优化<em&gt;前OVO</em&gt;鸡胚培养，以发展的高级阶段

摘要

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

摘要

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

引言

Ex ovo culturing has played an important role in the study of development of the chicken^{1, 2}. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes ^{3, 4, 5}.

There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup^6,7,8 or a glass bowl⁵. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate humidity⁶. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses.

The above challenges in the ex ovo method has meant that researchers turn to the windowing method ^9,10 to view embryonic chick development. In this technique, a hole or “window” is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax⁹ to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 – HH 27), viewing the entire embryo at late stages is not easily accomplished.

Here, we describe an improved and simple ex ovo culturing technique¹¹ that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

研究方案

注:所有用品都列在表1中。

1.存放鸡胚

1. 孵育应变鸡水平鸡在^37℃，大约40％的湿度的鸡蛋，把鸡蛋一次或每天两次。翻蛋重要的是要防止胚胎附着在蛋壳。
2. 不打开鸡蛋在24小时之前，建立培养另有胚胎将位于腹侧蛋黄的质量和在打开鸡蛋在步骤3除了将被损坏，保持蛋在^4℃下度孵化，以"叫停"开发前不超过一个星期，但这种不理想。

2.分期鸡胚

1. 舞台采用汉堡和汉密尔顿¹²临时表中的鸡胚。设立的前OVO培养的理想阶段是HH阶段19-20（约3-天的潜伏期），BEC澳洲英语这是头转向后不久，在53高倍视野。
   注意:HH阶段19-20的特征在于用以下形态学性状:体节延伸到大多数的尾部，但是尾部的最末端保持不分段，尾芽卷曲，尿囊小并且具有有限的脉管，腿芽比翼芽大，眼睛是未染色的，或有一个灰色的色调。

3.从外壳取下胚胎

1. 之前从外壳取出胚胎，喷壳用70％乙醇，并允许其干燥。不要把鸡蛋迅速，因为这会损坏胚胎。
2. 小心不要改变卵的取向，小心裂纹蛋上的下侧（即，侧那孵育期间腹侧），并释放该胚胎到无菌称量船（约88 x 88×23毫米）。擦拭称量皿，用70％的乙醇。检查胚胎，以确保黄麻袋不被损坏。如果蛋黄ħ作为破碎，放弃胚胎，因为它会无法生存。
3. 观察胚胎，以确保它是可行的;心脏跳动，血管显示正常，未见明显异常存在。确保称量船的边缘是干燥的。
4. 使用移液管，滴40微升青霉素/链霉素（5,000单位青霉素，每毫升5毫克链霉素）上的白蛋白的顶部，以防止感染。

4.准备恒温恒湿箱

1. 放置一小叠KIM-湿巾和/或在无菌的塑料容器（12×12×6厘米）的底部由棉的吸收垫擦拭用70％的乙醇。
2. 加无菌蒸馏水弄湿KIM-湿巾和/或棉（约150毫升水）。

5.装配防爆大毛文化

1. 将含在潮湿的填充顶部胚胎的权衡船。然后，将半方形无菌培养皿（9.5×9.5厘米）对T顶部他权衡船，形成一个松散的盖子。
2. 覆盖塑料容器，其盖子。向下压在盖的两个角，从而确保一局部密封，其仍然允许该腔室内部的良好的气流。
3. 小心地将设置到^37℃培养箱，直到所需的阶段。
4. 放置的水的容器，在培养箱中以帮助控制湿度，如果一个受控湿度培养箱不可用。消毒所有的水在湿室和孵化器和发病期间根据需要及时补充。 40％的湿度是理想的。

结果

这当然大毛方法可以从开发的初期阶段（HH 19/20），观察胚胎的发展（ 图1B和1A），后期（HH 40-41）。建立文化的HH 19-20增加了文化的胚胎存活。在此之前的头部转动（前53 HPF）生存能力是很低的文化和级21之后，胚胎往往如此少完整胚获得更粘到上除去外壳。在一般情况下， 前OVO文化的胚胎生存能力更是高达35-36 HH（90-100％），但它确实下降的发展更高级阶?...

讨论

前大毛培养和窗口都有优点和挑战。在这里，我们比较一下优点和泡沫塑料杯前卵内的方法和开窗的方法对我们的优化卵内的方法当然在这里显示的挑战。我们的方法能够操纵和便于观察小鸡胚胎在发育的后期阶段，我们的改进传统的前卵方法^1，2，3，使其附加地很容易在本科教学实验室类使用。

虽然许多研究人员更加窗方法来研?...

材料

Name	Company	Catalog Number	Comments
Penicillin/Streptomycin	Sigma	P4458	Make small aliquots to avoid freeze/thaw events
Square Petri Dish			9.5 cm x 9.5 cm
Weigh Boat	Fischer Scientific	8732113	88 x 88 x 23 mm
Ziplock container	Ziplock	N/A	12 cm x 12 cm x 6 cm