Techniques routinely used in our lab for analysing the X-inactivation process in differentiating ES cells are described. We focus particularly on chromatin changes (histone modifications, protein association...) during X inactivation, using immunofluorescence (IF) combined with RNA FISH or DNA FISH on interphase nuclei. These should provide a tool for defining potential causal relationships between different events not only during X inactivation but also during the establishment of other patterns of gene activity.
The main purpose of a combined IF and FISH analysis is, on the one hand, to preserve nuclear architecture and the antibody's epitope as far as possible but, on the other hand, to allow the penetration of the FISH probe for detection of nuclear transcripts, gene location or chromosome territories. The optimal conditions for IF are usually poorly compatible with those for FISH. We have therefore tested a variety of methods and conditions and we describe here those that we find optimal for the immuno-detection of histone modifications combined with RNA or DNA FISH on mouse fibroblasts or embryonic stem cells. For more details, the reader is referred to Chaumeil et al., 2002 and Chaumeil et al., 2004.
Mammalian Development Epigenetics Group
UMR 218 Institute Curie - 26, rue d'Ulm - 75231 Paris Cedec 05, France