이 작품은 이스라엘 과학 재단, 건강의 이스라엘 사역 및 DFG (독일 학술 진흥 재단)에서 AK로 보조금에 의해 지원되었다.

I. Electroporation 에그 처리 <ol><li> 37-38 락을 쟁반과 함께 ° C, 바람직 인큐베이터에 humidified 보육 세트에 계란을 넣으십시오. </li><li> 그들이 햄버거 &amp; 해밀턴 (HH) 단계에 18-20에 도달하면 배아는 부화의 66 시간 정도 후에 electroporated 있습니다. 이 단계에서는 머리 트렁크 (경추 굴곡라는 조건)와 같은 앞쪽에, 사후, 오른쪽과 왼쪽 vitelline의 정맥을 볼 수 정상적으로 엑스트라 배아 혈관의 광대한 설정 직각 놓여 있습니다. 이것은 확장기에 의존 특정 표현 1,3,4에 대한 최적의 무대입니다. 가능한 및 동기 배아를 얻기 위해서는, 온도와 보육의 습도를 모니터하는 것이 중요합니다. </li><li> 부화의 66 시간 후에, 인큐베이터에서 계란을 제거합니다. 수평 계란을 넣으십시오. 50~10분 기다립니다. 배아 이제 계란의 위쪽 기둥에 있습니다. </li></ol> 준비 <ol><li> 페니실린 / 스트렙토 마이신 (P / S) (1:100 희석)와 행크의 솔루션을 준비합니다. </li><li> microcapillary에 유리 모세관 (0.5 mm 직경) 당기세요. </li><li> micromanipulator에 전극 (텅스텐)을 장소와 펄스 발생기 (ECM 830)에 전극을 연결합니다. </li><li> - 3, 펄스 50 MS의 길이 전압 30V, 펄스의 수를 다음과 같은 매개 변수를 사용하여 펄스 발생기를 조정합니다. </li><li> 입 피펫, 주사기 바늘과 테이프를 준비합니다. </li><li> DNA (2-5μg/μl)의 원하는 혼합을 준비하고 빠른 그린 염료를 추가합니다. </li></ol> 윈도우와 electroporation <ol><li> 주사기를 사용하여 각각의 난자에서 알부민의 5-6 ML를 제거합니다. </li><li> 작은 가위는 계란의 상단에 타원형 창을 열고. </li><li> 0.5 - 1ml 행크 + P / S 솔루션으로 젖은 배아. </li><li> 유리가 DNA 혼합물로 microcapillary 로드할 수, 입 피펫을 사용합니다. </li><li> 당신 향해 꼬리로 배아를 놓습니다. 이 단계에서 배아의 척수는 분명하게 볼 수 있습니다. 따라서, 어떤 대조 기술이 필요하지 않습니다. 척수는 양쪽의 머리와 꼬리를 봉인이지만, microcapillary 충분히 얇은 경우, 찔린 작은 구멍을하실 수 있으며 얕은 각도에 신경 튜브를 관통합니다. 오른쪽 직경 microcapillary는 DNA와 그것을 손상하지 않고 튜브를 침투하고 정기적으로 폭발로 DNA를 풀어 쉽게 로드할 수 있습니다. </li><li> 입 피펫을 사용하여 DNA를 주사. 녹색 염료 개발 두뇌의 vesicles까지 꼬리의 끝부분에서 전파해야합니다. </li><li> 튜브 자체 및 펄스을 건드리지 않고,로 할 수있는 근처의 신경 튜브에 병렬 전극을 삽입합니다. 펄스의 당시 전극은 전기 전도성을 허용에 대한 액체로 덮여 있어야합니다. 보통 행크의 구걸에 추가의 양은 충분합니다. 그렇지 않다면 펄스로 행크의 직전 한 방울을 추가합니다. </li><li> 조심스럽게 전극을 제거하고 테이프로 창문을 밀봉. 다음 잠복기 동안 배아의 건조 방지하기 위해 완전히 계란을 밀봉하는 것이 중요합니다. </li><li> 인큐베이터에있는 계란을 돌려 놓습니다. </li></ol> II. 척수 오픈 책을 준비 배아에서 척수의 분리 <ol><li> 계란에서 E6에서 eletroporated 배아를 제거하고 PBS를 포함하는 실리콘과 코팅 페트리 접시에 놓으십시오. </li><li> 세포막을 버려야하고 개최 핀을 사용하여 자신의 복부 측면에있는 배아를 스트레치. </li><li> 날카로운 텅스텐을 사용하면 아래 꼬리로 hindbrain에서 지붕 판을 따라 세로 절개를 microcapillary. </li><li> 멀리 척수의 지느러미 루트 신경 (DRGs)를 분리, 척수의 양쪽에서 추가이 세로 incisions하십시오. </li><li> 척수는 그대로두고, 꼬리에서 hindbrain 시작 조직에서 바닥 플레이트를 분리합니다. </li><li> 좋은 가위를 사용하여 hindbrain에서 가로 섹션에서 척수를 잘라 본체에서 분리. </li></ol> 정치 <ol><li> 실리콘은 hindbrain에서 평면 장착 준비를 생산 꼬리에 그것을 잡아 핀을 사용하여 PBS를 포함하는 코팅 새로운 배양 접시에있는 분리된 척수를 벌리고. </li><li> 접시에서 PBS를 붓고 및 인산염 버퍼 호수의 4 % paraformaldehyde로 바꾸십시오. </li><li> 1 시간 동안 실온에서 알을 품다. </li></ol> Immunohistochemistry <ol><li> PBS의 기본 항체가 들어있는 유리병에 고정 척수를 전송합니다. </li><li> 4 박 이상의 부드러운 agitations로 품어 ° C. </li><li> PBS로 항체 X5 회, 부드러운 agitations 1 시간 동안 각 씻어 씻으십시오. </li><li> 이차 항체를 추가하고 4 박 이상의 부드러운 agitations로 품어 ° C. </li><li> 각 PBS와 항체 X5 번 씻으십시오부드러운 agitations 1 시간 동안 씻어. </li></ol> 이미징 장착 공개돼 <ol><li> 스미어 그리스이나 실리콘 슬라이드에 사각형 모양과 내에 PBS 드립. </li><li> 핀셋과 파스퇴르 피펫으로 주위의 액체를 밖으로 무승부를 사용하여 사각형의 중앙에 뻗어 척수를 놓습니다. </li><li> 척수에 장착 매체에서 한두 방울을 넣고 커버 전표로 커버. 스쿼시는 기포를 방지합니다. </li><li> 4 ° C.에 어두운 슬라이드를 유지 </li><li> 공촛점 현미경을 사용하여 척수를 검사합니다. </li></ol>

<ol>
	<li>Zisman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+and+membrane+capture+of+F-spondin+generates+combinatorial+guidance+cues+from+a+single+molecule.">Proteolysis and membrane capture of F-spondin generates combinatorial guidance cues from a single molecule.</a> J Cell Biol. 178 (7), 1237-1249 (2007).</li><li>Arakawa, T., Iwashita, M., Matsuzaki, F., Suzuki, T., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paths,+elongation,+and+projections+of+ascending+chick+embryonic+spinal+commissural+neurons+after+crossing+the+floor+plate.">Paths, elongation, and projections of ascending chick embryonic spinal commissural neurons after crossing the floor plate.</a> Brain Res. 1223, 25-33 (2008).</li><li>Avraham, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+axonal+guidance+and+sorting+in+dorsal+interneurons+by+the+Lim-HD+proteins+Lhx9+and+Lhx1.">Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.</a> Neural Dev. 4 (1), 21 (2009).</li><li>Timmer, J., Johnson, J., Niswander, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+in+ovo+electroporation+for+the+rapid+analysis+of+neural-specific+murine+enhancers.">The use of in ovo electroporation for the rapid analysis of neural-specific murine enhancers.</a> Genesis. 29 (3), 123-132 (2001).</li><li>Reeber, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+Robo+expression+in+vivo+perturbs+commissural+axon+pathfinding+in+the+chick+spinal+cord.">Manipulating Robo expression in vivo perturbs commissural axon pathfinding in the chick spinal cord.</a> J Neurosci. 28 (35), 8698-8708 (2008).</li></ol>

여자의 배아에 플라스미드 DNA의 Electroporation은 생체내 이소성 표현을위한 강력한 기술로 발전. 특정 강화하고, 여자의 electroporation의 결합 뉴런 1,3,5의 유전자 정의된 그룹의 axonal 경로를 파악을 위해 신속하고 효율적인 도구를 제공합니다. Cre 호텔 / LoxP 및 Gal4/UAS의 증폭 시스템을 활용하는 것은 표현의 수준 및 기간을 확대하실 수 있습니다. 새로운 그림들은 axonal 계획의 방향에 의해 정?...

deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation

뉴런에서 기자 유전자의 표현 드라이브 향상제 요소의 고​​용은 axonal 투영를 추적하기위한 널리 사용되는 패러다임이다. 척추 동물에서 척추 interneurons의 axonal 투영을 추적하는 경우, 세균 라인 타겟 기자의 유전자가 좌우 대칭으로 대칭 상표를 얻을 수 있습니다. 따라서, 그것은 ipsi 및 콘트라 옆으로 돌출 axons을 구별하기 어렵습니다. 여자가 신경 튜브로 일방적 electroporation은 중추 신경계의 한쪽으로 리포터 유전자의 표현을 제한하고, 양쪽에 axonal 영사에 따라 유용한 수단을 제공<sup&gt; 1 ,2 - 5</sup&gt;. 이 동영상은 주사하기 전에 달걀을 처리하는 방법 첫째 보여줍니다. HH 단계 18-20에서 DNA는 신경 튜브의 성례의 수준으로 주입 후, 텅스텐 전극은 배아에 병렬 배치하고 짧은 전기 펄스는 펄스 발생기와 함께 관리됩니다. 계란은 테이프로 봉인과 발전을위한 인큐베이터에 다시 배치됩니다. 사흘 후 (E6) 척수는 배아에서 열린 도서 준비로 제거 고정하고, 전체 마운트 항체 염색법에 대한 처리됩니다. 스테인드 척수가 슬라이드 장착과 공촛점 현미경을 사용하여 시각입니다.

Watch this Scientific Journal Video about Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation at JoVE.com

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

이 동영상은 이용 배아 여자 척수에서 뉴런의 유전자 정의된 그룹의 axonal 경로를 시각화하는 방법을 보여줍니다<em&gt; 비켜에서</em특정 향상제 요소의 통제하에 기자 유전자&gt; electroporation.

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 ...

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

9.2K 조회수.  Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School. 이 동영상은 이용 배아 여자 척수에서 뉴런의 유전자 정의된 그룹의 axonal 경로를 시각화하는 방법을 보여줍니다 비켜에서 electroporation.

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

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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Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Mycobacteria의 Electroporation

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

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

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

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 ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

세포 운동성 및 Zebrafish 태아의 개별 뉴런과 신경 크레스트 세포의 굴지의 Cytoskeleton의 라이브 영상

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

의 정화 M. magneticum 스트레인 AMB - 1 Magnetosome 관련 단백질 MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

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. ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...