JoVE Encyclopedia of Experiments
Biological Techniques
0 views • 7:16 min • July 8th, 2025
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 t
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