The In Vitro Single Oocyte Reporter Assay: A Technique to Study Target mRNA Translation Regulation During In Vitro Oocyte Maturation
Published: April 30, 2023
Abstract
Source: Costermans, N. G. J. et al., Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay. J. Vis. Exp. (2021).
In this video, we demonstrate that in the presence of a phosphodiester inhibitor, the oocyte cell cycle is arrested at prophase-I, during which transcribed mRNAs are stored in the absence of translation. After the release of the inhibitor, oocyte maturation resumes, and the cells enter metaphase. The signal of reporter mRNA fused with the target gene in metaphase-I indicates the activation of translational machinery and protein expression.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of media Add all components, as described in Table 1, to make the basic oocyte collection medium and oocyte maturation medium. For the basic collection medium, set the pH to 7.4. For both collection and maturation medium, add 3 mg/mL of bovine serum albumin (BSA) and 1 µM cilostamide on…
Representative Results
Figure 1: Example of a single oocyte time-lapse recording. Brightfield, YFP, and mCherry recordings of a single oocyte injected with mRNAs encoding Ypet/Interleukin-7 3' UTR and polyadenylated mCherry at prophase I, MI (6 h after cilostamide release), and MII (15 h after cilostamide release). Scale bar = 25 µm. Abbreviations: YFP = yellow fluorescent protein; GV = germinal vesicle; MI = …
Declarações
The authors have nothing to disclose.
Materials
| Preparation of media | |||
| Bovine Serum Albumin Powder Bioxtra | Sigma-Aldrich | SIAL-A3311 | |
| Cilostamide | EMD Millipore | 231085 | |
| MEM alpha | Gibco | 12561-056 | |
| Minimum Essential Medium Eagle | Sigma-Aldrich | M2645 | |
| Penicillin-Streptomycin 100x Solution, Sterile Filtered | Genesee Scientific Corporation (GenClone) | 25-512 | |
| Sodium Bicarbonate | JT-Baker | 3506-1 | |
| Sodium Pyruvate | Gibco | 11360-070 | |
| Ultrapure distilled water | Invitrogen | 10977-015 | |
| Preparation of mRNA encoding YFP/3' UTR and mCherry | |||
| Agarose | Apex Biomedical | 20-102QD | |
| Carbenicillin disodium salt | Sigma-Aldrich | C1389-1G | |
| Choo-Choo Cloning Kit | McLab | CCK-20 | |
| CutSmart Buffer (10x) | New England Biolabs | B7204 | |
| DNA loading dye (6x) | Thermo Scientific | R0611 | |
| dNTP Solution | New England Biolabs | N0447S | |
| DpnI | New England Biolabs | R0176 | |
| GeneRuler 1 kb DNA ladder | Thermo Fisher | SM1333 | |
| LB Agar Plates with 100 µg/mL Carbenicillin, Teknova | Teknova | L1010 | |
| LB Medium (Capsules) | MP Biomedicals | 3002-021 | |
| MEGAclear Transcription Clean-Up Kit | Life Technologies | AM1908 | |
| MfeI-HF restriction enzyme | New England Biolabs | R3589 | |
| mMESSAGE mMACHINE T7 Transcription Kit | Invitrogen | AM1344 | |
| Phusion High Fidelity DNA polymerase | New England Biolabs | M0530 | |
| Poly(A) Tailing kit | Invitrogen | AM1350 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | |
| S.O.C. medium | Thermo Fisher | 15544034 | |
| TAE buffer | Apex Biomedical | 20-193 | |
| Ultrapure Ethidium Bromide Solution | Life Technologies | 15585011 | |
| Oocyte collection | |||
| Aspirator tube assembly for calibrated micro-pipettes | Sigma-Aldrich | A5177-5EA | |
| Calibrated micro-pipettes | Drummond Scientific Company | 2-000-025 | |
| PMSG- 5000 | Mybiosource | MBS142665 | |
| PrecisionGlide Needle 26 G x 1/2 | BD | 305111 | |
| Syringe 1 ml | BD | 309659 | |
| Oocyte micro-injection | |||
| 35 mm Dish | No. 0 Coverslip | 20 mm Glass Diameter | Uncoated | MatTek | P35G-0-20-C | For time-lapse microscopy |
| Borosilicate glass with filament | Sutter Instrument | BF100-78-10 | |
| Oil for Embryo Culture | Irvine Scientific | 9305 | |
| Petri Dish | Falcon | 351006 | For micro-injection |
| Tissue Culture Dish | Falcon | 353001 | For oocyte incubation |
| VacuTip Holding Capillary | Eppendorf | 5195000036 | |
| Software | |||
| Biorender | BioRender | Preparation of Figure 1S | |
| MetaMorph, version 7.8.13.0 | Molecular Devices | For time-lapse microscopy, analysis of 3' UTR translation |
Tags
Citar este artigo
The In Vitro Single Oocyte Reporter Assay: A Technique to Study Target mRNA Translation Regulation During In Vitro Oocyte Maturation. J. Vis. Exp. (Pending Publication), e21237, doi: (2023).