Nolte (Engel), Zechner

Project Title: Characterization of the putative pluripotency-regulating genes Lrrc34 and Zfp819

During the first funding period of SPP1356, our aim was to further substantiate the pluripotency of the multipotent adult Germline Stem Cells (maGSCs) that we derived in vitro from Spermatogonial Stem Cells (SSCs) by performing several comparative analyses with other pluripotent stem cells. We could detect no significant differences between all the analysed pluripotent cell types so that there is no doubt left that maGSCs are indeed pluripotent and have the same potential as Embryonic Stem Cells (ESCs), the gold standard of pluripotency. As a consequence from all our studies, and especially from the comparative analysis of the transcriptomes of undifferentiated and differentiated maGSCs and ESCs, we obtained several candidate genes for the regulation and/or maintenance of the pluripotent state. For two of these candidate genes, Lrrc34 and Zfp819, we already have preliminary results which strongly support their involvement in the pluripotency network. For these two genes we want to perform a detailed functional characterization by generating stable knockdown and stable overexpressing ESCs as well as conditional knock out mice and transgenic mice.

In addition, we want to check the ability of these two genes to support reprogramming of somatic cells to pluripotency. For selected experimental stages, we will further carry out genespecific (by bisulphite pyrosequencing and ChIP-Real-time-PCR) and genome-wide (by highthroughput reduced representation bisulphite sequencing (RRBS) and ChIP-Seq) epigenetic analysis to get a global picture of the epigenetic effects of the two genes during maintenance and establishment of pluripotency.