What Was Observed? (Introduction)

• Xenopus laevis is a type of frog often used in scientific studies about how embryos develop and regenerate.
• Researchers wanted to find a way to control when and where genes are activated during development, without causing unwanted side effects.
• Injecting mRNA (the molecule that helps make proteins) into early-stage embryos can cause problems because it can turn on genes at the wrong time and place.
• To solve this, scientists used electroporation to deliver mRNA to specific cells at specific times during development. Electroporation uses electric pulses to help mRNA enter cells.
• The method works on embryos at the gastrula-to-tailbud stages, which is the part of development where important structures start to form.
• By using this method, scientists can study gene function with more precision than traditional injections.

What is Electroporation?

• Electroporation is a technique that uses electrical pulses to make holes in the cell membrane, allowing substances like mRNA to enter cells.
• This method is useful because it can target specific cells or regions without affecting the whole embryo.
• Electroporation can deliver mRNA into cells at any stage of development, providing better control over when genes are activated.

Materials Needed

• Marc’s Modified Ringer’s (MMR) solution: Used to maintain a healthy environment for the embryos.
• mRNA encoding a protein of interest: This is the genetic material that will be introduced into the embryos.
• Tricaine Methanesulfonate (MS222): Used to anesthetize the embryos to prevent movement during the procedure.
• Agarose solution (1% in MMR): To create a stable surface where the embryos can be held during electroporation.
• Injection needles: Used to inject mRNA into the embryos.
• Electroporation chamber: Equipment used to apply electrical pulses to the embryos.

Preparing the Electroporation Setup

• Cover the bottom of a dish with parafilm to prevent leakage of liquids.
• Pour 1% agarose into the dish and let it cool to form a solid bed.
• Create small pockets in the agarose using rubber dimples or micropipette tips to hold the embryos in place during the procedure.
• While the agarose is cooling, set up the micromanipulator (used to position the injection needle) and electroporator (used to apply the electrical pulses).
• Prepare the injection needle and fill it with mRNA solution. Use a micromanipulator to control the needle and inject a small amount of mRNA into the target cells.
• Prepare the electroporator for the next step, ensuring it is ready to apply the correct electrical pulses.

Determining Electroporation Parameters

• Measure the resistance of the solution to determine the correct strength and duration of the electrical pulses.
• Based on the resistance, adjust the electroporation parameters (voltage and pulse length) to achieve efficient mRNA delivery.
• Use pre-set values to make adjustments easily based on the measured resistance.

Injecting and Electroporating the Embryos

• Anesthetize the embryos using MS222 to prevent movement during the procedure.
• Place the embryos in the agarose bed pockets and inject them with a small volume of mRNA solution (10-20 nl per embryo).
• After injection, apply the electrical pulses to help the mRNA enter the cells. This is done by positioning the cathode close to the injection site and applying the appropriate pulses.
• Repeat the injection and electroporation process for each embryo, ensuring they are all treated with the same method.
• After electroporation, transfer the embryos to fresh MMR solution and allow them to recover for one hour.
• Transfer the embryos to a diluted MMR solution and incubate them overnight at the appropriate temperature (14-18°C).

Scoring Protein Expression and Imaging

• After the embryos have been incubated, anesthetize them again using MS222.
• Mount the embryos on a microscope stage to observe their development and look for protein expression.
• Use special filters to visualize fluorescent proteins (like GFP or tdTomato) that were expressed from the mRNA introduced into the cells.

Troubleshooting

• If the resistance values are too high or too low, check the electrodes for proper connection or adjust the medium to alter the resistance.
• If the protein expression is not in the correct location, make sure the mRNA was injected properly and that the electrical pulses were applied correctly.
• If no protein expression is observed, try increasing the mRNA concentration or injection volume.

Key Results and Findings

• This method achieves high transfection efficiency (86-93%) with 100% viability in embryos.
• Targeted mRNA expression can be achieved in difficult-to-target tissues, such as the tail and flank.
• The electroporation method is effective for delivering both mRNA and other genetic materials like DNA or morpholinos.

Conclusion (Discussion)

• Electroporation provides a precise and efficient way to introduce mRNA into Xenopus embryos at specific developmental stages.
• This technique can be used to study gene function and development, enabling researchers to control when and where genes are activated.
• With high efficiency and low toxicity, electroporation is a valuable tool in developmental biology research.

观察到了什么？ (引言)

• 非洲爪蟾 (Xenopus laevis) 是一种常用于研究胚胎发育和再生机制的青蛙。
• 研究人员希望找到一种方法，可以控制基因在发育过程中的激活时间和位置，而不会引起不必要的副作用。
• 将 mRNA（帮助生成蛋白质的分子）注入早期胚胎可能会出现问题，因为它可能在错误的时间和位置激活基因。
• 为了解决这个问题，科学家使用了电穿孔技术将 mRNA精确地送入特定细胞。电穿孔通过电脉冲帮助 mRNA进入细胞。
• 这种方法适用于从胚胎的胃内期到尾芽期，这个阶段是胚胎发育中重要结构开始形成的阶段。
• 通过使用这种方法，科学家可以比传统注射方法更精确地研究基因功能。

什么是电穿孔？

• 电穿孔是一种使用电脉冲在细胞膜上打孔的技术，从而使像 mRNA 这样的物质能够进入细胞。
• 这种方法非常有用，因为它可以精确地将物质送入特定的细胞或区域，而不会影响整个胚胎。
• 电穿孔可以在任何发育阶段将 mRNA送入细胞，从而更好地控制基因的激活时间。

所需材料

• Marc 改良 Ringer 溶液（MMR）：用于为胚胎提供健康的环境。
• 编码目标蛋白的 mRNA：这是将要引入胚胎的基因物质。
• Tricaine Methanesulfonate (MS222)：用于麻醉胚胎，防止其在操作过程中移动。
• 琼脂糖溶液（1% 在 MMR 中）：用来创建稳定的表面，在该表面上固定胚胎进行电穿孔。
• 注射针：用来将 mRNA 注入胚胎。
• 电穿孔室：用于施加电脉冲的设备。

准备电穿孔设置

• 在一个盘子底部涂上一层 parafilm，以防液体泄漏。
• 将 1% 的琼脂糖倒入盘子中，并让它冷却，形成一个固体床。
• 使用橡胶小圆点或微量移液器的尖端在琼脂糖中创建小口袋，以固定胚胎。
• 在琼脂糖冷却时，设置显微操作器（用于控制注射针）和电穿孔仪（用于施加电脉冲）。
• 准备注射针，并用 mRNA 溶液填充它。通过显微操作器控制针头，精确地将少量 mRNA 注射到目标细胞中。
• 为下一步准备电穿孔仪，确保其能够施加正确的电脉冲。

确定电穿孔参数

• 测量溶液的电阻，以确定电脉冲的强度和持续时间。
• 根据电阻值调整电穿孔的参数（电压和脉冲长度），以确保有效的 mRNA 传递。
• 根据测量的电阻值，调整设备的参数并进行必要的调整。

注射并进行电穿孔

• 使用 MS222 麻醉胚胎，防止其在操作中移动。
• 将胚胎放入琼脂糖床小口袋中，并将 mRNA 溶液注射到目标位置。
• 注射后，施加电脉冲帮助 mRNA 进入细胞。通过将阴极放置在注射点附近并施加适当的电脉冲来完成此步骤。
• 对每个胚胎重复注射和电穿孔过程，确保它们都按照相同的方法处理。
• 电穿孔后，将胚胎转移到新鲜的 MMR 溶液中，并让它们恢复一小时。
• 将胚胎转移到稀释的 MMR 溶液中，并在适当的温度下（14-18°C）过夜培养。

评分蛋白表达并成像

• 胚胎培养过夜后，再次使用 MS222 进行麻醉。
• 将胚胎安装到显微镜的舞台上，观察它们的发育情况并检查蛋白表达情况。
• 使用特殊的滤光片来观察荧光蛋白的表达（如 GFP 或 tdTomato），这些荧光蛋白是通过注入的 mRNA 表达出来的。

故障排除

• 如果电阻值过高或过低，检查电极连接是否正确，或者调整介质的浓度来改变电阻值。
• 如果蛋白表达没有出现在正确的位置，确保 mRNA 已正确注射，并且电脉冲已正确施加。
• 如果没有观察到蛋白表达，尝试增加 mRNA 的浓度或注射量。

主要结果与发现

• 该方法在胚胎中实现了 86-93% 的转染效率，并且 100% 存活。
• 能够在困难的组织（如尾部和侧翼）中实现靶向的 mRNA 表达。
• 电穿孔方法对于传递 mRNA 以及其他遗传材料（如 DNA 或 morpholinos）也非常有效。

结论 (讨论)

• 电穿孔为将 mRNA 精确引入 Xenopus 胚胎提供了一种有效的方法，能够精确控制基因激活的时间和位置。
• 该技术可以用来研究基因功能和发育过程，允许研究人员在特定的时间和位置激活基因。
• 电穿孔方法具有高效率、低毒性，是发育生物学研究中的一项重要工具。