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Mouse Oocyte Isolation, Cultivation and RNA Microinjection

小鼠卵母细胞分离、培养和RNA显微注射

Anna Tetkova Anna Tetkova
Marketa Hancova Marketa Hancova

发布: 2016年02月05日第6卷第3期 DOI: 10.21769/BioProtoc.1729 浏览次数: 16834

评审: Anonymous reviewer(s)

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Abstract

Mammalian oocyte is a highly specialized cell, characterized by synthesis and storage of maternal proteins and RNAs that contributes to the meiotic cell cycle and early embryo development. The fully grown oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during the growing phase. Mouse oocytes are often used as a mammalian model for the study of molecular biology of the cell or biomedical research. Microinjection technique is a useful tool to deliver RNA coding for fluorescently tagged proteins to determine their subcellular localization or function, delivering biosensors for the study of various metabolic pathways or downregulation of specific targets by RNAi or oligo morpholinos to study gene function. Here, we describe a protocol for isolation, cultivation and microinjection of oocytes that might contribute to research or educational purposes.

Keywords: Oocyte (oocyte) search Microinjection (显微注射) search RNA (RNA) search Meiosis (减数) search

Materials and Reagents

  1. Petri dishes (90 mm) (GAMA GROUP, catalog number: 400974 )
  2. Needles Omnifix F Duo (B. Braun Melsungen AG, catalog number: 9161465V )
  3. Petri dishes 3 ml, 8.8 cm2 (Thermo Fisher Scientific, catalog number: 153066 )
  4. Holding micropipette (Microtech IVF, catalog number: 001-120-30 )
  5. Borosilicate Thin Wall with Filament, 1.0 mm OD 0.78 mm ID, 150 mml (Harvard Apparatus, catalog number: 300039 )
  6. Microloader TM, tip for filling Femtotips and other glass microcapillaries, Sterile, 0.5-20 µl, 100 mm (Eppendorf, catalog number: 5242956003 )
  7. Cultivation medium M16 (Merck Millipore Corporation, catalog number: MR016D )
  8. 4 well cell culture plate (SPLLIFESCIENCES, catalog number: 30004 )
  9. µ-Slide 4 Well Glass Bottom (Ibidi, catalog number: 80427 )
  10. Nunc Lab-Tek II Chamber Slide System (Thermo Fisher Scientific, catalog number: 154534 )
  11. Capillaries for oocyte manipulation with tip 100 µm in diameter
  12. mMESSAGE mMACHINE Kit (Life Technologies, catalog number: AM1344 )
    Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM1344”.
  13. Stimulated mouse (Mus musculus, CD1) at least 6 weeks old; stimulation via pregnant mare serum gonadotropin (PMSG)-Folligon (MSD Animal Health) and human chorionic gonadotropin (hCG) (Sigma-Aldrich)
  14. 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: 28822584 )
  15. Poly(A) Tailing Kit (Life Technologies, catalog number: AM1350 )
    Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM1350”.
  16. RNeasy Mini Kit (QIAGEN, catalog number: 74104 )
  17. Mineral oil (Sigma-Aldrich, catalog number: M8410 )
  18. RNase-free water (Life Technologies, Ambion®, catalog number: AM9932 )
    Note: Currently, it is “Thermo Fisher Scientific, Ambion™, catalog number: AM9932”.
  19. Dyes for monitoring fluid injection into oocyte,
    1. Fast Green (Sigma-Aldrich, catalog number: F1252 )
    2. Tetramethylrhodamine isothiocyanate-Dextran (Sigma-Aldrich, catalog number: T1287 ) 
  20. NaCl
  21. KCl
  22. CaCl2.2H2O
  23. KH2PO4
  24. MgSO4.7H2O
  25. Glucose
  26. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
  27. Polyvinyl alcohol (PVA)
  28. Destilated water
  29. Bovine serum albumin (BSA)
  30. Transfer medium (see Recipes)

Equipment

  1. Incubator Hera Cell 150 (Heraeus Holding)
  2. Stereo microscope Stemi 2000 (ZEISS)
  3. NanoDrop ND-1000 (Thermo Fisher Scientific)
  4. Centrifuge 5418 (Eppendorf)
  5. Pulling capillaries (Sutter Instrument Company, model: P-97 )
  6. Microforge for bending capillaries (NARISHIGE Group, model: MF-79 )
  7. Inverted microscope (OLYMPUS, model: CKX41 and Leica, model: DMI 6000B )
  8. Channel Pressure Injector (MicroData Instrument, model: PM2000B 4 )
  9. Joystick MIS-5000 Series Microinjection Manipulation Systems (Burleigh)
  10. Pressurized nitrogen gas or FemtoJet (Eppendorf)
  11. Confocal microscope (Leica, model: SP5 )
  12. EMBL stage incubator
  13. Water corrected objectives HCX PL APO 20x/0.7 IMM CORR λBL and HCX PL APO 40/1.1

Software

  1. Image J (http://rsbweb.nih.gov/ij)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Tetkova, A. and Hancova, M. (2016). Mouse Oocyte Isolation, Cultivation and RNA Microinjection. Bio-protocol 6(3): e1729. DOI: 10.21769/BioProtoc.1729.

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分类

细胞生物学 > 细胞分离和培养 > 细胞分离

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