최적화<em> 전 비켜</em> 개발의 고급 단계에 병아리 배아의 배양

Summary

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).

Abstract

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.

Introduction

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.

Protocol

참고 : 모든 소모품은 표 1에 나열되어 있습니다. 1. 닭 배아를 저장 약 40 %의 습도 루스가 37 ℃로 수평 루스 균주의 닭 알을 품어 한 번 또는 두 번 매일 계란을 켭니다. 달걀을 돌리면 껍질에 부착 배아를 방지하는 것이 중요하다. 4 O C에서 계란을 유지, 이전에, 그렇지 않으면 배아는 노른자 질량에 복부를 위치 할 것이며, 또한 3 ?…

Representative Results

방법 비켜이 전 늦게 개발의 단계 (HH 40 ~ 41) (그림 1A 및 1B)에 개발의 초기 단계 (HH 19/20)에서 배아의 관찰이 가능합니다. HH 19 ~ 20에서 문화를 설정하는 것은 문화의 배아의 생존을 증가시킨다. 생존의 문화와 단계 (21) 후 매우 낮은 (53 HPF 전) 선회 머리 이전에, 배아 적은 그대로 배아가 얻어진다 있도록 분리에 쉘에 더 집착하는 경향이있다. (약 40 % HH 40-41로 생존) 일?…

Discussion

배양을 모두 윈도 비켜 예 장점과 도전이있다. 여기에서 우리는 장점과 방법 비켜 스티로폼 컵 전 아래에 표시된 방법 비켜 우리의 최적화 된 예에 윈도우 방법의 문제를 비교합니다. 우리의 방법은 늦게 개발의 단계 및 방법 ^{1, 2} 비켜 전통적인 예에 대한 우리의 개선에 조작과 병아리 배아를 관찰 할 수 ^{있도록, 3} 학부 교육 실험실 ?…

Materials

Penicillin/Streptomycin	Sigma	P4458	Make small aliquots to avoid freeze/thaw events
Square Petri Dish	N/A	N/A	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