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Publication type: Diploma Thesis

Year: 2012

Author(s): Steiner, Susanne

Title: Tetraploid Embryo Complementation.

Other title: Tetraploide Embryo-Komplementierung

Source: Diplomarbeit, Vet. Med. Univ. Wien, pp. 54.


Advisor(s):

Kolbe Thomas
Rülicke Thomas

Reviewer(s):
Walter Ingrid

Vetmed Research Units:
Institute of Laboratory Animal Science


Graduation date: 22.11.12


Abstract:
Tetraploid embryo complementation is a method for generating mice originating only from embryonic stem cells. Through combination of embryonic stem cells and tetraploid embryos, embryos are produced where the embryo proper originates from embryonic stem cells while the embryonic parts of the placenta and the yolk sac develop from tetraploid cells. The method of tetraploid embryo complementation is used to produce mice from genetically modified embryonic stem cells or from induced pluripotent stem cells (iPS), as an alternative to cloning by somatic cell nuclear transfer. Moreover, tetraploid embryo complementation is accepted as the most reliable proof for pluripotency of embryonic stem cells (Nagy et al., 1990; Boland et al., 2009). The goal of this diploma thesis was the establishment of tetraploid embryo complementation at the Institute for Laboratory Animals and the proof of the embryonic stem cell origin in the mice produced. Tetraploid mouse embryos were produced by electrofusion of embryos in the two-cell-stage, and tetraploidy of these embryos was demonstrated by karyotyping. Embryonic stem cells were then injected into the blastocoel of tetraploid blastocysts on ED 3.5 and embryo transfer into pseudopregnant mice was performed. In total 17 mice produced by tetraploid embryo complementation were delivered naturally or by hysterectomy. Two of these mice were monitored through their development after birth and both appeared physiologically normal and proofed to be fertile. Moreover, the mice from tetraploid embryo complementation were analysed histologically and by flow cytometry for contribution of tetraploid cells. As expected, placenta and yolk sac contained massive amounts of tetraploid cells, while no tetraploid contributions could be found in 11 out of 15 mice analysed. However, in four mice minor contributions of tetraploid cells were detected in various organs.


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