Induction of pluripotency by protein gain of function in cloned mouse embryos

• Applicant: Privatdozent Dr. Michele Boiani
• Subject Area: Developmental Biology ; Cell Biology
• Term: from 2008 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 66209674

Compared to the other reprogramming methods, somatic cell nuclear transfer into oocytes relies on a naturally existing cell, and is fast and totipotent. Since the resultant pluripotent stem cells are of superior quality, and many oocytic mRNAs find no match in the oocyte proteome, we put forward the hypotheses that: 1) the oocytic reprogramming factors differ from those known in ES and iPS cells; and 2) oocyte-mediated reprogramming uses also new proteins that are General information synthesized immediately after oocyte activation. We predict these oocytic factors to synergize with the known iPS reprogramming factors, leading to a more complete epigenetic 'resetting' of the somatic genome. Accordingly, we propose two sets of experiments. Firstly, we will combine biochemistry, mass spectrometry and bioinformatics to identify candidate reprogramming factors present in the pronuclei of activated mouse oocytes. Objective screening criteria have already been tested on the proteome of metaphase II oocytes, retaining 29 of 3699 proteins. Secondly, we will express the old (29) and new candidates in mouse fibroblasts during their conversion to iPS cells. The expected gain in reprogramming efficiency will be assessed by comparing rates of Nanog gene induction and pluripotent cell colony formation in the presence vs absence of the oocytic factor. Our project is very innovative on at least three fronts: firstly, it will define the catalog of natural reprogramming factors that operate in the oocyte. Secondly, our project will be the first oocyteeducated attempt to find the limiting stage of iPS reprogramming. Thirdly, independently of reprogramming function, our list of proteins will bring new insight in the molecular landscape of the mature female germ cell.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1356: Pluripotency and Cellular Reprogramming