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

Play Video

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).

View Video