Name: chick ex ovo culture and ex ovo cam assay: how it really works
Uploaded: 2009-11-30T14:45:00
Description: Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

저자는 아낌없이 종양 물질을 공급하는 우수한 기술 지원 및 교수 Ruebben에 대한 J. Kueper 및 J. Wittschier 감사하고 싶습니다.

모든 장비와 시약은 멸균 구입해야하거나 열 또는 증기 소독 또는 70 % ETOH와 소독을해야합니다. 동물 실험 기관에 의해 규정된 동물 실험 절차와 규정에 대한 동물의 보호 및 사용에 관한 NIH의 지침에 따라, 유럽 커뮤니티 이사회 지침 (86/609/EEC)에 따라 수행다고 저자 상태 관리 및 뒤스부르크 - 에센 대학 (독일)에서위원회 (IACUC)를 사용합니다. 1 부 : 달걀 ...

<ol>
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Sci. 49, 3289-3298 (2008).</li><li>Oppitz, M., Busch, C., Shriek, G., Metzger, M., Just, L., Drews, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-malignant+migration+of+B16+mouse+melanoma+cells+in+the+neural+crest+and+invasive+growth+in+the+eye+cup+of+the+chick+embryo.">Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo.</a> Melanoma Res. 17, 17-30 (2007).</li><li>Uchibayashi, T., Egawa, M., Nakajima, K., Hisazumi, H., Tanaka, M., Endo, Y., Sasaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+tumour+cell+lines+implanted+onto+the+chorioallantoic+membrane+of+chick+embryo+to+anticancer+agents+in+combination+with+hyperthermia.">Responses of tumour cell lines implanted onto the chorioallantoic membrane of chick embryo to anticancer agents in combination with hyperthermia.</a> Urol. Res. 20, 237-239 (1992).</li><li>Demir, R., Naschberger, L., Demir, I., Melling, N., Dimmler, A., Papadopoulus, T., Sturzl, M., Klein, P., Hohenberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+generates+a+more+invasive+phenotype+of+tumour+cells:+an+in+vivo+experimental+setup+based+on+the+chorioallantoic+membrane.">Hypoxia generates a more invasive phenotype of tumour cells: an in vivo experimental setup based on the chorioallantoic membrane.</a> Pathol. Oncol. Res. , (2008).</li><li>Kunzi-Rapp, K., Genze, F., Kufer, R., Reich, E., Hautmann, R. E., Gschwend, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioallantoic+membrane+assay:+vascularized+3-dimensional+cell+culture+system+for+human+prostate+cancer+cells+as+an+animal+substitute+model.">Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model.</a> J. Urol. 166, 1502-1507 (2001).</li><li>Hall, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+of+organs+and+tissues+to+the+chorioallantoic+membrane+of+the+embryonic+chick.">Grafting of organs and tissues to the chorioallantoic membrane of the embryonic chick.</a> TCA Manual. 4 (3), 881-883 (1978).</li><li>Kunzi-Rapp, K., Kaskel, P., Steiner, R., Peter, R. U., Krahn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+blood+levels+of+human+S100+in+melanoma+chick+embryo+xenografts'+circulation.">Increased blood levels of human S100 in melanoma chick embryo xenografts' circulation.</a> Pigment Cell Res. 14, 9-13 (2001).</li><li>McFall, R. C., Sery, T. W., Makadon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+continuous+cell+line+derived+from+a+human+retinoblastoma.">Characterization of a new continuous cell line derived from a human retinoblastoma.</a> Cancer Research. 37, 1003-1010 (1977).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Research. 65, 10959-10969 (2005).</li><li>Kim, J., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li></ol>

혁신 아니면 그냥 다른 아가씨 문화 프로토콜? 전 쉘 적은 문화 프로토콜과 함께 여자는 태아 1-4 전체 생존 요금은, 예를 들어 CA 낮은되었습니다. 30% 1. 이 비디오 문서에 설명되어있는 세련된 전부 비켜 문화 프로토콜은, 대조적으로, 50 % 이상 생존 속도를 허용합니다. 비켜 문화의 고전에 비해 여자는 배아의 전 비켜 문화 크게 CAM과 여자?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fertilized Eggs</td>
<td>Animal</td>
<td>S&ouml;ries Trockels, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator Type 3000 with rotating egg tray</td>
<td>Tools</td>
<td>Siepmann GmbH Germany</td>
<td>9503</td>
<td>Maintain at 37&deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>Egg incubator BSS 160</td>
<td>Tools</td>
<td>Grumbach, Germany</td>
<td>8101</td>
<td>Maintain at 37-387deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile
</td>
</tr>
<tr>
<td>Dulbecco's modified eagle's medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 6429</td>
<td>DMEM culture medium</td>
</tr>
<tr>
<td>Leibovitz's L-15 Medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>31415-029</td>
<td>here: collection medium for tumor samples</td>
</tr>
<tr>
<td>Tumor cell -specific medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz's Medium or Dulbecco's modified eagle's medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 &mu;M insulin</td>
</tr>
<tr>
<td>70% EtOH</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic stir bar, triangled 80 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>442-0391</td>
<td>A triangled magnetic stir bar serves well to crack the eggs shell.</td>
</tr>
<tr>
<td>Forceps DUMONT #5 </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors, straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Fine Iris scissors, straight</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14094-11</td>
<td>Use to cut our the CAM</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>microliter syringe 1702 TLLX, 25 &mu;l</td>
<td>Tool</td>
<td>Hamilton</td>
<td>80222</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Hamilton 33-G needle (15 mm)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>18073-15</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Sterile scalpels</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use for dissecting tumor samples</td>
</tr>
<tr>
<td>Small drain spoon</td>
<td>Tool</td>
<td>Geuder, Germany</td>
<td>15758</td>
<td>Use to transfer of small chick embryos</td>
</tr>
<tr>
<td>Wax board with fixing pins</td>
<td>Tool</td>
<td>Self made</td>
<td>&nbsp;</td>
<td>Use to fix of animals for dissection</td>
</tr>
<tr>
<td>Petri dishes 60 x 15 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>628102</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 92 x 16 mm</td>
<td>Tool</td>
<td>Sarstedt</td>
<td>82.1472</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Petri dish 100 x 20 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>664160</td>
<td>extra deep, with spacers for ventilation</td>
</tr>
<tr>
<td>Beaker</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>300-600 ml</td>
</tr>
<tr>
<td>FALCON tubes</td>
<td>Tool</td>
<td>FALCON</td>
<td>&nbsp;</td>
<td>15 ml and 50 ml</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>1.5 ml and 2 ml</td>
</tr>
<tr>
<td>1ml pipette tips</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use to cut plastic rings for application of substances / cells</td>
</tr>
<tr>
<td>Peripheral venous catheter (PVL)</td>
<td>Tool</td>
<td>Viggo&reg;</td>
<td>&nbsp;</td>
<td>Use to cut silicon rings from the tube for application of substances / cells</td>
</tr>
<tr>
<td>Pipettes and tips</td>
<td>Tool</td>
<td>Eppendorf / Abimed</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Autoclaved folded Filters 595 1/2, 110 mm diameter</td>
<td>Tool</td>
<td>Schleicher &amp; Schuell</td>
<td>311643</td>
<td>Carrier, which caused least irritation of the CAM; used in the paper</td>
</tr>
<tr>
<td>PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm</td>
<td>Tool</td>
<td>santec-medical</td>
<td>REF 277, LOT 832/511-1</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Hxdroxyethylcellulose Tylose H 100000</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Carrier mixed with Kollidon 17 PF; caused false positive results</td>
</tr>
<tr>
<td>Kollidon 17 PF</td>
<td>Reagent</td>
<td>BASF</td>
<td>S30200</td>
<td>mixed with Hydroxyethylcellulose; caused false positive results</td>
</tr>
<tr>
<td>Collagen Biomatrix TissuDura</td>
<td>Tool</td>
<td>Baxter Healthcare S.A.</td>
<td>REF B2246000999999</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Round glass cover slips, 11 mm</td>
<td>Tool</td>
<td>Assistent Germany</td>
<td>1001/0011</td>
<td>Carrier; causes false positive results</td>
</tr>
<tr>
<td>Bovine Collagen Sponge</td>
<td>Tool</td>
<td>Wyeth</td>
<td>&nbsp;</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Resodont Absorbable Collagen Membrane</td>
<td>Tool</td>
<td>Resorba Woundcare Germany</td>
<td>LOT 280303</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Non-Woven Swabs</td>
<td>Tool</td>
<td>Fink &amp; Walter GmbH</td>
<td>REF 328132</td>
<td>Carrier; caused false positive results</td>
</tr>		</tbody>
		</table>
		</div>

chick ex ovo culture and ex ovo cam assay: how it really works

번식의 초기 단계에 치킨 계란은 체외에서 생체내 시스템의 사이에 있으며, angiogenesis를 공부뿐만 아니라 tumorigenesis를 공부뿐만 아니라 혈관 테스트 환경을 제공합니다. 여자 chorioallantoic 막 (CAM)가 개발되면, 그 혈관 네트워크는 쉽게 액세스할 수 조작하고 관찰하고 따라서 angiogenesis의 assays에 대한 최적의 설정을 제공합니다. 림프 시스템이 완전히 부화의 늦은 단계까지 개발이 아니므로, 여자의 난자는 종 특정 제한없이 이식할 조직 및 세포를 유지 수있는 자연 immunodeficient 호스트로 제공하고 있습니다. 개발 육성 이외에 allo을하고 xenografts, CAM의 혈관 네트워크는 종양 세포 intravasation, 보급, 그리고 혈관 체포와 체포 세포 마이크로 전이성 foci를 형성 extravasate 저장소를위한 고유 지원 환경을 제공합니다. 실험 목적을 위해, 최근 연구의 대부분의 CAM은 알 껍질을 통해 창문을 절단하고 실험 관찰과 효과의 사진을 문서에 대한 CAM과 가능성의 접근에 상당한 제한의 결과로, 비켜에서 실시되었다하여 노출되었습니다. 전혀 게시된 경우 여자는 배아의 쉘 적은 문화가 1-4 사용했을 때, 어떤 실험 자세한 내용은, 제공하지 않으며 있었다 이러한 문화의 생존 요금이 낮은되었습니다. 우리는 계란 내용의 이성적으로 제어 압출을 도입하여 크게 여자는 배아의 전 비켜 문화의 방법을 정제. 이 전 비켜 문화는 효과의 생체내 문서에 쉽게 활성화 및 배아의 실험 조작을 촉진, CAM 및 여자 태아의 접근을 향상시킵니다. 이것은 과학적인 질문이 다수를 성공적으로 응용 프로그램을 수행할 수 있습니다 (1) 개선 angiogenesis 분석 5,6로, (2) 실험 (3), 여자는 배아 아이 7-9의 유리체에서 촉진 주사에 대한 설정 의 보급과 intravasation위한 테스트 환경 (5 닭고기와 마우스 13은 잘 사지 꽃봉오리의 성공적인 이식과 문화에 대한 개선 유지 시스템으로 CAM 10-12에 주사를 설립 세포 라인에서 종양 세포 (4) 분산으로 )에 대한, 접목 전파 및 CAM 14 표면에 biopsies에서 얻은 탄탄한 기본 종양 조직의 다시 접목. 이 비디오를 문서에서 우리는 세련된 여자는 전 비켜 문화와 50 % 이상 생존 속도 CAM 분석의 설립을 설명합니다. 게다가 우리는 과학 응용 프로그램의 많은에 대한 예 비켜 문화의 성공적인 응용 프로그램의 단계 시범에 의해 단계를 제공합니다. 다니엘 S. 도일, 수잔느 D. 파사, 그리고 세바스찬 Gustmann이 연구에 동등하게 공헌했습니다.

Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

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

병아리 chorioallantoic 막 (CAM)은 angiogenesis와 tumorigenesis을 연구하는 독특한 자연 immunodeficient 지원 문화 환경입니다. 이 비디오 기사는 여자의 서로 다른 단계를 보여줍니다<em&gt; 예 비켜</em&gt; 문화, 가능성 angiogenic 물질 및 CAM의 표면에 종양 세포와 조직을 성공적으로 접종의 응용 프로그램입니다.

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 ...

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

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 ...

연구

생물학

Method for Culture of Early Chick Embryos ex vivo (New Culture)

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 patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

조기 칙 태아 예 생체내 (새로운 문화)의 문화에 대한 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 ...

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 ...

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

기본 인간 난관 상피 세포의 전의 VIVO 문화

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

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 ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

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 ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

소설<em&gt; 생체</em배아 마우스 사랑을&gt; 문화 방법

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

마우스 배아의 피부와 Melanoblast 이주의 라이브 영상의 생체 문화

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.