The oocyte is the only cell of the body that can reprogram transplanted somatic nuclei and sets the gold standard for all reprogramming methods. Therefore, an in-depth characterization of its proteome holds promise to advance our understanding of reprogramming and germ cell biology. To date, limitations on oocyte numbers and proteomic technology have impeded this task, and the search for reprogramming factors has been conducted in embryonic stem (ES) cells instead. Here, we present the proteome of metaphase II mouse oocytes to a depth of 3699 proteins, which substantially extends the number of proteins identified until now in mouse oocytes and is comparable by size to the proteome of undifferentiated mouse ES cells. Twenty-eight oocyte proteins, also detected in ES cells, match the criteria of our multilevel approach to screen for reprogramming factors, namely nuclear localization, chromatin modification, and catalytic activity. Our oocyte proteome catalog thus advances the definition of the "reprogrammome", the set of molecules--proteins, RNAs, lipids, and small molecules--that enable reprogramming.

Chimeric antigen receptor (CAR) engineering of T cells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside G D2 , but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces G D2 surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of G D2 biosynthesis, and EZH2 inhibition enhances expression of these genes. G D2 surface expression in Ewing sarcoma cells is not associated with distinct in vitro proliferation, colony formation, chemosensitivity, or in vivo tumorigenicity. Moreover, disruption of G D2 synthesis by gene editing does not affect its in vitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by G D2 -specific CAR gene-modified T cells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred G D2 -redirected T cells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains < 5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell's genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in > or = 3 months.

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An important question in oocyte-mediated nuclear reprogramming is whether gene expression of the donor nucleus changes randomly or follows a pattern. Since cloned embryos are very heterogeneous and arrest frequently during preimplantation development, a random scenario is generally accepted. In the present study, we resolve the heterogeneity of cumulus cell-derived mouse clones by recognizing structured subsets, and we analyze their relationship to reprogramming of donor nuclei. We utilize live cell imaging of the Oct4 promoter-driven GFP transgene to resolve the populations of cloned and ICSI-fertilized morulae, and we sort them both into three subsets based on different GFP expression. Functionally, subsets of cloned but not ICSI morulae form blastocysts and ES cells proportional to Oct4-GFP expression. Regulatively, the subsets of cloned morulae are characterized by small differences of transcript level for the pluripotency-associated genes Oct4, Nanog and Sox2. Small differences of the level of select mRNAs across subsets suggest a uniform rather than random course of reprogramming from the morula stage on. Since these small differences correspond with substantial differences in developmental competence, we propose that developmental potential of clones relates to levels of gene expression in a different way than fertilized embryos.