The purpose of this project is to map the locations at which specific genes are expressed in pre-implantation mouse embryos. Dr. Minoru Ko's group in LG has used expression profiling with microarrays to identify 500-1000 genes that are differentially expressed at various stages of early mouse embryo development. During the past year, several Fluorescence in-situ hybridization (FISH) techniques and probes were evaluated for their ability to locate the transcripts of interest in whole mount early development embryos using small oligonucleotide probes attached to fluorescent molecules. Evaluation of these probes presented several challenges that we were unable to completely solve before Dr. Yoshikawa had to return to Japan. Dr. Yoshikawa was successful at developing medium-throughput techniques for processing whole-mount embryos for microscopy, however problems with probe penetration, bleaching, and reproducibility were never completely overcome despite some tantalizing initial results. It must be pointed out that this was a very ambitious project that required the development of novel techniques at several steps along the way - from the physical processing of the embryos to the manipulations required for producing the FISH signal, to the fluorescent probes themselves. We have worked with the companies that make these specialized probes - 3DNA from Genisphere and Qdot from Quantum Dot - to address our special needs: small size, bright signal, resistance to bleaching, and appropriate chemistry for attachment to oligonucleotides. Given more time, I believe Dr. Yoshikawa would have been able to solve these problems because of his great talent and dedication. It may be that FISH on whole-mount mouse embryos may be too difficult to accomplish at this time. However several less ambitious possibilities remain including probing for protein products using antibodies as they become available, GFP expression through transfection or microinjection, and general cellular morphology stains like phaloidin and DAPI. An atlas of these very early stages of development has never been undertaken, and this is something that is quite feasible with the tools and techniques developed by Dr. Yoshikawa.
| Yoshikawa, Toshiyuki; Piao, Yulan; Zhong, Jinhui et al. (2006) High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization. Gene Expr Patterns 6:213-24 |
