cells irrespective of the cell origin or type and no close similarities in the patterns of this reprogramming was observed between in vivo and nuclear transfer embryos. Embryos cloned from MSC A/B differed from all others in the way that H3K4m2/3 was generally lower and remained in the range of the donor cells. This suggests that reprogramming of H3K4m2/3 modification is more aberrant with MSC A/B cells than with any other cell type used in this study. However, with MSC A/B cells, a significantly higher or similar proportion of cloned embryos developed to blastocysts indicating that reprogramming of H3K4m2/3 modification does not correlate with developmental potential of donor cells. In conclusion, our study provides evidence that histone modifications for heterochromatin are faithfully reprogrammed during NT of rabbit somatic cells, while patterns of epigenetic reprogramming of euchromatic histone modifications differ between individual cell lines irrespective of their origin or type and are not correlated with their developmental potential. Although MSCs were not superior to RAFs in respect to the criteria tested in our study they may be suitable nuclear donors for generating transgenic cloned rabbits due to their high developmental plasticity.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: rabbit somatic cells, mexenchymal stem cells, nuclear transfer, histone methylation
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 590 Tiere (Zoologie)
• Fakultäten: Tierärztliche Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 6. Februar 2009
• 1. Bericht­erstatter:in: Zakhartchenko, Valeri