우리는 그의의 HD 카메라 이용 (캐논 VIXIA HFS100) 동안 집게 Maaz Enver 감사드립니다. 이 작품은 NIH (EY018738), 척수 연구에서 뉴저지위원회 (08-3074-SCR-e-0과 10-3091-SCR-E-0) 및 부시 바이오 메디컬 연구 대상에서 교부금에 의해 부분적으로 지원되었다.

이전에 사소한 수정과 함께 다른 주피터 동영상 2, 서류 3 설명된대로 프로토콜의 일부 지역이 수행되므로 우리는 여기에 자세히들을 논의하지 않습니다. 1. 에그 처리 및 니들 준비 <ol><li> 계란이 최대 1 일주일 동안 약 13도 ° C에서 쿨러 와인에 저장할 수 있습니다. 온도가 너무 높은 경우에는 낮은 온도가 높은 사망률을 초래하면서, 태아는 비정상적으로 개발하기 시작합니다. 일단 와인 쿨러에서 알을 꺼내 큰 엔드와 세로로 계란을 설정할 품어 준비가 된 것입니다. 37.5 ° C 배양기에서 그들을 투입하기 전에 최소한 2 시간 동안 실내 온도에 계란을 보관. </li><li> 햄버거와 해밀턴 단계 22 4-5에 나와 있습니다 배아를 얻기 위해 배아 4 일 약 96-1백시간 대한 알을 품어. </li><li> 뽑아 유리 모세관 튜브에서 micropipette의 바늘을 준비하고 해부 마이크로 미만의 팁을하다0.1 μm의 직경과 20mm 테이퍼에 관한 팁 오프닝을 얻을 수있는 핀셋과 범위. 더 큰 팁을 바늘은 어려움이 작은 조언 DNA의 솔루션을로드하고 제공하는 데 문제가있는 동안 vitelline 막 시작 한거지 있습니다. </li><li> micromanipulator에 탑재된 0.1 ML 해밀턴 Gastight 주사기에 바늘을 연결합니다. 주사기에 바늘을 부착할 수 Masterplex 실리콘 튜브의 작은 조각을 사용합니다. 연결 때문에 꼭 어떤이 첨부 파일을 봉인하기 위해 미네랄 오일을 추가할 필요가 없습니다. </li><li> 3-6 μg / μl와 parafilm의 조각에 빠른 그린 (0.025 %)의 0.2 μl를 이르는 농도와 리포터 플라스미드 DNA 용액 2 μl를 섞는다. 빠른 녹색 염료는 주사를 시각화하는 데 도움이 될 것입니다. 천천히, 혼합물과 바늘을로드합니다. </li><li> 180 °에 대해 부드럽게 계란을 회전하고 몇 분 후 원래 위치로 다시 회전하고 electroporation을 위해 설정을 기다릴 내부의 멤브레인에서 vitelline 멤브레인을 풀고있다. </li><li> 그렇게해야 닦아배아에 감염을 피하기 위해 70 %의 에탄올과 EPS 및 난각. </li><li> 즉시 AA 크기의 포셉 한 쌍을 가진 에어 셀 위에 계란에 작은 구멍을 만듭니다. 알 껍질을 깰하지 않도록주의하십시오. 작은 조각에게 작은 창을 만드는 한 번에 하나씩 제거합니다. 조심스럽게 vitelline 막에 닿지 않고 집게를 사용하여 내부 멤브레인를 제거합니다. </li></ol> 2. 사출 및 Electroporation <ol><li> 시선을 입력하고 부리쪽으로 향하고 혈관의 기본 번들로 측면 뇌 또는 심장, 위치 바늘 콘트라 손상을 방지합니다. </li><li> 바늘의 갑작스런 약한 추진하여 vitelline 막, 공막엔, 망막 및 유리체 유머를 통해 피어스. 바늘은 안구의 다른 쪽 끝을 통해 pierces 경우 어떤 주요 혈액 혈관을 손상하지 않는 한 그냥가요이어야합니다. </li><li> 천천히 개방의 가장자리에 바늘을 뒤로 물러나게하고 안구의 외벽에 거의 미동 놓으십시오. </li><li> Insert 공막엔와 망막 사이의 하위 망막 우주로 바늘. </li><li> 당신은 안구의 측면을 복잡하게하고 볼록한 만들어 망막의 안쪽으로 밀어 녹색 솔루션을 시각화 때까지 DNA를 주입. </li><li> 귀하의 바늘 배치가 올바르지 않은 경우 다음의 눈을 볼의 중간을 채우는 유리체 유머 내부 확산의 DNA 용액을 볼 수 있습니다. 당신이 망막에 손상을 경우에도 너무 많이 나서 유전자 솔루션은 안구 밖으로오고있다 볼 수 있습니다. </li><li> 천천히 바늘을 제거하고 즉시 PBS에 몸을 담글 후 계란 내부에 병렬 전극 배치합니다. 주입 눈이 전극 사이에 위치되도록하는 방식으로 양수로 잠수함에 전극을 누르십시오. 그들을 배치하는 동안 전극과 함께 모든 주요 혈관이나 심장을 만지는 피하십시오. 그 DNA는 하위 망막 우주에서 긍정적인 전극에 대한 망막에 이송 될 수 있도록 마이너스 전극은 주입 측면에 있어야합니다. 전기950 MS 간격으로 50 MS위한 15V의 5 펄스와 망막을 porate. </li><li> 조심스럽게 전극을 제거하고 투명 스카치 테이프의 조각과 계란의 창문을 봉쇄. </li><li> 라벨 및 인큐베이터에 다시 투입하기 전에 주입 계란 날짜를 기입하십시오. 일반적으로, 그것은 전체 electroporation 프로세스를 완료하는 데 5-3 분 정도 소요됩니다. </li><li> GFP 발현은 electroporation 후 8시간 초부터 볼 수 있습니다. 배아는 수확하기 전에 원하는 단계에 도달할 때까지 그러나 기다릴 수 있습니다. </li></ol> 3. 대표 결과 우리의 연구에서 우리는 망막 세포 개발에 관여하는 유전자 발현의 조절을 연구하기 위해 다양한 플라스미드 구조를 사용합니다. 이 비디오에서는 pCAG-GFP (transfection 제어)이 성공적인 분사와 electroporation을 수행하는 데 사용되었다. 그러나 리포터 유전자 (GFP, RFP 등)와의 플라스미드 구조를 사용할 수 있습니다. GFP 발현은 ele 후 8시간 초부터 볼 수있다하더라도ctroporation, 우리는 일반적으로 하루 6 달걀 (E6)와 일부터 수확 시작합니다. Electroporated 망막은 배아 밖으로 해부하고 내장과 sectioning 전에 형광 해부 현미경 하에서 분석했다. 일반적으로 리포터 유전자 발현은 성공적인 electroporation (그림 1) 후 최소 망막의 4 분의 1에서 볼 수 있습니다. transfected 망막 조직은 더 이상 세포 morphologies의 명확한 시각화를 위해 sectioning을 통해 분석되었다. 셀 유형 특정 마커 (Brn3a, Pax6 등) 셀 - 특정 GFP의 표현의 허용 특성화 (그림 2)를 사용하여 Immunohistochemistry. <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> 1 그림. 기자 플라스미드 결과 긍정적인 GFP의 표현의 성공적인 electroporation. 닭 배아 망막은 주입과 electroporated 배아 일 4에서와 배아 일 6 시에 수확했다. 망막의 최소 25 %는 성공적으로 (= 탑 뷰, B transfected되었다= 아래보기). 스케일 바 = 1mm. <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> 그림 2. GFP의 특성화는 immunohistochemistry를 사용하여 망막 세포를 표현. E7 단계에서 망막 조직을 표현 GFP는 고정하고 sectioned했다. 이러한 섹션은 다음 각 셀을 특정 마커 물들일되었다. Brn3a은 Pax6가 수평 amacrine 및 신경절 세포 (B)를 결정하는 데 사용하는 도중에 신경절 세포 (A)를 결정하는 데 사용되었다. 스케일 막대 = 50 μm의.

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

동물 실험의 모든 Rutgers 대학 기관 동물 보호 시설위원회에 의해 승인되었다.

망막 주사를 타겟 및 E4 단계에서 배아 망막에서 비켜 electroporation에 특별히 단일 세포 수준에서 모두 6 개 주요 망막 세포 유형을 시각화하는 능력의 결과로 망막 전구 세포를 타겟팅할 수 있습니다. HH10에서 비켜 electroporation에서 (~ E1.5)을 대상으로 광 소포는 눈을 형성 발전 전지를 transfect 수 있습니다. 그러나 이러한 세포는 현재 매우 높은 매출액을 가지고 있으며이 방법은 망막 세...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 수정된 병원체 프리 (SPF) 흰색 레그혼종 닭고기 달걀 </td><td> 일출 농장 (Catskill, NY) </td><td></td><td></td></tr><tr><td> 치킨 계란 인큐베이터 </td><td> GQF 제조, 사바나, 조지아 </td><td> 모델-1550 </td><td> 60 %의 습도와 37.5 ° C.로 설정 </td></tr><tr><td> 유리 모세관 튜브 </td><td> 세계 정밀 악기 주식 회사 </td><td> TW150F-4 </td><td></td></tr><tr><td> 0.1 ML의 주사기 </td><td> 해밀턴 (주) 리노, 네바다 </td><td> Gastight 1710 </td><td> 또는 1ml 주사기 잘으로 사용할 수 있습니다 </td></tr><tr><td> Masterflex 실리콘 (과산화수소) 튜브 </td><td> 콜 - Parmer </td><td> HV-96400-13 </td&gt; <td> 주사기로 유리 바늘을 부착을위한 작은 조각 (1 센티미터) 잘라내기 </td></tr><tr><td> 족집게 </td><td> 뒤몽 </td><td> AA </td><td></td></tr><tr><td> Micromanipulator </td><td> 세계 정밀 악기 주식 회사 </td><td> M3301-M3 </td><td></td></tr><tr><td> 플라스미드 DNA와 </td><td></td><td></td><td> pCAG-GFP는 박사 코니 Cepko로부터 차용되었다 </td></tr><tr><td> 빠른 그린 FCF </td><td> 시그마 - 올드 리치 </td><td> F7252 </td><td> PBS와 0.025 %로 희석 </td></tr><tr><td> 펄스 발생기 </td><td> 하버드 장치, MA </td><td> BTX ECM 830 </td><td> 스퀘어 웨이브 제너레이터 </td></tr><tr><td> 전극 </td><td> 하버드 장치, MA </td><td> BTX 모델 514 </td><td> 우리 전극은 3-5mm 떨어져 잊어버리고 있었다 </td></tr><tr><td> 흑백 디지털 카메라 </td><td> 자이스 혈구, 독일 </td><td>xiocam MRM </td><td></td></tr><tr><td> 형광등 해부 현미경 </td><td> Leica 이크로 시스템즈, 독일 </td><td> Leica MZ16FA </td><td></td></tr><tr><td> 입식 형광 현미경 </td><td> 자이스 혈구, 독일 </td><td> 자이스 혈구 Axio 영상기 A1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

닭 배아 망막 높은 척추 동물의 망막 개발을 공부하는 훌륭한 도구입니다. 때문에 대형 및 외부 발전으로 인해, 그것은 재조합 DNA / RNA 기술을 사용하여 병아리 배아 망막를 조작하는 비교적 매우 쉽습니다. 태아의 망막에 DNA / RNA 구조의 Electroporation은 망막 개발하는 동안 망막 줄기 / 전구 세포에서의 유전자 조절을 연구하는 큰 잇점을 가지고있다. 이러한 리포터 유전자 분석, 표현을 통해, 유전자 등 (shRNA) 허물고 유전자 등 assays의 다른 유형은 electroporation 기술을 사용하여 수행할 수 있습니다. 이 비디오는 타겟 망막 주사를 보여주고 배아 일 4 (E4)에 관한 햄버거와 해밀턴 무대 22-23,에서 배아 여자의 망막에 비켜 electroporation 인치 마커로 녹색 형광 단백질 (GFP)을 표현하는 플라스미드 DNA를 직접으로 전달됩니다 의하여 여기 비켜 electroporation 기술에 신속하고 편리하게 보여줄병아리 배아 subretinal 공간과 망막 줄기 / 전구 세포로 DNA의 이해를 촉진하기 위해 전기 펄스에 의해 따라갔다. E4에서 망막 주입과 electroporation의 새로운 방법은 E1.5 2 시에 주입과 electroporation의 전통적인 방법으로 어렵게되었습니다 후반 태생의 뉴런 1을 포함한 모든 망막 세포 유형의 시각화 수 있습니다.

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

이 비디오의 전반적인 목표는 타겟 망막 주사를 수행하는 방법을 보여주기입니다<em&gt; 비켜에서</em배아 일 4 (E4)에 관한 햄버거와 해밀턴 단계 22-23,에서 병아리 배아 망막으로의 DNA / RNA 구조의&gt; electroporation. 이 기술은 유전자 발현, 유전자 조절, 그리고 여자의 망막 개발에 형태학의 변화를 연구하는 것은 매우 유용합니다.

배아 칙의 망막에서 비켜 Electroporation에

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

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Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K 조회수.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. 이 비디오의 전반적인 목표는 타겟 망막 주사를 수행하는 방법을 보여주기입니다 비켜에서 electroporation. 이 기술은 유전자 발현, 유전자 조절, 그리고 여자의 망막 개발에 형태학의 변화를 연구하는 것은 매우 유용합니다.

비디오: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HH 스테이지 10 치킨 태아의 비켜 Electroporations에서

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

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Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

칙의 전체 마운트 항체 스테 이닝을위한 방법

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

칙의 배아에서 신경 유도에 대한 분석

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

병아리 예 비켜 문화와 예 비켜 CAM 어세이 : 그게 정말로 작동하는 방법

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

차 문화와 Corti의 Murine 오르간의 플라스미드 Electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

활용 칙 신경 튜브에서 뉴런의 유전 정의된 그룹 Axonal 경로를 벗기기 비켜에서 Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

칙 태아의 단일 세포 Transfection

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

초기 여자는 배아에서 Morphogenetic 조직 Deformations 추적

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...