우리는 DRS 감사하고 싶습니다. 미라 Saxena 및 원고에 도움이 덧글에 대한 조나단 마타. 우리는 DRS 감사합니다. 형질 라인을 제공하는 마이클 파슨스, 파멜라 레이먼, 그리고 레이첼 웡. 동물 실험 프로토콜 07-12-003을 승인 MCG IACUC에 의해 명시된 지침과 규정에 따라 수행되었습니다 * B. 이 작품은 JSM에 NIH R21 MH083614과 천 바실 O 코너 초보 학술 상을 월 지원했습니다

<ol>
	<li>K&#246;ster, R. W., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+microscopy+of+brain+development.">Time-lapse microscopy of brain development.</a> Methods Cell Biol. 76, 207-235 (2004).</li><li>Mumm, J. S., Williams, P. R., Godinho, L., Koerber, A., Pittman, A. J., Roeser, T., Chien, C. B., Baier, H., Wong, R. O. <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+imaging+reveals+dendritic+targeting+of+laminated+afferents+by+zebrafish+retinal+ganglion+cells.">In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells.</a> Neuron. 52, 609-621 (2006).</li><li>Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> Dev Dyn. 236, 1025-1035 (2007).</li><li>Ren, J. Q., McCarthy, W. R., Zhang, H., Adolph, A. R., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+visual+responses+of+wild-type+and+hypopigmented+zebrafish.">Behavioral visual responses of wild-type and hypopigmented zebrafish.</a> Vision Res. 42, 293-299 (2002).</li><li>Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+ablation+of+beta+cells+in+the+embryonic+zebrafish+pancreas+using+E.+Coli+nitroreductase.">Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. Coli nitroreductase.</a> Mech Dev. 124, 218-229 (2007).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Vis Exp. , (2010).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Vis Exp. , (2009).</li></ol>

JSM은 zebrafish의 세포 절제의 nitroreductase 기반 시스템 (멈 JS, 그리고 Schroeter, Zebrafish &quot;미국 특허 7,514,595에서 EH 타겟 지역 박리 세포 절제. 2009년 4월 7일)의 사용에 대한 특허를 획득했습니다.이 기법은 도구 및 JSM은 설립과 서비스를 생명 공학 회사, Luminomics 주식 회사에 대한 일반적인 플랫폼으로 사용하는 것은 인치 이해 관계자 남아

이 비디오에서는, 우리는 여러 가지 빛깔의 공촛점 시간 저속 이미징 및 타겟 세포 절제에 사용 기술의 개요가 제공됩니다. 타겟 세포 절제가 신경 회로 기능과 세포 고유의 연결을 재생 패러다임을 연구하는 데 사용하는 동안 우리는 생체내에 대한 관심의 여러 세포 유형의 동작을 모니터하는 시간 저속 영상을 사용합니다. 예제는 이러한 방식에서 gleaned 수있는 몇 가지 장점을 강조 표시됩니다. 아마도 가장 중요한 시간 경과 영상은 내부 통제 패러다임을 제공 관찰 모든 현상은 이전 상태로 다시 관련되어 있습니다. 이것은 직접보다는 유추 비교가 수치 쉽게 상대적인 변화를 만들고 인구 내에서 변화와 관련된 실험 &#39;소음&#39;을 감소 실험 시간이 지점에 걸쳐 만들 수 있습니다. 여러 가지 빛깔의 이미지의 장점은 상호 작용 및 / 또는 인접 세포 유형의 변화를 시각화하는 능력입니다. 예를 들어, 멀러 glia를 라벨 유전자 변형 라인을 사용하여, 우리는 이제 개별 망막 세포 subpopulations의 손실이 잠재적인 부상을 유발 줄기 세포의 반응을 특성화하고 있습니다. 우리는 또한 신경 회로 형성 2 소설 메커니즘을 정의하기 위해이 방법을 사용했습니다. 마지막으로, 세포 재생 연구 이외에, 우리는 최근의 연결 subtypes의 기능 역할을 다양한 행동 패러다임과 electrophysiological assays을 사용하므로 개별 신경 회로를 조사하기 위해 NTR 기반 절제 시스템을 사용하기 시작했습니다. 이러한 연구 zebrafish을 고용의 독특한 장점은 자신의 재생 능력으로 인해, 유도의 적자가 일시 그입니다. 따라서, 시간 저속 영상 assays과 연관하여 우리는 reinnervation의 메커니즘을 초래할뿐만 아니라, 기능 회복에 필요한 reinnervation의 범위를 typify 수 있습니다. 제공 방법은 여러 가지 질문에 적용할 수 있습니다. 예를 들어, 절제와 중생의 유사한 분석은 어떤 transgenically 정의 셀룰러 subtype에 적용할 수 있습니다. 이하 이러한 연구가 개별 세포 유형의 수준과 이산 줄기 세포 틈새 내에서 재생에 대한 우리의 이해를 확장 가능성이있다.

1. 시약 : Danieau의 솔루션 : <ul><li> 나트륨 염화물 (피셔, S271 1) </li><li> 염화칼륨 (피셔, P217 500) </li><li> HEPES (피셔, BP310500) </li><li> 마그네슘 황산 (피셔, M65 500) </li><li> 칼슘 질산 (피셔, C109500) </li><li> 페니실린 - 스트렙토 마이신 (피셔, ICN1670049) </li></ul> 기타 시약 : <ul><li> 낮은 용융 아가로 오스 (피셔, BP165 - 25) </li><li> Tricane methanesulfonate (피셔, NC9435281) </li><li> N - phenylthiourea (피셔, NC9968868) </li><li> 메트로니다졸 (피셔, AC21034 - 1000) </li></ul> 2. 소모품 : <ul><li> 딥 배양 - 요리 (피셔 과학 / 코닝, 08-772-32) </li><li> 더몬트 # 5 멋진 포셉 (피셔, NC9404145) </li></ul> 3. 장비 : <ul><li> 줌 입체경 (올림푸스, SZ51) </li><li> 형광 줌 입체경 (올림푸스, SZX16) </li><li> 공촛점 현미경 (올림푸스 FV1000) </li><li> 디지털 블록 히터 - Isotemp (피셔, 11-715 - 125DQ) </li><li> 반스 테드 / Thermolyne Benchtop 보육 (피셔, 11-702-4). </li></ul>

multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation

살아있는 zebrafish의 애벌레의 고해상도 시간 경과 영상은 방법을 생물 학적 과정이 (검토를 위해 1 참조) 펼쳐 시각화에 활용하실 수 있습니다. 이웃 세포 ​​유형에서 다른 형광 기자를 표현 화합물 유전자 변형 물고기는 시간이 지남에 따라 실험적인 조작을 세포 상호 작용이 및 / 또는 조직 수준의 답변을 다음과 같은 수단을 제공합니다. 이 비디오에서는, 우리는 며칠 분 거리에 걸쳐 수있는 시간이 코스를 통해 각각의 물고기에 순차적으로 이미징 여러 transgenically 표시 셀 유형에 대해 사용할 수있는 방법을 보여줍니다. ) 분리에 대한 단순화된 접근 방식 1) 직렬 &#39;잡으 및 릴리스&#39;이미징을위한 방법 연속 일, 2 이상의 물고기가 많은 : 설명 기술을 포함하여, 시간이 지남에 인접한 세포 유형의 &quot;행동&quot;을 상호 연관하고자하는 모든 연구에 적용 중복 여기 / 방출 프로파일 (예 : GFP 및 YFP), 3) 개발, 4 후반 애벌레 단계로 고해상도 이미징을위한 시간 창 사용할 수 연장 hypopigmented 돌연변이 라인의 사용) 형광 기자가 공개를 타겟으로 막의 사용 fluorophores 개별 세포뿐만 아니라 세포의 큰 인구에있는 세포 자세한 내용 및 transgenically 타겟 세포 유형의 화학을 이용한 절제 5) 이전에 설명한 방법을 잘 형태학의 세부 사항; 같은 메트로니다졸 같은 즉, nitroreductase (NTR) prodrug 기판의 중재 변환, (MTZ), 세포 독성 파생 3,5 수 있습니다. 이러한 접근 방법의 예를 들어, 우리는 며칠 동안 개별 생선 이내에 망막 신경 세포 양극성의 subtype의 절제와 재생을 시각화합니다. 동시에 우리는 이웃 아닌 타겟 바이폴라 전지 및 잠재적인 변성 자극 망막 줄기 세포 (즉, Mϋller의 glia)을 포함한 여러 다른 망막 세포 유형을 모니터합니다. 이 전략은 타겟의 연결을 세포 유형의 선택 상실과 재생을 세포 및 조직 수준 (예를 들어, 줄기 세포 틈새) 응답을 특성화하기 위해 실험실에 적용되고 있습니다.

Watch this Scientific Journal Video about Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation at JoVE.com

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

이 비디오에서는 여러 가지 빛깔의 공촛점 시간 저속 이미징 및 타겟 세포 절제에 대한 기술이 제공됩니다. 시간 저속 영상은 관심의 여러 세포 유형의 동작을 모니터하는 데 사용됩니다<em&gt; 생체내에</em&gt;. 타겟 세포 절제는 학습 신경 회로 기능과 세포 고유의 연결을 재생 패러다임을 용이하게합니다.

트렌스 제닉 Zebrafish의 여러 가지 빛깔의 시간 저속 영상 : 타겟의 연결을 세포 절제에 의해 활성화 떠올리 망막 줄기 세포

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

multicolor-time-lapse-imaging-transgenic-zebrafish-visualizing

Research

JoVE Journal

Biology

16.4K 조회수.  Medical College of Georgia. 이 비디오에서는 여러 가지 빛깔의 공촛점 시간 저속 이미징 및 타겟 세포 절제에 대한 기술이 제공됩니다. 시간 저속 영상은 관심의 여러 세포 유형의 동작을 모니터하는 데 사용됩니다 생체내에. 타겟 세포 절제는 학습 신경 회로 기능과 세포 고유의 연결을 재생 패러다임을 용이하게합니다.

비디오: Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

성인 Zebrafish의 전체 신장 마로우의 이식

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

연구

생물학

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery에 의해 인간 배아 줄기 세포의 유도

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

라이브 zebrafish의 배아에서 두 광자의 axotomy하고 시간이 경과 공촛점 영상

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

인간 CD40 - 활성화된 B 세포의 생성

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

조직 타겟 배아 Chimeras : Zebrafish Gastrula 세포 이식

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

기형 종의 형성에 의해 인간 배아 줄기 세포의 운명 매핑

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

sox10의 두개 안면 개발의 시각화 : 단풍 나무 형질 전환 제브라 피쉬의 선은 시간 경과 공 초점 현미경을 사용하여

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.