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).

<ol>
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著者らは、利害の衝突にJoveのの方針を読み、開示する競合を持っていませんでした。

CRISPR / Cas9媒介ゲノム編集技術は、ゲノム改変のための、簡単で迅速かつ安価な方法を提供します。機能的エンハンサー特徴付けのために1アレルエンハンサーの削除を生成し、分析するために、ここで具体的な方法は、F1マウス細胞におけるSNPを利用しています。このタイプのアプローチの利点は次のとおりです。1）1アレルエンハンサー欠失は重要なエンハンサーは、 すなわち 、両?...

転写調節エレメントは、異常な遺伝子発現2に起因する疾患をもたらし得る発展1およびこれらの要素の変更時の遺伝子発現の時空間微調整のために重要です。ゲノムワイド関連解析により同定された多くの疾患関連領域は非コード領域にあり、転写エンハンサー3-4の機能を備えています。エンハンサーを特定し、彼らはしばしば、数キロベース離れて、彼らが調節する遺伝子から位置しており、組織特異的様式5-6で活性化することができるように複雑である調節する遺伝子とそれらをマッチング。エンハンサー予測は一般にヒストン修飾マーク、メディエーターコヒーシン複合体に基づいており、細胞型特異的転写因子7-10の結合されています。予測エンハンサーの検証は、ほとんどの場合、エンハンサーは、レポーター遺伝子11-12の発現を活性化するベクトルベースのアッセイを介して行われます。これらのデータは、Vを提供します推定されるエンハンサー配列の規制の可能性についてaluable情報が、それらの内因性のゲノムのコンテキストでそれらの機能を明らかにするまたはそれらが調節する遺伝子を特定するものではありません。ゲノム編集が機能喪失分析によってそれらの内因性コンテキスト内の転写調節エレメントの機能を研究するための強力なツールとして機能します。 ゲノム編集、すなわち、CRISPR / Cas9ゲノム編集システムにおける最近の進歩は、ゲノム機能の研究を促進します。 CRISPR / Cas9システムは使いやすく、多くの生物学的システムに適応可能です。 Cas9タンパク質は、ガイドRNA（gRNA）13によるゲノムの特定部位に標的化されます。 SpCas9 / gRNA複合体はprotospacer隣接モチーフ（PAM）シーケンス、NGG 14-15に5 &#39;である必要があり、その標的ゲノム配列のためのゲノムをスキャンします。そのターゲットにgRNA、gRNAに相補的な20ヌクレオチド（nt）の配列の塩基対形成は、doublその結果SpCas9ヌクレアーゼ活性を活性化します電子鎖切断（DSB）PAM配列の上流の3bp。特異性はgRNAシード領域における完全な塩基対形成を介して達成され、6月12日は、PAMに隣接NT;逆に、シードのミスマッチが5 &#39;は通常16-17を許容しています。導入されたDSBは、いずれかの修理非相同末端結合（NHEJ）DNA修復または相同性によって中断されることが標的部位で数塩基対の修復を（HDR）mechanisms.NHEJのDNA修復がしばしば挿入/削除を作成します（インデル）向けることができます遺伝子のオープンリーディングフレーム（ORF）。関心領域に隣接するゲノム2 gRNAs、より大きな欠失を生成するために、18〜19を使用することができます。このアプローチは、従来のエンハンサー9,18,20-22よりも大きい遺伝子座制御領域または超エンハンサーにクラスタ化転写エンハンサーの研究のために特に有用です。 1アレルの欠失は、転写のシス -regulationを研究するための貴重なモデルです。観察チャンエンハンサーの1アレル削除後の転写レベルでの電子は、両方の対立遺伝子の転写は、細胞の適応に影響を与える潜在的影響を受けているときに発生する可能性の交絡効果のない遺伝子調節におけるそのエンハンサーの役割と相関します。発現低下を評価する野生型対立遺伝子から削除を区別する能力なしには困難です。さらに、2つの対立遺伝子を識別する能力なしに各対立遺伝子に欠失を遺伝子型決定することは、特にPCRにより全体の野生型領域を増幅することは困難である1メガビット23&gt; 10キロバイトの大きな欠失のために、困難です。 ハツカネズミのcastaneusと交差ハツカネズミ 129によって生成されたF1 ES細胞の使用は、二つの対立遺伝子は、対立遺伝子特異的PCR 18,24によって区別されることを可能にします。これらの細胞におけるハイブリッドゲノムは、対立遺伝子特異的欠失スクリーニングおよび発現分析を容易にします。平均的にSNPは、これら2つのゲノムの間のすべての125bpがあります発現および遺伝子型決定のためのプライマーの設計に柔軟性を提供する分析します。 1つのSNPの存在は、プライマーの融解温度（T m）に影響し、二つの対立遺伝子25の識別を可能にするリアルタイム定量PCR（定量PCR）増幅に特異性を標的とすることができます。さらに、プライマーの3 &#39;末端内のミスマッチが大きく、望ましくない対立遺伝子ターゲット26の増幅を防止するプライマーから伸長するDNAポリメラーゼの能力に影響を与えます。 CRISPR / Cas9ゲノム編集システム（ 図1）を使用して、1より大きいκBの対立遺伝子特異的エンハンサーの欠失、およびその後の発現解析のためにF1 ES細胞の使用は、以下の手順で説明します。 <img alt="図1" src="/files/ftp_upload/53552/53552fig1.jpg" /> シス -regを 研究するためにCRISPR / Cas9を使用して、図1エンハンサーの削除遺伝子発現のピュレーション。 ハツカネズミ 129とハツカネズミのcastaneus間の交差によって生成された（A）F1 ES細胞は、対立遺伝子特異的欠失を可能にするために使用されます。 （B）2ガイドRNA（gRNA）は、エンハンサー領域の大Cas9媒介性欠失を誘導するために使用されます。 （C）プライマーセットは、大モノおよび二対立遺伝子の欠失を同定するために使用されます。オレンジ色のプライマーは内側プライマーである、紫色のプライマーは、外部プライマーであり、緑のプライマーはgRNAフランキングプライマーです。遺伝子発現の（D）の変更は、対立遺伝子特異的定量PCRを使用して監視しています。 RFUは相対蛍光単位を示している。 <a href="https://www.jove.com/files/ftp_upload/53552/53552fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</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.

ここで説明するプロトコルは、CRISPR / Cas9ゲノム編集（ 図1）を使用して生成された1アレルエンハンサー削除細胞における遺伝子発現のシス -regulationを研究するためにF1 ES細胞を使用しています。遺伝子型決定および遺伝子発現のためのgRNAおよび対立遺伝子特異的プライマーの設計は、このアプローチにおける重要な要因です。各対立遺伝子特異的?...

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).

生成CRISPR / Cas9媒介1アレル欠失マウス胚性幹細胞でエンハンサー機能を研究するために、

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 ビュー.  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).

ビデオ: 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.

無血清単層培養中のマウス胚性幹細胞の神経分化

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

発生生物学

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.

幹細胞様<em&gt;アフリカツメガエル</em&gt;胚外植片を早期神経発達の特徴を研究するために<em&gt;インビトロ</em&gt;と<em&gt;インビボ</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.

リコンビナーゼ媒介カセット交換を用いて、マウス胚性幹細胞における構造と機能の研究

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.

生活そのままゼブラフィッシュの単一セルの光電

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.

核の分布、蛋白質および細胞骨格を研究する線虫生殖細胞の解析

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.

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

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由来筋前駆体におけるジストロフィン発現の回復に関するCRISPR/Cas9技術

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.

紫外C(UV-C)損傷に対する主なマウス眼表面細胞/幹細胞における酸化的損傷の評価

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.

ヒト胚性幹細胞から脳オルガノイドを生成するための静的自己指向法

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.

初期マウス胚からのシーケンシングのための小型RNAライブラリーの調製

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