Manipulation of the epigenetic mark histone 3 lysine 9 tri-methylation (H3K9me3) in donor cells prior to nuclear transfer

Abstract

Despite the production of live cloned animals from a range of mammalian species, the efficiency of (nuclear transfer) NT still remains low. Inadequate reprogramming of epigenetic marks in the donor cells has been identified as the key cause of this inefficiency. One repressive epigenetic mark which is known to be very stable and difficult to remove during reprogramming is H3K9Me3. The aim of this project has been to test the hypothesis that removal of H3K9me3 via over expression of its specific demethylase, jmjd2b, in donor cells prior to NT would enhance reprogramming. To test this hypothesis, mouse embryonic stem (ES) cells with tet inducible expression of jmjd2b were generated. Induction of jmjd2b in these ES cells resulted in 62-65% reduction in H3K9me3 as determined by immunofluorescence and western analysis. Use of ES cells that were induced for jmjd2b as donors during NT resulted in 8% higher number of blastocysts and 10% higher overall in vitro development compared to non-induced ES cells. However, following embryo transfers no live cloned mice could be generated. To test the pluripotency of the jmjd2b ES cells, one live germ-line chimera was generated. The overall efficiency of generating a live germline chimera was very low of 0.5%. However, the generation of this chimera enabled the generation of jmjd2b inducible mice and subsequent derivation of somatic mouse embryonic fibroblasts (MEFs), which enabled the examination of the effects of using somatic donor cells with reduced level of H3K9me3 during NT. Induction of jmjd2b in MEFs resulted in a dramatic decrease in H3K9me3 by 85-90% and 99% as determined by immunofluorescence and western analysis respectively. In contrast to the results seen with jmjd2b ES cells, use of MEFs induced for jmjd2b as donors in NT, resulted in 10 fold higher number of blastocysts and 3 fold higher overall in vitro development compared to non-induced MEFs. Although, following embryo transfers no live clones were generated, this was probably because of the small number of reconstructs (33) transferred. Thus it appears that reduction of H3K9me3 via over-expression of jmjd2b in donor cells improves in vitro reprogramming efficiency of NT reconstructs, but the greater effect seen with MEFs suggests that this type of chromatin therapy is more suitable for somatic NT.