이 작품은 독일 학술 진흥 재단 (; LU1455/1-1 DFG)에서 교부금에 의해 지원되었다.

1. 예 비켜 문화 <ol><li> 신선한 수정된 달걀이 부화 60 %의 습도와 37.5에서 강제 통풍 인큐베이터 (BSS160, Ehret, 독일) ° C에서 그들의 긴 측면에 놓여있다. 이상 1 주일 동안 12 ° C에서 저장된 계란을 사용하지 않아야합니다. </li><li> 2.5 일 (무대 HH17에 대한)에 대한 인큐베이션 후, 계란을 제거하고 해독을위한 방향을 나타내는 연필로 상단에 라벨을 달고. </li><li> 크래킹하기 전에 깨끗한 대형 배양 접시 (; Greiner 생물 하나 게엠베하, 독일 145mm의 직경)로 약 20 ML 멸균 증류수를 부어. </li><li> 대형 배양 접시에, 또 다른 작은 멸균 페트리 접시를 (Greiner 바이오 - 하나 게엠베하 94mm의 직경) 놓습니다. </li><li> 날카로운 금속 모서리에 대한 바닥에 알을 약쟁이, 조심스럽게 달걀을 열고 작은 배양 접시에 각 계란의 전체 내용을 전송합니다. </li><li> 뚜껑과 함께 큰 페트리 접시를 커버하여 다른 인큐베이터 (바인더 게엠베하, Tuttlinge, 독일)에 넣어추가로 문화의 37.5에서 ° C에서 약 60 %의 습도. 일 원하는 번호 부화 후 배아가 electroporation을 위해 사용될 수 있습니다. </li></ol> 2. 전 비켜 Electroporation을위한 준비 <ol><li> 1.0 밀리미터 직경 (TW100F-4, 세계 정밀 계측기)의 유리 튜브를 사용하는 microelectrode의 풀러 (세계 정밀 계측기, 베를린, 독일 PUL-100 Micropipette 풀러)와 유리 모세관을 당겨와 적절한로 유리 모세관의 끝부분을하다 지름. </li><li> electroporation에 대한 적절한 매개 변수 (즉, 배아의 독특한 조직 및 크기에 대해 다른 전압) 설정 및 electroporator (CUY21 - 편집, Nepa 진, 치바, 일본)에 전극을 연결 배아의 대상 조직 및 크기에 따라. </li><li> 그리고 마커 유전자; 대상 유전자를 인코딩 pCAGGS 벡터의 plasmids, 예를 들어, cadherin7 (2.0 μg / μl의 농도로 pCAGGS-Cad7 Cad7)를 포함하는 플라스미드 솔루션을 준비예를 들어, 녹색 형광 단백질 (GFP, 0.25 μg / μl의 농도로 pCAGGS-GFP). 그렇다면 녹색 색상 플라스미드 솔루션을 라벨에 0.1 %의 최종 농도 (시그마)와 빠른 그린을 추가합니다. </li><li> 입 피펫을 사용하여 플라스미드 솔루션 모세관 유리를로드합니다. </li></ol> 3. 전 비켜 Electroporation에 의한 치킨 광학 Tectum로 진 전송 <ol><li> 전 비켜 문화 후 예는 인큐베이션 일 (E) 6으로, 닭 배아는 전 비켜 electroporation을 위해 사용됩니다. </li><li> 조심스럽게 미세 집게로 광학 tectum 위에 vitelline 및 양막 세포막을 찢어하고 tectum의 표면에 멸균 0.9 %의 나트륨 염화물 용액 3 방울을 추가합니다. </li><li> 입 피펫을 사용하여 모세관 유리하여 tectum의 캐비티에 플라스미드 솔루션을 주사. </li><li> tectum 옆에 텅스텐 바늘 음극과 직사각형 플레이트 양극 (CUY 661-3X7, Nepa 유전자)와 전극을 배치하고,즉시 tectum에 electroporator에 의해 생산된 전기 펄스를 (6 펄스, 25 V, 60 MS 펄스 길이, 각각의 경우 100 MS 간격)이 적용됩니다. </li><li> 뚜껑이있는 큰 페트리 접시를 덮어 인큐베이터로 반환합니다. 적절한 일 (예, 2 또는 삼일 electroporation 이후)을위한 인큐베이션 후, tectum는 immunostaining (그림 1)과 생물 학적 검출을 위해 수집하고 해결됩니다. </li></ol> 4. 대표 결과 전 비켜 electroporation에 의해 Cad7와 GFP의 외인성 단백질의 성공 overexpression은 예를 들어 그림 1에 표시됩니다. 플라스미드 GFP와 Cad7 플라스미드 함께이 E6에서 tectum에 electroporated되었을 때, 2-3 일 electroporation 후 배아를 모으고 수정되었습니다. E8에서 GFP 단백질은 강력 전체 마운트 이미지 (그림 1A-C)에 tectum에 표시됩니다. 또한, E9의 tectum의 섹션에서GFP 단백질 (그림 1D 녹색) 및 Cad7 단백질 (그림 1E에서 빨간색) 전 비켜 electroporation은 생체내에 오래된 닭 배아에 유전자 전달을위한 성공적인 방법이라고 제안하고, (그림 1 층에서 노란색) coexpressed 있습니다. <img src="/files/ftp_upload/4078/4078fig1.jpg" alt="그림 1"/> 그림 1. plasmids 인코딩 GFP와 Cad7은 E6에서 치킨 tectum로 cotransfected되면 2-3 일 전 비켜 electroporation 후, GFP 단백질은 (녹색) 강하게 같은 E8에 wholemount 이미지 (AC)과 같이 표시됩니다. E9의 tectum의 섹션에서 GFP 단백질 (D 녹색) 및 Cad7 단백질 (E에서 적색) (F에서 노란색) coexpressed 있습니다. BF, 명시야 이미지입니다. 스케일 바 : AC 용 200 μm의, DF위한 D 100 μm의.

<ol>
	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., 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=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</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.">A series of normal stages in the development of the chick embryo.</a> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

이 프로토콜은 전 비켜 electroporation에 의해 기존의 닭 배아 (예 : E6의 광학 tectum로)로 유전자 전달을위한 가이드를 제공합니다. 이 방법은 기존의 닭 배아에 electroporation의 사용을 확장하고 쉽게 나중 단계에서 생체내의 유전자 기능을 연구를 위해 많은 실험실에 적용할 수 있습니다. E4에서 최소한 E7에 태아도 성공적으로이 방법에 의해 electroporated 수 있으며 electroporated 배아는 E15 ...

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> Electroporator </td><td> Nepa 진, 일본 </td><td> CUY21 - 편집 </td></tr><tr><td> 전극 </td><td> Nepa 진, 일본 </td><td> CUY661-3X7 </td></tr><tr><td> 계란 인큐베이터 </td><td> Ehret, 독일 </td><td> BSS160 </td></tr><tr><td> 배아 인큐베이터 </td><td> 바인더 게엠베하, 독일 </td><td></td></tr><tr><td> 형광 현미경 </td><td> Keyence, 독일 게엠베하 </td><td> BZ-8000 </td></tr><tr><td> 페트리 접시 </td><td> Greiner 바이오 하나 게엠베하, 독일 </td><td></td></tr><tr><td> Microelectrode의 풀러 </td><td> 세계 정밀 계측기, 독일 </td><td> PUL-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

닭 배아가 배아 발달 동안 유전자 기능과 규제를 공부를위한 훌륭한 모델 시스템을 제공합니다. 비켜 electroporation에는 강력한 오버 특급 외인성 유전자에 대한 방법이나 닭 배아 1 생체내의 내생 유전자를 다운 조절입니다. 이러한 유전자 plasmids 인코딩 유전자 2-4, 작은 간섭 RNA (siRNA) plasmids 5, 작은 합성 RNA oligos 6 및 morpholino 안티 센스 oligonucleotides 7 등 다양한 건축물에 쉽게 electroporation에 의해 닭 배아로 transfected 수 있습니다. 그러나 비켜 electroporation에의 응용은 초기 배양 단계 (단계 HH20 미만 - 함부르크와 해밀턴에 따라)에 배아로 제한됩니다 8, 나중 단계에서 배아에서의 응용을위한 몇 가지 단점은 (무대 HH22보다 오래된 없습니다 - 약 3.5 개발 일). 예를 들어, 나중 단계에 vitelline 막이 우스입니다조 막에 붙어 있으며 껍질에 창을 열지 앨리 배아의 죽음에 그 결과 혈관의 파열을 초래, 이전 배아는 그것이 배아를 액세스하고 조작하기가 어렵습니다 vitelline 및 allantoic 혈관에 의해 적용되며, 이전 배아 움직이지 셸의 상대적으로 작은 창을 통해 방향을 제어하기 어려운 강력하고 있습니다. 이 프로토콜에서는 늦은 단계 (단계 HH22보다 오래된)에서 닭 배아에 유전자 전달을위한 전직 비켜 electroporation 방법을 보여줍니다. 전 비켜 electroporation의 경우 배아가 페트리 접시 9 배양해하고 vitelline 및 allantoic 혈관이 널리 퍼져있다. 이러한 조건에서 기존의 닭 배아 쉽게 액세스하고 조작합니다. 따라서이 방법은 기존의 닭 배아에 적용 비켜 electroporation에의 단점을 극복. 이 방법을 사용 plasmids 쉽게 여러 부분으로 transfected 수오래된 닭 배아 10-12니다.

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

나중에 인큐베이션 단계 (햄버거와 해밀턴 무대 (HH) 22 세 이상)에서 닭 배아에 유전자 전달의 방법을 설명합니다. 이 방법은 단점을 극복<em&gt; 비켜에서</em&gt; electroporation은 나이 닭 배아에 적용하여 유전자 기능과 기존의 개발 단계에서의 규제를 공부하는 유용한 기술입니다.

에 의한 이전 치킨 태아에 유전자 이체<em&gt; 전 비켜</em&gt; Electroporation

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

gene-transfer-into-older-chicken-embryos-by-ex-ovo-electroporation

Research

JoVE Journal

Biology

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

14.5K 조회수.  School of Medicine University of Rostock. 나중에 인큐베이션 단계 (햄버거와 해밀턴 무대 (HH) 22 세 이상)에서 닭 배아에 유전자 전달의 방법을 설명합니다. 이 방법은 단점을 극복 비켜에서 electroporation은 나이 닭 배아에 적용하여 유전자 기능과 기존의 개발 단계에서의 규제를 공부하는 유용한 기술입니다.

비디오: Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

마우스 Oocytes에 핵 전송

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

연구

생물학

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15 마우스 태아의 Utero의 심실 주입 및 Electroporation에

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

초기 치킨 태아의 성장 팩터 - 코팅 비즈 배치

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

마우스 망막 신경절 세포의 유전자 발현에 대한 utero 및 전직 생체내의 Electroporation에

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

치킨의 배아에 인간 줄기 세포의 Xenotransplantation

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

전 비켜</em이미징 및 미세 애플 리케이션에 적합> 치킨의 배아 문화 시스템

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...