We would like to thank all the members of the Mitchell lab for helpful discussions. This work was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation (operating and infrastructure grants held by JAM).

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저자는 이해 충돌에 조브의 정책을 읽고 공개 충돌이 더이 없다.

CRISPR / Cas9 매개 게놈 편집 기술은 게놈 수정에 대한 간단 빠르고 저렴한 방법을 제공한다. 기능 증강 특성에 대한 monoallelic 증강 삭제를 생성하고 분석하기 위해 여기에 설명 된 방법은 F1 마우스 세포에서 SNP를 활용합니다. 이러한 유형의 접근법의 이점은 : 1) monoallelic 인핸서 삭제가 중요한 증강제, 즉 두 대립 유전자에서 치사 셀에 이르는 조절 유전자의 단백질 수준에서 큰 감소를 삭제하...

전사 조절 요소에 의한 비정상적인 유전자 발현이 질병을 초래할 수있다 이러한 요소 개발 1 변형시 유전자 발현 시공간 미세 조정이 중요하다. 게놈 넓은 협회 연구에 의해 확인 된 많은 질병 관련 지역 비 코딩 지역에 있고 전사 증강 3-4의 기능을 가지고 있습니다. 강화제를 파악하고 그들이 자주 몇 킬로베이스 거리가 조절하는 유전자의 위치와 조직 - 특이 적 방식으로 5-6으로 활성화 될 수 있으므로 복잡 조절하는 유전자로 일치. 증강 예측은 일반적으로 히스톤 수정 마크, 중재자-cohesin 단지를 기반으로 세포 유형 특정 전사의 결합은 7-10 요인 있습니다. 예측 증강의 유효성 검사는 가장 자주 인핸서는 리포터 유전자 11 ~ 12의 발현을 활성화하는 벡터 기반의 분석을 통해 이루어집니다. 이러한 데이터는 V를 제공추정 인핸서 서열의 규제 가능성에 대한 aluable 정보는하지만 자신의 내생 게놈 맥락에서 그 기능을 공개하거나 조절하는 유전자를 식별하지 않습니다. 게놈 편집 기능 상실 분석에 의해 자신의 내생 적 맥락에서 전사 조절 인자의 기능을 연구하는 강력한 도구 역할을한다. 게놈 편집, 즉 CRISPR / Cas9 게놈 편집 시스템의 최근의 진보는, 게놈 기능의 조사를 용이하게한다. CRISPR / Cas9 시스템은 많은 생물학적 시스템에 사용하기 쉽고 적용 할 수 있습니다. Cas9 단백질 가이드 RNA (gRNA) (13)에 의해 게놈의 특정 사이트를 대상으로합니다. SpCas9 / gRNA 단지는 protospacer 인접 모티브 (PAM) 시퀀스, NGG 14 ~ 15에 &#39;5이어야 목표 게놈 시퀀스의 게놈을 스캔합니다. 목표에 gRNA의 gRNA에 상보적인 20 뉴클레오티드 (NT) 시퀀스의 염기쌍는 doubl 결과 SpCas9 클레아 활동을 활성화전자 가닥 브레이크 (DSB)가 PAM 시퀀스의 상류 3 염기쌍. 특이도가 gRNA 종자 지역의 전체 염기 쌍을 통해 달성되면, 6-12은 PAM에 인접한 NT; 반대로, 종자의 보통 16-17을 허용하는 &#39;5 불일치. 가입 비 동종 말 (NHEJ)의 DNA 수리 또는 상동 감독 수리 (HDR) mechanisms.NHEJ의 DNA 수리가 종종 방해 할 수있는 대상 사이트에서 몇 bp의 삽입 / 삭제 (삽입과 삭제)을 작성 중 하나를 소개 DSB는 복구 할 수 있습니다 유전자의 오픈 리딩 프레임 (ORF). 관심 영역의 측면 게놈 두 gRNAs에서 큰 결실을 생성하기 위해, 18 ~ 19을 사용할 수있다. 이 접근법은 궤적 제어 영역 또는 종래 증진제 9,18,20-22보다 큰 수퍼 증강제로 클러스터 전사 인핸서의 연구에 유용하다. Monoallelic 삭제는 전사의 시스 -regulation 연구를위한 귀중한 모델입니다. 관찰 장증강의 monoallelic 삭제 후 성적 수준에서 전자는 두 대립 유전자의 전사가 세포 적합성에 영향을 미치는 잠재적 영향을 때 발생할 수있는 혼란 효과없이 유전자 조절에서 해당 증강의 역할에 상관 관계. 감소 된 발현을 평가하기 그러나, 야생형 대립 유전자에서 삭제를 구별하는 능력없이 어렵다. 또한, 두 개의 대립 유전자를 구별하는 능력없이 각 대립 유전자의 결실을 유전형 특히이 PCR에 의해 전체 야생형 영역을 증폭하기 어렵다되는 1 메가 23&gt; 10킬로바이트 큰 결실에 대한 도전이다. 뮤스 castaneus으로 건너 뮤스의 musculus (129)에 의해 생성 된 F1 ES 세포의 사용은 두 가지 대립 유전자가 대립 유전자 특이 PCR 18,24에 의해 구별 할 수 있습니다. 이러한 세포의 하이브리드 유전자는 대립 유전자 특정 삭제 검사 및 발현 분석을 용이하게한다. 평균적으로이 두 게놈 사이의 모든 125 BP는 SNP에게있다, 표현과 유전자형에 대한 프라이머 설계 유연성을 제공하는 분석한다. 하나의 SNP의 존재는 프라이머 융해 온도 (Tm)에 영향을 미치는 두 대립 (25)의 식별을 허용 실시간 정량 PCR (qPCR에) 증폭 특이성을 타겟팅 할 수있다. 또한, 프라이머의 3 &#39;말단 내의 불일치 크게 원하지 대립 타겟 (26)의 증폭을 방지 프라이머로부터 연장하는 DNA 폴리머 라 아제의 능력에 영향을 미친다. CRISPR / Cas9 게놈 편집 시스템 (도 1)를 사용하여보다 큰 1킬로바이트의 특정 대립 유전자 인핸서 삭제 이후 발현 분석 F1 ES 세포의 사용은 다음의 프로토콜에 설명한다. <img alt="그림 1" src="/files/ftp_upload/53552/53552fig1.jpg" /> 시스 -reg을 연구하는 CRISPR / Cas9를 사용하여 그림 1. 증강 삭제유전자 발현의 위험률. 뮤스의 musculus 129 뮤스 castaneus 사이의 교차에 의해 생성 (A) F1 ES 세포는 대립 유전자 특정 삭제를 허용하는 데 사용됩니다. (B)는 두 개의 RNA를 가이드 (gRNA)는 인핸서 영역의 큰 Cas9 매개 결실을 유도하기 위해 사용된다. (C) 프라이머 세트가 많은 모노 - 및 이중 대립 유전자 결실을 식별하는 데 사용된다. 오렌지 프라이머 내부 프라이머이며, 보라색 프라이머는 외부 프라이머 및 녹색 프라이머는 gRNA 측면 프라이머되어 있습니다. 유전자 발현의 (D)의 변화는 대립 유전자 특정 qPCR에를 사용하여 모니터링됩니다. RFU 상대 형광 단위를 의미한다. <a href="https://www.jove.com/files/ftp_upload/53552/53552fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:200px;">NEB</td>
			<td style="width:119px;">M0530S</td>
			<td style="width:395px;">high fidelity DNA polymerase used in gRNA assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Gibson Assembly Master Mix</td>
			<td style="width:200px;">NEB</td>
			<td>E2611L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">gRNA_Cloning Vector</td>
			<td style="width:200px;">Addgene</td>
			<td style="width:119px;">41824</td>
			<td>A target sequence is cloned into this vector to create the gRNA plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">pCas9_GFP</td>
			<td style="width:200px;">Addgene</td>
			<td style="width:119px;">44719</td>
			<td>Codon-optimized SpCas9 and EGFP co-expression plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">AflII</td>
			<td style="width:200px;">NEB</td>
			<td style="width:119px;">R0520S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">EcoRI</td>
			<td style="width:200px;">NEB</td>
			<td style="width:119px;">R3101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Neon Transfection System 100 &#181;L Kit</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">MPK10096</td>
			<td>Microporator transfection technology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">prepGEM</td>
			<td style="width:200px;">ZyGEM</td>
			<td style="width:119px;">PT10500</td>
			<td>genomic DNA extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Nucleo Spin Gel &#38; PCR Clean-up</td>
			<td style="width:200px;">Macherey-Nagel</td>
			<td style="width:119px;">740609.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">High-Speed Plasmid Mini Kit</td>
			<td style="width:200px;">Geneaid</td>
			<td style="width:119px;">PD300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Maxi Plasmid Kit Endotoxin Free&#160;</td>
			<td style="width:200px;">Geneaid</td>
			<td style="width:119px;">PME25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">SYBR select mix for CFX</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">4472942</td>
			<td>qPCR reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">iScript cDNA synthesis kit</td>
			<td style="width:200px;">Bio-rad</td>
			<td style="width:119px;">170-8891</td>
			<td>Reverse transcription reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">0.25% Trypsin with EDTA</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:253px;">PBS without Ca/Mg2+</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">0.5M EDTA</td>
			<td style="width:200px;">Bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">HBSS</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">1M HEPES</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">13630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">BSA fraction V (7.5%)</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">15260037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Max Efficiency DH5&#945; competent cells</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">18258012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">FBS</td>
			<td style="width:200px;">ES cell qualified</td>
			<td style="width:119px;"></td>
			<td>FBS is subjected to a prior testing in mouse ES cells for pluripotency</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">DMSO</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Glutamax</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">35050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">DMEM</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">11960069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Pencillin/Streptomycin</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sodium pyruvate</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">11360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Non-essential aminoacid</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">11140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">&#946;-mercaptoethanol</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:119px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">96-well plate</td>
			<td style="width:200px;">Sarstedt</td>
			<td style="width:119px;">83.3924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sealing tape</td>
			<td style="width:200px;">Sarstedt</td>
			<td style="width:119px;">95.1994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">CoolCell LX</td>
			<td style="width:200px;">Biocision</td>
			<td style="width:119px;">BCS-405</td>
			<td>alcohol-free cell freezing container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">CHIR99021</td>
			<td style="width:200px;">Biovision</td>
			<td style="width:119px;">1748-5</td>
			<td>Inhibitor for F1 ES cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">PD0325901</td>
			<td style="width:200px;">Invivogen</td>
			<td style="width:119px;">inh-pd32</td>
			<td>Inhibitor for F1 ES cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">LIF</td>
			<td style="width:200px;">Chemicon</td>
			<td style="width:119px;">ESG1107</td>
			<td>Inhibitor for F1 ES cell culture</td>
		</tr>
	</tbody>
</table>

generating crispr/cas9 mediated monoallelic deletions to study enhancer function in mouse embryonic stem cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

여기에 설명 된 프로토콜 CRISPR / Cas9 게놈 편집 (도 1)를 이용하여 생성 monoallelic 인핸서 삭제 세포에서 유전자 발현의 시스 -regulation 공부 F1 ES 세포를 사용한다. 유전자형 유전자 발현의 gRNA 및 대립 유전자 특이 적 프라이머를 디자인이 방법에서 중요한 요소이다. 각 대립 유전자 특이 프라이머 세트는 대립 유전자 특이성을 확인하는 qPCR에 의해 검증되?...

Watch this Scientific Journal Video about Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells at JoVE.com

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

생성 CRISPR은 / Cas9 중재 Monoallelic 삭제할 경우는 마우스 배아 줄기 세포의 증강 기능을 연구하는

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

generating-crisprcas9-mediated-monoallelic-deletions-to-study

Research

JoVE Journal

Developmental Biology

13.9K 조회수.  University of Toronto. Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

비디오: Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

혈청이없는 단층 문화 마우스 배아 줄기 세포의 신경 분화

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

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Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

줄기 세포처럼<em&gt; Xenopus의</em&gt; 배아의 Explants는 초기 신경 발달의 특징을 연구하는<em&gt; 체외</em&gt; 및<em&gt; 생체</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

재조합 효소 매개 카세트 교환을 사용하여 마우스 배아 줄기 세포의 구조 - 기능 연구

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

생활 그대로 Zebrafish에 단일 셀 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

꼬마 선 충 생식 핵의 분포, 단백질, 및 골격을 전산 분석

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

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 ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

iPSC 유래 근육 전조제에서 디스트로핀 발현 복원에 있는 CRISPR/Cas9 기술

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

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 ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

인간 배아 줄기 세포에서 뇌 오르가노이드를 생성하기위한 정적 자기 지향 방법

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

초기 마우스 배아에서 시퀀싱을 위한 작은 RNA 라이브러리 의 준비

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...