We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.
Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.
非洲爪蟾是一种广泛使用的,功能强大的模式生物研究开发1。这是因为, 爪蟾卵是非常大(约1.2-1.4毫米,相较于哺乳动物对口为约0.1毫米)和丰富。鸡蛋中含有母体合成和存储组件,这是足以驱动胚胎发育至中期囊胚转换(MBT,在舞台8-8.5发生与4,000-8,000细胞)。 MBT伴随合子基因的激活,然后产生了直接的进一步发展胚胎的基因产物。
许多研究旨在确定为发展的重要母体因素。许多研究依靠注射反义寡核苷酸(寡)包括吗啉代寡核苷酸为受精胚胎,其中产妇蛋白质的降解情况下,可在原肠期2-4被观察到。可替代地,的mRNA注射到受精的青霉bryos干扰基因的功能或跟踪过表达的蛋白质的命运。然而,注射入单细胞阶段的胚胎通常不影响母体蛋白质的表达水平在MBT前非常早期胚胎阶段。
Heasman和怀利建立主机传输方法来克服这个问题,5。在他们的方法中,手动defolliculated卵母细胞被注射以反义寡聚物,并转移到宿主雌性6。蛋白质受精之前下调,使早期胚胎发育下调蛋白的作用可以检查。该产妇消耗的方法导致产妇蛋白质的几个独特的发展角色的身份,在7审查。
在这份报告中,我们详细介绍我们最近开发的方法,其中受精前母体消耗mRNA的表达,或在没有人工defolliculation和主机传输实现8。手动defolliculation需要大量的时间和主机传输往往需要熟练的技术和动物手术特定许可证,从而妨碍了经常使用的产妇消耗的方法。 Defolliculation和主机传输是通过酶defolliculation 9,10和胞浆内单精子注射(ICSI)11-13,分别取代。 ICSI到鸡蛋最初用于产生转基因青蛙12。精子和胚胎细胞核也移植到体外成熟的卵母细胞的两栖11,14,15。这里,我们显示了一步一步方法注入精子在体外成熟的卵母细胞即进行预注射反义寡聚物或mRNA。
我们在这里详细的新方法来消耗母体因素或过表达外源性因素受精前。该系统需要两个显微注射,而是跳过青蛙的手术,在主机传送方法7,17使用。它是最佳的以除去蛙手术步骤在动物护理方面。此外,我们也没有考虑到对主机雌蛙的转移实验的质量,这意味着我们可以摆脱影响的实验的成功的一个生物因素。因此,在这个系统中,从PMSG打底的蛙卵母细胞得到的质量是一个重要的生物因子是关键成功实验。卵母细胞的质量可以收集卵巢或之后defolliculation后进行判断。如果有任何异常时,在这些阶段观察到的,这是最佳的,从一个新的蛙收集另一个卵巢。当你可以检查卵母细胞质量的另一点是卵母细胞成熟后。如果小于80%的孕激素电子处理卵母细胞成熟表现的迹象,你可能无法获得足够数量的胚胎进行进一步的分析。使用酶促defolliculation代替手工defolliculation也是使用这种系统,以减少所需的defolliculation劳动的另一优点。然而,它仍然是可能的,手动defolliculation可以给出比酶处理更好的发展。
如图3中 , 在体外几乎10%的成熟卵母细胞可到达游泳蝌蚪阶段。这些数据是从13个独立实验包括那些报告在8和新注射实验收集。主机传输方法需要在每个治疗获得有意义的数据,因为转移的卵母细胞的30-60%,可以达到神经胚阶段,17 75-150卵母细胞。我们的方法通常与200-300的卵母细胞中各实验组启动自裂解的胚胎的约40-60%可达到肌肉反应的阶段。这些数据表明,这两种方法都支持一个合理的发育率。到目前为止,我们已获得来自对照的mRNA注射的和控制的反义寡 – 注射的卵母细胞用这种技术10活着青蛙,表明该方法支持开发通过变态。
我们的卵母细胞操纵ICSI方法提供了一个机会,很早期胚胎发育过程中,测试的母体因素的作用下,受精后不久。此外,染色质修饰因子的受精前过表达使得可以重塑染色质母体受精前对理解所必需的开发产妇染色质状态。这一战略也可能会与目前的基因编辑系统,如转录激活因子样效应核酸酶(TALEN)18,19和CRISPR / CAS9 20,21因为这些即使受精前可表现正常工作。因此,我们的新方法有势的人要用于在未来的许多应用。
The authors have nothing to disclose.
We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Pregnant Mare Serum Gonadotropin (PMSG) | MSD Animal Health | Vm 01708/4309 | PMSG-Intervet 5000iu powder and solvent for solution for injection |
Ethyl 3-aminobenzoate methanesulfonate salt (MS222) | Sigma | A5040 | For anesthesia |
Liberase TM Research Grade | Roche | 05 401 127 001 | For oocyte defolliculation. Store at -20oC |
Drummond Nanoject | Drummond Scientific Company | 3-000-205/206 | For microinjection |
glass capillary | Alpha laboratories | 7” Drummond #3-000-203-G/XL | For microinjection |
Micropipette Puller | SUTTER Instrument | Model D-97 | For microinjection |
UltraPure Agarose | Invitrogen | 16500-500 | For invitro maturation and ICSI |
14 ml ultra-clear centrifuge tube | Beckman Coulter United Kingdom Ltd | 344060 | For sperm purification |
OptiPrep Density Gradient Medium (Iodixanol) | Sigma | D1556 | For sperm purification |
Digitonin | Sigma | D141 | Cell permeabilization reagent |
Shaker | Hybaid | HB-SHK-1 | For oocyte defolliculation. |
Dissecting microscope (Stereo zoom microscope) | ZEISS | Semi SV6 | For oocyte collection and microinjection |
50 μm pore filter | CellTrics | 04-0042-2317 | For sperm purification |
Ultracentrifuge | Beckman Coulter | Optima L-100XP | For sperm purification |
50 ml centrifuge tube | Cellstar Greiner Bio-One | 227261 or 210261 | Oocyte collection and defolliculation reaction |
15 ml centrifuge tube | FALCON | 352097 | For sperm purification |
90 mm Petri dish | Thermo Scientific | 101VR20/C | |
Easy-Grip Cell Culture Dish, 60×15 mm | FALCON | 353004 | |
Easy-Grip Cell Culture Dish, 35×15 mm | FALCON | 351008 |