In the Jones laboratory we investigate the roles of various proteins (in the past most notably Cdh1 and Emi2) in development of the mouse oocyte by modifying their transcription.
We usually achieve this by microinjection of morpholinos and/or cRNAs.
Morpholinos are synthetic oligonucleotides, designed to bind to specific RNA molecules and block transcription of proteins. This approach allows robust knockdown of proteins with little off target effect.
We also construct cRNAs of genes of interest, usually tagged with a fluorescent gene such as GFP, YFP or Venus. Once microinjected into oocytes the RNA is expressed as protein and it is possible to follow the fate of these proteins by fluorescence microscopy. This gives valuable information about a proteins localization and destruction in real time. This technique is extremely valuable when studying oocyte maturation because as in mitosis, a proteins temporal and spatial localization and its destruction are crucial for its regulation during the cell cycle.
To complement these techniques we have a range of high end microscopes including three inverted microscopes, equipped with micromanipulation apparatus for injection of oocytes and fluorescent lamps, cameras and imaging software for real-time recordings of oocyte maturation. In addition we have a laser scanning confocal microscope equipped for live cell imaging and immunofluorescence studies and a Nikon Biostation IM for long term imaging.
Because the goal of meiosis is production of euploid gametes, it has been important for us to be able to reliably count the chromosome compliment of mature oocytes. We have therefore developed an in-situ assay for counting chromosomes, utilizing staining of kinetochores by CREST anti-serum and DNA by Hoechst. 3D confocal imaging allows us to count the CREST foci within and oocyte and within polar bodies to reveal any chromosome segregation errors, without having to spread the chromosomes by traditional methods, which run the risk of chromosome losses during the procedure.
Microinjection of cRNA into a GV stage oocyte.
In-situ chromosome count. Chromatin (green) and Kinetochores (pink).