The In Vitro Single Oocyte Reporter Assay: A Technique to Study Target mRNA Translation Regulation During In Vitro Oocyte Maturation
Transcrição
Oocytes in prophase-I of meiosis are transcriptionally active; they produce mRNAs and store them in a dormant form. During maturation, as the oocyte enters metaphase, the stored mRNAs are translated into proteins.
To study the translation of mRNA, begin with prophase-I-arrested mouse oocytes suspended in a drop of medium. Position the holding pipette near a single oocyte to hold it in position.
Take a micro-injection pipette containing a reporter mix. This mix contains two distinct types of mRNA, one encoding yellow fluorescent protein, or YFP – a reporter fused with the 3'-untranslated region of the target gene – and a polyadenylated mRNA encoding red fluorescent protein.
Inject the reporter mix into the oocyte. Incubate the microinjected oocytes in a maturation medium supplemented with cilostamide - a chemical inhibitor that maintains prophase-I arrest.
The oocyte translates polyadenylated mRNA until the red fluorescence intensity reaches a plateau, while the levels of YFP-encoding mRNA lacking a polyadenylation tail remain as such.
Transfer the treated oocytes into a maturation medium without cilostamide, which helps to resume meiosis; and the target mRNA undergoes translation to produce the target protein fused with yellow fluorescent reporter.
Perform time-lapse imaging to visualize the fluorescence signals at regular intervals.
Injected oocytes show constant levels of red fluorescence, while the yellow fluorescence increases over time, indicating translation and gradual accumulation of the reporter proteins.
