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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   

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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), Microinjection(显微注射), RNA(RNA), Meiosis(减数)

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

  1. Preparation of RNA/morpholinos
    1. Perform the in vitro RNA transcription of sequence of your interest (your RNA, or morpholino oligo for blocking selected specific RNA targets) using mMESSAGE mMACHINE Kit and add poly(A) tail with Poly(A) Tailing Kit.
    2. Purify the RNA sample by RNeasy Mini Kit.
    3. Measure RNA concentration by NanoDrop.
    4. Store the RNA in -80 °C before usage.
    5. Prepare working solution with a 20-50 ng/µl concentration of RNA or 1-10 µM concentration of morpholino. Dilute in RNase free water. Important: Centrifuge for 5 min/17,000 x g and use supernatant only to get rid of particles which then might seal the tip of micropipette.
    6. Keep working solution on ice.

  2. Micropipette preparation
    1. Set parameters on micropipette puller: heat 300, pull 80, velocity 70, time 150.
    2. Fix the capillary into the groove, pull firmly the holder, close the cover and press PULL. Thus you get the capillary with thin end suitable for microinjection (see Video 1).
    3. Then fix the capillary into the manipulator MF-79. Magnify and focus the end of the capillary and close it to the hot fiber which bends the capillary into the proper angle, approximately 30 degrees (see Video 1).
    4. Fill the prepared injection capillary with working solution (approx 1.5 µl) using microloader tip.

      Video 1. Micropipette preparation

  3. Oocyte isolation and cultivation
    1. Obtain mouse ovaries from laboratory mice (Mus musculus, CD1) at least 6 weeks old (from 6 to 10 weeks old), which were stimulated by Folligon (PMSG; 5 IU per one mouse) 46 h before collection to get oocytes in the stage of germinal vesicle (GV stage). For zygotes, administer hCG (5 IU per one mouse) 48 h after PMSG and mate stimulated females with males. Isolate zygotes 17 h after mating.
    2. Euthanize the mice by cervical dislocation. Cervical dislocation is the recommended method of mice euthanasia due to its speed and reliability (of course after the acquisition of proper technique). Moreover any chemicals that could potentially affect the experiment are not added.
    3. Remove the ovaries and clean them from the fat tissue.
    4. Isolate oocytes immediately after collection ovaries using two needles: disrupt the follicles in transfer medium (TM) supplemented with IBMX (100 µM) (see Video 2).
    5. Transfer isolated oocytes into the cultivation dish with M16 medium with IBMX (100 µM) and incubate at 37.5 °C, 5% CO2. The dishes with medium have to be equilibrated before for at least 2 h in 37.5 °C, 5% CO2.
    6. After 15 min in the incubator, denude the oocytes by gentle pipetting (suction and discharge several times) the medium in the well. The pipetting will mechanically remove cumulus cells from the oocyte. Then let the oocytes in the incubator for other 15 min.
    7. Then select the best oocytes visually (rounded, nucleolus in the middle, fully grown oocytes ~ 70 µm in diameter; Figure 1).

      Video 2. Oocyte isolation


      Figure 1. Comparison of good and bad oocytes. Green arrow marks a good oocyte. Scale bar 40 µm

  4. Microinjection
    1. Turn on the microscope, microinjector, joystick and the gas (pressure up to 500 kPa). Set the injection pressure: FILL 0.1 psi, INJ 10.1 psi, BAL 2.9 psi, HOLD 14.5 psi.
    2. Put the holding capillary and the loaded injection capillary to the relevant holders and close them slowly to the petri dish with a drop of TM + IBMX and the oocytes.
    3. Then immediately inject: Hold the oocyte using the holding capillary (holding capillary creates a negative pressure by which the oocyte is held) and inject ~5 pl of your working solution from the injection capillary into oocyte, not close to the nucleus (Figure 2).
    4. Transfer oocytes with constructs to incubator in M16 + IBMX.
    5. To verify the success of the microinjection use fluorescent microscopy.
    6. See also Video 3.


      Figure 2. Microinjection. Inject working solution not close to the nucleus.

      Video 3. Microinjection

  5. Live-cell imaging
    1. 1-2 h after microinjection wash the oocytes from IBMX in TM and then in M16.
    2. Transfer the oocytes into 2 µl of M16 covered with oil in Lab-tek Chamber Slide System. The dish with medium has to be equilibrated in 37.5 °C, 5% CO2. Raised coverslip is not suitable. We also use dishes from Ibidi (No. 1.5 H), similar to Labtek. Or, for cell culture (not for oocytes) you can choose for example a dish from µ-Slide I Luer Family.
    3. Place your sample in the confocal microscope equipped with EMBL stage incubator (37.5 °C, 5% CO2). Be sure that the incubator is closed.
    4. Turn on the appropriate lasers and set the software for video capturing (image each 5-15 min). We usually let the mouse oocytes cultivate overnight, but cultivations of embryos over a week long were published, depending on the chemistry of respective dyes, bleaching etc.
    5. Assembly the movie using Image J (http://rsbweb.nih.gov/ij).
    6. See also Figure 3 and Video 4 showing visualization of meiotic maturation of mouse oocyte using H2B-GFP mRNA.


      Figure 3. Visualization of meiosis in mouse oocyte. Visualization using microinjected H2B-GFP (green). A. Fully grown oocyte in GV stage. B. Oocyte after nuclear envelope breakdown (NEBD) and typical chromosomal spread. C. Oocyte in metaphase I (MI) stage. D. Oocyte in anaphase I. E. Oocyte after first meiotic division with extruded first polar body.

      Video 4. Mouse oocytes during meiotic maturation with microinjected H2B-GFP. Control oocytes are not microinjected (yellow arrows), H2B-GFP is shown in green. Growing oocyte (blue arrow) has no meiotic competence and remains in GV stage.

Recipes

  1. Transfer medium
    NaCl 12.8 g
    KCl 0.8 g
    CaCl2.2H2O 0.6 g
    KH2PO4 0.14 g
    MgSO4.7H2O 0.194 g
    Glucose 4 g
    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 4 g
    Polyvinyl alcohol (PVA) 2 g
    Distilled water 2 L
    Bovine serum albumin (BSA) 5 g

Acknowledgments

This work was supported by research grant GACR 13-12291S to Andrej Susor. The original work was published in Karabinova et al., (2011) and Susor et al., (2015). Thanks to colleagues from Laboratory of Biochemistry and Molecular Biology of Germ Cells for support.

References

  1. Gagnon, J. A. and Mowry, K. L. (2010). Visualizing RNA localization in Xenopus oocytes. J Vis Exp (35): e1704.
  2. Karabinova, P., Kubelka, M. and Susor, A. (2011). Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals. Cell Tissue Res 346(1): 1-9.
  3. Layden, M. J., Rottinger, E., Wolenski, F. S., Gilmore, T. D. and Martindale, M. Q. (2013). Microinjection of mRNA or morpholinos for reverse genetic analysis in the starlet sea anemone, Nematostella vectensis. Nat Protoc 8(5): 924-934.
  4. Susor, A., Jansova, D., Cerna, R., Danylevska, A., Anger, M., Toralova, T., Malik, R., Supolikova, J., Cook, M. S., Oh, J. S. and Kubelka, M. (2015). Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR-eIF4F pathway. Nat Commun 6: 6078.
  5. Yuan, S. and Sun, Z. (2009). Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J Vis Exp (27): e1113.

简介

哺乳动物卵母细胞是高度特化的细胞,其特征在于合成和储存母体蛋白质和RNA,其有助于减数分裂细胞周期和早期胚胎发育。 完全生长的卵母细胞是转录性静止的,并且仅利用在生长期期间合成和储存的转录物。 小鼠卵母细胞通常用作哺乳动物模型用于研究细胞的分子生物学或生物医学研究。 显微注射技术是递送编码荧光标记蛋白的RNA以确定其亚细胞定位或功能,递送生物传感器用于研究各种代谢途径或通过RNAi或寡聚morpholinos下调特定靶标以研究基因功能的有用工具。 在这里,我们描述了可能有助于研究或教育目的的卵母细胞的分离,培养和显微注射的协议。

关键字:oocyte, 显微注射, RNA, 减数

材料和试剂

  1. 培养皿(90mm)(GAMA GROUP,目录号:400974)
  2. Needles Omnifix F Duo(B.Braun Melsungen AG,目录号:9161465V)
  3. 培养皿3ml,8.8cm 2(Thermo Fisher Scientific,目录号:153066)
  4. 保持微量移液管(Microtech IVF,目录号:001-120-30)
  5. 具有长丝的硼硅酸盐薄壁,1.0mm OD 0.78mm ID,150mml(Harvard Apparatus,目录号:300039)
  6. Microloader TM,用于填充Femtotips和其他玻璃微毛细管的尖端,Sterile,0.5-20μl,100mm(Eppendorf,目录号:5242956003)
  7. 培养基M16(Merck Millipore Corporation,目录号:MR016D)
  8. 4孔细胞培养板(SPLLIFESCIENCES,目录号:30004)
  9. μ-Slide 4井玻璃底部(Ibidi,目录号:80427)
  10. Nunc Lab-Tek II室滑动系统(Thermo Fisher Scientific,目录号:154534)
  11. 用于卵母细胞操作的毛细管,尖端直径为100μm
  12. mMESSAGE mMACHINE Kit(Life Technologies,目录号:AM1344)
    注意:目前,它是"Thermo Fisher Scientific,Ambion?,目录号:
  13. 至少6周龄的刺激小鼠( Mus musculus ,CD1)通过怀孕母马血清促性腺激素(PMSG)-Folligon(MSD动物健康)和人绒毛膜促性腺激素(hCG)(Sigma-Aldrich)刺激。
  14. 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma-Aldrich,目录号:28822584)
  15. Poly(A)尾部套件(Life Technologies,目录号:AM1350)
    注意:目前,它是"Thermo Fisher Scientific,Ambion?,目录号: AM1350 "。
  16. RNeasy迷你包(QIAGEN,目录号:74104)
  17. 矿物油(Sigma-Aldrich,目录号:M8410)
  18. 无RNase的水(Life Technologies,Ambion ,目录号:AM9932)
    注意:目前,它是"Thermo Fisher Scientific,Ambion?,目录号:
  19. 用于监测液体注入卵母细胞的染料,
    1. Fast Green(Sigma-Aldrich,目录号:F1252)
    2. 四甲基罗丹明异硫氰酸酯 - 葡聚糖(Sigma-Aldrich,目录号:T1287) 设备

      1. 孵化器Hera Cell 150(Heraeus Holding)
      2. 立体显微镜Stemi 2000(ZEISS)
      3. NanoDrop ND-1000(Thermo Fisher Scientific)
      4. 离心机5418(Eppendorf)
      5. 拉拔毛细管(Sutter Instrument Company,型号:P-97)
      6. 用于弯曲毛细管的微型锻造(NARISHIGE Group,型号:MF-79)
      7. 倒置显微镜(OLYMPUS,型号:CKX41和Leica,型号:DMI 6000B)
      8. 通道压力注射器(MicroData仪器,型号:PM2000B 4)
      9. 操纵杆MIS-5000系列显微注射操作系统(Burleigh)
      10. 加压氮气或FemtoJet(Eppendorf)
      11. 共焦显微镜(Leica,型号:SP5)
      12. EMBL阶段培养箱
      13. 水校正目标HCX PL APO 20x/0.7 IMM CORRλBL和HCX PL APO 40/1.1

      软件

      1. 图片J( http://rsbweb.nih.gov/ij

      程序

      1. 制备RNA/morpholinos
        1. 使用mMESSAGE mMACHINE Kit进行您感兴趣的序列(您的RNA或吗啉代寡核苷酸用于阻断选定的特定RNA靶标)的体外 RNA转录,并添加Poly(A) 。
        2. 通过RNeasy Mini Kit纯化RNA样品
        3. 通过NanoDrop测量RNA浓度
        4. 使用前将RNA储存于-80℃。
        5. 准备工作溶液与20-50 ng /μl浓度的RNA或1-10μM浓度的吗啉代。在无RNA酶的水中稀释。重要:离心5分钟/17,000×g,并且使用上清液仅除去可能密封微量移液管吸头的颗粒。
        6. 将工作溶液保持在冰上。

      2. 微量移液器准备
        1. 设置微量移液器拉杆上的参数:加热300,拉80,速度70,时间150.
        2. 将毛细管固定到凹槽中,用力拉动支架,关闭盖子并按下拉杆。因此,您可以得到适合显微注射的薄端毛细管(见视频1)。
        3. 然后将毛细管固定到机械手MF-79。放大并聚焦毛细管的端部,并将其关闭到热纤维,将毛细管弯曲到适当的角度,大约30度(见视频1)。
        4. 使用微量加样器吸头向准备好的进样毛细管注入工作溶液(约1.5μl)
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          视频1.微量移液器准备
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      3. 卵母细胞分离和培养
        1. 从至少6周龄(6至10周龄)的实验室小鼠(小鼠肌肉,CD1)获得小鼠卵巢,其由Folligon(PMSG;每只小鼠5IU)刺激46小时收集在胚泡(GV阶段)阶段获得卵母细胞。对于受精卵,在PMSG后48小时施用hCG(每只小鼠5IU),并且用男性施用配偶刺激的雌性。分离合子17小时后交配
        2. 安乐死小鼠颈椎脱位。颈部脱位是小鼠安乐死的推荐方法,因为其速度和可靠性(当然在获得适当的技术后)。此外,不会添加任何可能影响实验的化学物质。
        3. 取出卵巢并清除脂肪组织。
        4. 使用两个针在收集卵巢后立即分离卵母细胞:破坏补充有IBMX(100μM)的转移培养基(TM)中的卵泡(参见视频2)。
        5. 将分离的卵母细胞转移到具有IBMX(100μM)的M16培养基的培养皿中,并在37.5℃,5%CO 2下孵育。具有培养基的培养皿必须在37.5℃,5%CO 2中之前平衡至少2小时。
        6. 在孵育器中15分钟后,通过轻轻吹打(吸入和排出数次)孔中的培养基来破坏卵母细胞。移液将机械地从卵母细胞去除卵丘细胞。然后让孵化器中的卵母细胞再培养15分钟。
        7. 然后选择最好的卵母细胞视觉(圆形,核仁在中间,完全生长的卵母细胞直径约70微米;图1)。

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          视频2.卵母细胞隔离
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          图1.良好和不良卵母细胞的比较。绿色箭头表示良好的卵母细胞。比例尺40μm

      4. 微量注射
        1. 打开显微镜,显微注射器,操纵杆和气体(压力高达500 kPa)。设置注射压力:FILL 0.1 psi,INJ 10.1 psi,BAL 2.9 psi,保持14.5 psi
        2. 将保持毛细管和加载的注射毛细管放入相关的持有人,并用一滴TM + IBMX和卵母细胞慢慢关闭到培养皿。
        3. 然后立即注射:使用保持毛细管(保持毛细管产生负压保持卵母细胞)保持卵母细胞,并注射?5 pl的工作溶液从注射毛细管卵母细胞,不靠近细胞核(图2) 。
        4. 转移卵母细胞与构造到孵化器在M16 + IBMX
        5. 验证显微注射的成功使用荧光显微镜
        6. 参见视频3.


          图2.显微注射。注射工作溶液不靠近细胞核。

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          视频3.微注射
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      5. 活细胞成像
        1. 显微注射后1-2小时从TMX中的IBMX洗涤卵母细胞,然后在M16中洗涤
        2. 将卵母细胞转移到2微升M16覆盖油在Lab-tek室幻灯片系统。具有培养基的培养皿必须在37.5℃,5%CO 2中平衡。抬高的盖玻片不合适。我们也使用来自Ibidi的菜(1.5H),类似于Labtek。或者,对于细胞培养(不用于卵母细胞),您可以选择例如μ-Slide I Luer Family的培养皿。
        3. 将您的样品在配备有EMBL阶段培养箱(37.5℃,5%CO 2)的共聚焦显微镜。确保孵化器关闭。
        4. 打开相应的激光器并设置视频捕获软件(图像每5-15分钟)。我们通常让小鼠卵母细胞培养过夜,但是根据各染料的化学性质,漂白等,公布了一个星期以上的胚胎培养。。
        5. 使用Image J汇编影片( http://rsbweb.nih.gov/ij )。
        6. 参见图3和视频4,显示使用H2B-GFP mRNA的小鼠卵母细胞减数分裂成熟的可视化

          图3.小鼠卵母细胞减数分裂的可视化。使用显微注射的H2B-GFP(绿色)进行可视化。 A.在GV阶段完全成熟的卵母细胞。 B.核细胞包膜破坏(NEBD)和典型的染色体扩散后的卵母细胞。 C.卵母细胞在中期I(MI)阶段。 D.卵母细胞在后期I. E.卵母细胞在第一次减数分裂后挤出第一极体
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          视频4.小鼠卵母细胞在减数分裂成熟与显微注射H2B-GFP。 对照卵母细胞未显微注射(黄色箭头),显示H2B-GFP 在绿色。生长卵母细胞(蓝色箭头)没有减数分裂能力和 保持在GV阶段。
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      食谱

      1. 传输介质
        NaCl 12.8g
        KCl 0.8g
        CaCl 2 2 2H 2 O 0.6g

        MgSO 4 .0.747g。</b> 7H 2 O 0.194g< br / Glukose 4 g
        4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)4g / 聚乙烯醇(PVA)2 g
        蒸馏水2 L
        牛血清白蛋白(BSA)5g

      致谢

      这项工作是由研究基金GACR 13-12291S支持Andrej Susor。原始的工作发表在Karabinova等人(2011)和Susor等人(2015)。感谢生物化学和分子生物学实验室的同事支持。

      参考文献

      1. Gagnon,J.A。和Mowry,K.L。(2010)。 在"Xenopus卵母细胞"中可视化RNA定位。 J Vis Exp (35):e1704。
      2. Karabinova,P.,Kubelka,M。和Susor,A。(2011)。 哺乳动物卵母细胞减数分裂和受精中泛素化蛋白的蛋白酶体降解。细胞Tissue Res 346(1):1-9。
      3. Layden,M.J.,Rottinger,E.,Wolenski,F.S.,Gilmore,T.D.and Martindale,M.Q.(2013)。 在小星海葵中显微注射mRNA或morpholinos用于反向遗传分析,Nematostella vectensis/em> Nat Protoc 8(5):924-934。
      4. Susor,A.,Jansova,D.,Cerna,R.,Danylevska,A.,Anger,M.,Toralova,T.,Malik,R.,Supolikova,J.,Cook,MS,Oh,JSand Kubelka, (2015年)。 通过mTOR-eIF4F通路在哺乳动物卵母细胞中翻译的时空和空间调节。 Nat Commun 6:6078.
      5. Yuan,S.and Sun,Z.(2009)。 在斑马鱼胚胎中显微注射mRNA和吗啉代反义寡核苷酸。 (27):e1113。
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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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