We aim to understand how nuclei of adult somatic cells can be experimentally reprogrammed to express embryo or stem cell genes. This has been achieved by others using retroviral gene transfection to induce pluripotent stem cells. However we are interested in how this can be achieved by transplanting somatic cell nuclei to unfertilised eggs. By this route, reprogramming uses natural components that operate on sperm after fertilisation; it works at a higher efficiency than does gene transfection, and does not impose any genetic changes in the resulting cells. We wish to identify the reprogramming molecules and mechanisms used by eggs.

We use amphibian oocytes in first meiotic prophase to induce reprogramming in injected mammalian nuclei, because embryo or stem gene transcription can be readily seen in transplanted somatic cell nuclei in the absence of cell division or protein synthesis.

Fig.1. Modified histone H3T6Ph is increased in mammalian nuclei transplanted to Xenopus oocytes. (0 hours, left; 24 hours, right).

We find that nuclear reprogramming requires the removal of differentiation marks from somatic cell nuclei. These include the demethylation of promoter DNA, and the substitution of certain modifications on core histones. Particularly important is the exchange of somatic H1 linker histones for the oocyte-specific variant named B4 or H1foo. Surprisingly the transcriptional activation of some reprogrammed genes is independent of transcription factor supply. A special advantage of using oocytes to reprogramme somatic nuclei is that the process can be observed under the microscope in real time. This permits the application of photobleaching, and hence an analysis of the exchange of gene-specific proteins on their DNA-binding sites. In some cases we use nuclei of mammalian cells in which multiple copies of the bacterial lac repressor binding site has been integrated, in order to follow the real-time binding and dissociation of the GFP-lac repressor, as an example of a gene-specific protein.

Fig.2. Design of a FRAP experiment on somatic nuclei injected into an oocyte germinal vesicle.

Fig.3. Replacement of adult somatic linker histone H1o by oocyte-specific linker histone B4.

Plain English:
We are trying to find ways of obtaining embryo cells from the cells of an adult. The eventual aim is to provide replacement cells of all kinds starting from usually obtainable cells of an adult individual. For example, we would like to be able to find a way of obtaining spare heart or brain cells from skin or blood cells. The important point is that the replacement cells need to be from the same individual, to avoid problems of rejection and hence of the need for immunosuppression. We are trying to identify the molecules and mechanisms by which eggs can reverse the process of specialization, so as to derive embryo cells from adult skin cells.

Selected publications:

• Gurdon JB and Melton DA (2009) Nuclear reprogramming in cells. Science 322, 1811-1815

• Ng RK and Gurdon JB (2008) Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription. Nature Cell Biology, 10,102-109

• Gurdon JB (2006) From nuclear transfer to nuclear reprogramming: the reversal of cell differentiation. Ann.Rev. Cell Devel. Biol., 22,1-22

Fig.4. Reprogramming of nuclei from differentiated ES cells (T0,R) to an undifferentiated state (T15 or T36, U) as a result of injection into oocytes.