We would like to thank the Wellcome Trust Sanger Institute for the gifts of piggyBac transposon and piggyBac transposase. This work was supported by the Butcher grant of Colorado and the NIH R01 AG041801 (W.S.C).

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The authors have nothing to disclose.

Forward genetic analysis allows for an unbiased interrogation of the genome for genes responsible for a specific phenotype. This method is very powerful to uncover novel gene functions. It has been widely used in lower organisms but not in mammal, such as the mouse, mainly due to the extremely high cost associated with the infrastructure and logistics that would entail. Here, we moved the genetic screening process to the ES cell culture platform, greatly increasing the efficiency and throughput in generating mutants a...

장수 스트레스 저항과 친밀한 관계를 가지고있다. 일반적으로, 수명이 긴 종들은 과산화수소, 파라콰트 (PQ), UV, 열, 중금속 1,2- 여러 스트레스쪽으로 저항을 증가 보여준다. 대조적으로, 스트레스 증가 감도 짧아 수명 및 / 또는 더 발생하기 쉬운 질환 표현형을 예측하는 경향이있다. 항산화 소기 경로가 긴 동물에게 스트레스 저항성을 부여하는데 중요한 역할을하는 것으로 추측하고있다. 그러나, 거의 예외없이, 다양한 항산화 제 효소 (예를 들면, SOD)의 조작과 형질 전환 동물의 다양한 연구가 산화제 소거 효소의 수준을 증가 시키면 수명 또는 건강 스팬 3을 증가시키지 않음을 나타낸다. 이러한 데이터는 지속적으로 수명이 긴 동물에서 관찰 된 스트레스 저항 특성이 아직 발견되는 다른 세포 경로에 의해 매개되는 것이 좋습니다. 우리는 편견 전달했다방법은 돌연변이 유전자에 배양 배아 줄기 (ES) 세포에서 스트레스 내성 표현형을 부여 할 수있는 유전자를 동정을 행 하였다. ES 세포는이 연구에서 두 가지 장점을 제공한다 : (1) 유전자 정교한 조작이 ES 세포의 게놈을 수정은 가능하다; 및 (2) 스크린으로부터 회수 스트레스 내성 ES 세포는 직접 수명 및 건강 범위를 측정하기 위해 전체 동물 연구에 신속한 변환을 허용 마우스 생산에 사용될 수있다. 이보고에서는, 대립 유전자가 BLM 테트라 사이클린 반응성 소자에 의해 제어 된 C9에 ES 세포주의 사용을 설명했다. 독시 싸이클린 (D​​OX)의 치료는 일시적으로 자매 염색 분체 교환의 증가 사고로 이어지는 BLM의 발현을 해제. 이 단기적으로는 BLM 노크 아웃 이질 집단 내에서 동형 접합 돌연변이의 생성을 허용 스트레스 저항 열성 돌연변이가 심사 과정에서 캡처 할 수 있도록. 우리또한 무작위 게놈에서 유전자 돌연변이 폴리 트랩 카세트 (PB-UPA)를 삽입 돌연변이로 피기 백 (PB) 트랜스포존의 사용을 설명했다. 폴리 트랩에 의해 유전자의 파괴와 세포 G418 내성이되었고, 유전자 트랩 돌연변이 (유전자 트랩 라이브러리)의 컬렉션을 만들고, 이후에 강한 스트레스했다 돌연변이 클론 상영 될 수 있도록 복구 할 수 있습니다. 선택으로부터 회수 스트레스 내성 클론 삽입의 수 (qPCR에)의 점에서, 분자의 기술에 의해 다소 빠르게 특성화 될 수 삽입 사이트 (splinkerette PCR), 파쇄 유전자 (BLAST)의 신원, 및 그 발현 량 ( RT-qPCR에). PB 삽입은 야생형 DNA 서열을 복원하고, 따라서 응력의 저항 손실을 테스트 할 클론의 MPB 트랜스의 일시적 발현으로 remobilized 될 수있다. 이들은 이전에 수행해야 돌연변이의 인과 관계를 확인하는 강력한 방법이다비싼 마우스 생산. 이전의 연구는 스트레스에 노출 된 세포가 만능의 4,5을 잃은 것으로 나타났다. 따라서,이 프로토콜에서, 스트레스로 처리되지 않는다 돌연변이 세포의 집합 복제, 보존 성공적인 마우스 생산에 중요하다. 우리 연구소는 모두 요청에 따라 다른 연구자가 사용할 수있는, C9 ES 세포 라인과 PB-UPA 벡터를 설계하고있다. 여기에보고 된 프로토콜은 스트레스 내성 클론 (그림 1B)를 분리하기 위해 복제 도금과 스트레스의 선택에 따라 PB-UPA (그림 1A)와 유전자 갇혀 ES 세포의 드 노보 라이브러리의 생성에 의해 시작됩니다. 우리는 파라콰트, 세포 내부의 강력한 활성 산소 발생기 선택을 보여 주었다. 사실상, 예를 들어 임의의 세포 독성 화합물 또는 독소, ER 스트레스 요인 (예, 및 쌥시 가르 긴 tunicamycin), 신경 세포의 산화제 (예를 들어, MPP +, 6- 히드 록시 도파민 및 로테 논), 열, 또는 무거운금속 (예를 들면, CD, SE는) 각각 내성 돌연변이 체에 대해 선택하는 방법에 적용 할 수있다.

<table border="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vector</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PB-UPA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPBase</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mPBasePuro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500-ml Stericup filters</td>
			<td>EMD Millipore</td>
			<td>SCGPU05RE</td>
		</tr>
		<tr>
			<td>250-ml Stericup filters</td>
			<td>EMD Millipore</td>
			<td>SCGPU02RE</td>
		</tr>
		<tr>
			<td>50-ml Steriflip-GV filters</td>
			<td>EMD Millipore</td>
			<td>SE1M179M6</td>
		</tr>
		<tr>
			<td>KO DMEM</td>
			<td>Life Technologies</td>
			<td>10829-018</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Tissue Culture Biologicals</td>
			<td>104</td>
		</tr>
		<tr>
			<td>Heat Inactivated FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>EMD Millipore</td>
			<td>ESG1107</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140148</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
		</tr>
		<tr>
			<td>Methyl Viologen dichloride (Paraquat)</td>
			<td>Sigma-Aldrich</td>
			<td>856177</td>
		</tr>
		<tr>
			<td>Dimethyl Sulphoxide Hybri-MAX</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
		</tr>
		<tr>
			<td>EmbryoMAX 0.1% gelatin</td>
			<td>EMD Millipore</td>
			<td>ES006B</td>
		</tr>
		<tr>
			<td>DPBS/Modified</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
		</tr>
		<tr>
			<td>T25 Flask</td>
			<td>Corning</td>
			<td>353108</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Corning</td>
			<td>353135</td>
		</tr>
		<tr>
			<td>100-mm&#160; plate</td>
			<td>Corning</td>
			<td>353003</td>
		</tr>
		<tr>
			<td>150-mm&#160; plate</td>
			<td>Corning</td>
			<td>430599</td>
		</tr>
		<tr>
			<td>96-well&#160; plate</td>
			<td>Corning</td>
			<td>3585</td>
		</tr>
		<tr>
			<td>96-well U-bottom plate</td>
			<td>Corning</td>
			<td>3799</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning</td>
			<td>3526</td>
		</tr>
		<tr>
			<td>50-ml reservoir&#160;</td>
			<td>Corning</td>
			<td>4870</td>
		</tr>
		<tr>
			<td>15-ml tubes</td>
			<td>VWR International, LLC</td>
			<td>82050-276</td>
		</tr>
		<tr>
			<td>Primary Mouse Embryonic Fibroblasts</td>
			<td>EMD Millipore</td>
			<td>PMEF-NL</td>
		</tr>
		<tr>
			<td>DR4 Mouse Embryonic Fibroblasts</td>
			<td>Applied StemCell</td>
			<td>ASF-1001</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP25312</td>
		</tr>
		<tr>
			<td>Geneticin (G418)</td>
			<td>Life Technologies</td>
			<td>11811-023</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP26531</td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>377267</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5702</td>
		</tr>
		<tr>
			<td>TC10 cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting Slides (for TC10)</td>
			<td>Bio-Rad</td>
			<td>1450011</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporation&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell</td>
			<td>Bio-Rad</td>
			<td>1652611</td>
		</tr>
		<tr>
			<td>Gene Pulser Cuvettes (4 mm gap)</td>
			<td>Bio-Rad</td>
			<td>1652088</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>Mastercylcer ep Gradient&#160; S</td>
		</tr>
		<tr>
			<td>Puregene Core kit B</td>
			<td>Qiagen</td>
			<td>158745</td>
		</tr>
		<tr>
			<td>Topo-TA Cloning kit</td>
			<td>Life Technologies&#160;</td>
			<td>450030</td>
		</tr>
		<tr>
			<td>High Capacity cDNA synthesis kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP358-212</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2716</td>
		</tr>
		<tr>
			<td>Double Processed Tissue Culture Water</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
		</tr>
		<tr>
			<td>Sau3A1</td>
			<td>New England BioLabs</td>
			<td>R0169L</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202T</td>
		</tr>
		<tr>
			<td>EcoRV</td>
			<td>New England BioLabs</td>
			<td>R3195S</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Lysis Buffer (Ramires-Solis et al. 1992)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher BioReagents&#160;</td>
			<td>A144-212</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E5134</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>L5777</td>
		</tr>
		<tr>
			<td>Proteinase-K</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP1700</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT</td>
			<td>Bio-Rad</td>
			<td>170-4469EDU</td>
		</tr>
		<tr>
			<td>LE Agarose&#160;</td>
			<td>GeneMate</td>
			<td>E3120500</td>
		</tr>
		<tr>
			<td>Ethidium Bromide&#160;</td>
			<td>Fisher BioReagents&#160;</td>
			<td>BP1302</td>
		</tr>
		<tr>
			<td>100 BP DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3231S</td>
		</tr>
		<tr>
			<td>1Kb DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3232S</td>
		</tr>
		<tr>
			<td>2-log DNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N3200L</td>
		</tr>
	</tbody>
</table>

forward genetic approach to uncover stress resistance genes in mice &#8212; a high-throughput screen in es cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

전형적인 돌연변이 유발 실험에서는 트랜 PB-UPA 및 MPB의 트랜스와 3 × 10 7 ES 세포의 전체로 구성되어있다. 두 독립적 인 실험에서 생성 된 유전자 포획 ES 세포의 숫자는 표 1에 요약되어있다. 유전자의 포집 효율은 약 0.04 %이다. 결합 된 유전자 트랩 라이브러리는 22,400 독립적 인 돌연변이 마우스 게놈에 대한 전체 범위 (23,000 코딩 유전자)를 포함한다. 과도한 따르면 반복 돌연...

Watch this Scientific Journal Video about Forward Genetic Approach to Uncover Stress Resistance Genes in Mice â€” A High-throughput Screen in ES Cells at JoVE.com

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

스트레스 저항은 장수의 특징 중 하나이며 유 전적으로 규율하는 것으로 알려져있다. 여기, 우리는 장수 연구에 마우스 모델을 개발하기 위해있는 배아 줄기 세포에 스트레스 저항을 부여 돌연변이 선별 편견 높은 처리량 방법을 개발했다.

앞으로 유전 접근은 마우스의 스트레스 저항성 유전자를 밝히기 위해서 - ES 세포에서 높은 처리량 화면

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

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Research

JoVE Journal

Developmental Biology

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

8.1K 조회수.  University of Colorado Denver.  스트레스 저항은 장수의 특징 중 하나이며 유 전적으로 규율하는 것으로 알려져있다. 여기, 우리는 장수 연구에 마우스 모델을 개발하기 위해있는 배아 줄기 세포에 스트레스 저항을 부여 돌연변이 선별 편견 높은 처리량 방법을 개발했다. 

비디오: Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

공동 문화 시스템에 신경 연결의 형성을 평가하기위한 Optogenetic 접근

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

연구

발생학

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

개발에 미세 주입을 대상으로 기술<em&gt; Xenopus의</em&gt; 신장

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

높은 처리량 억제제 스크리닝에 의한 배아 줄기 세포의 다 능성을 조절하는 키나제를 확인하는 간단한 방법

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

혈관 내피 줄기 세포의 혈관 생성을 시각화하기위한 유방 팻 패드 이식 기술

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

기계 조직 해리 및 유동 세포 계측법에 의한 포유 동물 수컷 세포의 다중 종 정제에 대한 표준화 된 접근법

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

C. 선 충 에서 신경 세포를 세균 세포를 재 설 정할 RNAi 및 열 충격 유도 하는 전사 인자 식의 적용

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

노드 및 스캐닝 전자 현미경 검사 법, 면역 형광 산 전체를 사용 하 여 Gastrulating 마우스 배아에서 Notochordal 접시를 시각화

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

인간 유도에서 Somite 파생 상품의 체 외 발생에서 만능 줄기 세포

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

면역 결핍 마우스에서 인간 골격 근 이종이식 수행

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...