施肥爪蟾卵母细胞使用主机传输方法
Summary
对施肥的程序<em>爪蟾卵母细胞</em由主机传输方法。
Abstract
往往阻碍了必要的时间和费用，产生产妇效应突变动物研究母系遗传的分子脊椎动物的早期发展的贡献。此外，许多技术如爪蟾和斑马鱼的生物过表达或抑制基因功能未能充分针对危重孕产妇的信号转导通路，如Wnt信号。在非洲爪蟾 ，在培养的卵母细胞的基因功能和操纵，随后施肥可以在一定程度上改善这些问题。手动defolliculated卵母细胞是从供体卵巢组织，注射或视为文化，然后用黄体酮刺激诱导成熟。下一步，卵母细胞引入腔体内的雌蛙排卵主机，届时他们将通过主机的输卵管移位和掌握修改和果冻大衣施肥必要。由此产生的胚胎，然后将筹集到所需的阶段和任何实验扰动的影响分析。这台主机传输方法已发现早期发展的基本机制非常有效，并没有在任何其他脊椎动物模式生物的实验可能允许广泛。
Protocol
Discussion
一个成功的转移，将导致受精和正常的卵母细胞> 50％（图3）裂解。过去一般在30-60％的转让的卵母细胞存活的神经胚阶段。我们通常是75-150％实验组的卵母细胞，这将产生几种类型的分析足够的胚胎（原位RT – PCR）以及可视化的表型转移。植入大幅更多的卵母细胞似乎并不大大提高产量。此外，应至少有30％组的卵母细胞转移到保证，至少有一些通过原肠。重要的染料一般不影响这里描述的浓?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者想感谢休斯顿实验室的关键读的手稿成员。研究是支持的美国国立卫生研究院授予DWH（GM083999）
Materials
Solutions and recipes:
OCM (Oocyte culture medium; modified from Wylie et al., 1996)
| 70% Leibovitz’s L-15 medium (containing l-glutamine) | (Invitrogen 11415-064) |
| 0.4 mg/ml Bovine serum albumin (BSA; fraction V) | (Sigma A-9418-50g) |
| 1x Penicillin-Streptomycin | (Sigma P-0781) |
| Adjust to pH 7.6-7.8 with 5N NaOH | |
| Make fresh weekly, store at 18°C. | |
10X MMR (Marc’s Modified Ringer’s Solution; Zuck et al., 1998)
| 1M NaCl |
| 20 mM KCl |
| 20 mM CaCl2 |
| 10 mM MgCl2 |
| 150 mM HEPES |
| adjust to pH 7.6-7.8 |
| Store at 4°C |
Anesthetic solution
| 1 g/L 3-Aminobenzoic Acid Ethyl Ester (MS222) | (Sigma A-5040) |
| 0.7 g/L sodium bicarbonate | |
| Dissolve in Amquel-treated water | |
| Make fresh weekly | |
Progesterone stocks
| 10 mM Progesterone (Sigma P- 0130) in 100% Ethanol |
| Dilute to 1 mM in 100% EtOH for a 500x stock/working solution |
| Store at -20°C |
Vital dyes (modified from Heasman et al., 1991)
| Blue: 0.1% Nile Blue A | (Aldrich 121479-5G), |
| Red: 0.25% Neutral Red | (Sigma N-6634), |
| Brown: 1% Bismarck Brown | (Sigma B-2759), |
| Green (80 μl blue + 80 μl brown), | |
| Mauve (80 μl blue + 80 μl red). | |
| A sixth color, Orange (80 μl red+ 80 μl brown), can also be used but is often hard to distinguish from brown. | |
Stocks of Blue, Red and Brown are made up in deionized water in 50 ml tubes, incubated for 20 minutes with rocking and spun in a clinical centrifuge. The supernatants are aliquotted into microfuge tubes and stored at -20°C.
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