Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos

نویسندگان

  • Jens Popken
  • Daniela Koehler
  • Alessandro Brero
  • Annegret Wuensch
  • Tuna Guengoer
  • Tobias Thormeyer
  • Eckhard Wolf
  • Thomas Cremer
  • Valeri Zakhartchenko
چکیده

Cloned bovine preimplantation embryos were generated by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts with a silent copy of the pluripotency reporter gene GOF, integrated at a single site of a chromosome 13. GOF combines the regulatory Oct4/Pou5f1 sequence with the coding sequence for EGFP. EGFP expression served as a marker for pluripotency gene activation and was consistently detected in preimplantation embryos with 9 and more cells. Three-dimensional radial nuclear positions of GOF, its carrier chromosome territory and non-carrier homolog were measured in nuclei of fibroblasts, and of day 2 and day 4 embryos, carrying 2 to 9 and 15 to 22 cells, respectively. We tested, whether transcriptional activation was correlated with repositioning of GOF toward the nuclear interior either with a corresponding movement of its carrier chromosome territory 13 or via the formation of a giant chromatin loop. A significant shift of GOF away from the nuclear periphery was observed in day 2 embryos together with both carrier and non-carrier chromosome territories. At day 4, GOF, its carrier chromosome territory 13 and the non-carrier homolog had moved back toward the nuclear periphery. Similar movements of both chromosome territories ruled out a specific GOF effect. Pluripotency gene activation was preceded by a transient, radial shift of GOF toward the nuclear interior. The persistent co-localization of GOF with its carrier chromosome territory rules out the formation of a giant chromatin loop during GOF activation.

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عنوان ژورنال:

دوره 5  شماره 

صفحات  -

تاریخ انتشار 2014