이 작품은 NIH 보조금 R01-CA158283 및 NSFC에 의해 ZWYW에 31,400,726는 젊은 천 인재 프로그램 및 국립 자연 과학 중국의 재단 (보조금 31471235 및 81422038)에 의해 자금 지원 허용 지원되었다. XY 중국의 박사후 과학 재단 (2015M571612)에 의해 지원된다.

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+alternative+splicing+markers+for+breast+cancer.">Identification of alternative splicing markers for breast cancer.</a> Cancer Res. 68 (22), 9525-9531 (2008).</li><li>Shapiro, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+EMT-Driven+Alternative+Splicing+Program+Occurs+in+Human+Breast+Cancer.">An EMT-Driven Alternative Splicing Program Occurs in Human Breast Cancer.</a> PLoS Genet. 7 (8), 1002218 (2011).</li><li>David, C. J., Manley, J. 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저자가 공개하는 게 없다.

이 보고서는 구체적으로 표적 유전자의 대체 스 플라이 싱을 조작 할 수있는 인공 접합 요소의 설계 및 구성에 대한 상세한 설명을 제공한다. 이 방법은 사용자의 특이성을 갖는 RNA 결합 골격을 생성하기 위해 반복 PUF의 고유 모드 RNA 결합을 이용한다. 그것은 중 하나를 활성화하거나 접합을 억제하는 데 사용할 수 있습니다. 이 프로토콜의 핵심 단계는 ESFs의 특이성을 정의 r...

대부분의 인간 유전자는 스 플라이 싱 (AS)가 크게 게놈 (1, 2)의 부호화 복잡도를 증가 구별 활동 여러 이성체를 제조 겪는다. AS는 유전자의 기능을 조절하는 주요 메커니즘을 제공하며, 밀접 다른 셀룰러 및 발달 단계 3, 4에서 다양한 경로를 통해 조절된다. 접합 misregulation 인간의 질병 5, 6, 7, 8의 일반적인 원인이기 때문에, 접합 규제를 대상으로하는 것은 매력적인 치료 경로가되고있다. 주로 접합 촉진제 또는 대체 엑손 소음기로서 기능 mRNA의 사전에 규정 -Elements 시스 접합 (SRES)에 의해 제어되는 바와 같이, 스플 라이스 규제의 간략화 모델에 따라. 목SREs는 스 플라이 싱 반응을 촉진 또는 억제하는 다양한 트랜스 - 작용 단백질 인자 ( 즉, 스 플라이 싱 인자)를 구체적으로 모집한다 3 , 9 . 대부분의 trans- acting splicing factor는 splicing을 제어하기 위해 타겟과 이펙터 도메인을 인식하기 위해 별도의 서열 특이 적 RNA 결합 도메인을 가지고 있습니다. 가장 잘 알려진 예는 exon splicing enhancer를 결합하는 N- 말단 RNA 인식 모티프 (RRMs) 및 엑손 삽입을 촉진시키는 C- 말단 RS 도메인을 포함하는 세린 / 아르기닌이 풍부한 (SR) 단백질 군의 구성원이다 . 반대로, hnRNP A1은 RRM 도메인을 통해 엑소닉 스 플라이 싱 사일런서에 결합하고 C- 말단 글리신이 풍부한 도메인을 통해 엑손 삽입을 억제한다. 이러한 모듈 형 구성을 사용하여 연구자는 서로 다른 효과를 갖는 특정 RNA 결합 도메인 (RBD)을 결합함으로써 인공적인 접합 인자를 조작 할 수 있어야한다활성화 도메인을 TOR 또는 접합을 억제. 이러한 설계의 핵심은 동화 영역의 DNA 결합 모드 유사 프로그램 RNA 결합 특이성과 함께 대상을 인식하는 소정의 RBD를 사용하는 것이다. 그러나, 대부분의 천연 스플 라이스 인자는 RRM을 포함하거나 이와 친 화성이 약하고 짧은 RNA 요소를 인식하고 K 동성 (KH) 도메인, 예측 RNA 단백질 인식 &quot;코드&quot;(12)이 부족하다. PUF 반복 단백질의 RBD는 (즉, PUF 도메인) (14), 특히 다른 RNA를 인식하는 PUF 도메인의 재 설계를 허용, 독특한 RNA 인식 모드가 13 목표로하고있다. 정규 PUF 도메인은 각각 8 NT의 RNA 표적의 단일 염기를 인식하는 세 개의 α 나선 여덟 반복을 포함한다. t의 왓슨 - 크릭 가장자리 번째 α 나선 형태 특정 수소 결합의 특정 위치에서 아미노산의 측쇄그는 각각의 반복 ( 그림 1A )의 RNA 바인딩 특이성을 결정 RNA베이스. PUF 반복의 RNA 염기 인식을위한 코드는 놀라 울 정도로 간단하며 ( Figure 1A ), 가능한 8 염기 조합을 인식하는 PUF 도메인을 생성 할 수 있습니다 (Wei and Wang 15 ). 이 모듈 형 설계 원리는 맞춤화 된 PUF 도메인과 접합 변조 도메인 ( 예 : SR 도메인 또는 Gly- 풍부한 도메인)으로 구성된 공학적 접합 요소 (ESF)를 생성 할 수있게합니다. 이 ESFs는 스 플라이 싱 액티베이터 (splicing activator) 또는 다양한 유형의 스 플라이 싱 이벤트를 제어하기위한 억제제로서 기능 할 수 있으며, 인간 질병과 관련된 내인성 유전자의 스 플라이 싱을 조작하는 도구로서 유용하다. 예를 들어, Bcl-x 유전자의 스 플라이 싱을 구체적으로 변경하기 위해 PUF-Gly- 유형 ESF를 구축했다(Bcl-xL)을 프로 - 아폽토시스 (pro-apoptotic) 단 이소성 형태 (Bcl-xS)로 전환시키는 것을 특징으로하는 방법. Bcl-x isoform의 비율을 바꾸는 것은 여러 가지 암 세포를 여러 항암 화학 요법 약물에 민감하게 만들기에 충분했으며, 이러한 인공 인자가 잠재적 인 치료 시약으로 유용 할 수 있음을 시사한다. 공지 된 스 플라이 싱 이펙터 도메인 ( 예 : RS 또는 Gly- 리치 도메인)으로 스 플라이 싱을 제어하는 ​​것 외에도, 설계된 PUF 인자를 사용하여 새로운 스 플라이 싱 인자의 활성을 조사 할 수있다. 예를 들어,이 접근법을 사용하여 여러 SR 단백질의 C 말단 도메인이 다른 pre-mRNA 영역 18에 결합 할 때 접합을 활성화하거나 억제 할 수 있다는 것을 보여주었습니다. RBM4의 알라닌이 풍부한 주제는 접합 19 를 억제 할 수 있고, DAZAP1의 프롤린이 풍부한 모티프는 스 플라이 싱을 향상시킬 수있다 20 , 21 &lt;/ sup&gt;. 이러한 새로운 기능 영역은 추가적인 유형의 인위적 요인을 구성하여 접합을 미세 조정할 수 있습니다.

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			<td height="21" style="height:21px;width:420px;">High-fidelity DNA polymerase (Phusion High-Fidelity) with PCR buffer</td>
			<td style="width:155px;">New England Biolabs</td>
			<td style="width:103px;">M0530L</td>
			<td style="width:491px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA ligase (T4 DNA ligase)</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liposomal transfection reagent (Lipofectamine 2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reduced serum medium (Opti-MEM)</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA extraction buffer (TRIzol Reagent)</td>
			<td>ambion</td>
			<td>15596018</td>
			<td>TRIzol reagent includes phenol, which can cause burns. Wear gloves when handling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A7638-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td>Life Technologies</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III reverse transcriptase</td>
			<td>Invitrogen</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caspase-3 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PARP&#160;antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bcl-x antibody</td>
			<td>BD Bioscience</td>
			<td>610211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">beta-actin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A5441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alpha-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T5168</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FLAG antibody</td>
			<td>Sigma-Aldrich</td>
			<td>F4042</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>Amersham-Pharmacia</td>
			<td>RPN203D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECL Western Blotting detection reagents</td>
			<td>Invitrogen</td>
			<td>WP20005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA55021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence-activated cell sorter</td>
			<td>BD Bioscience</td>
			<td>FACSCalibur&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>GE Healthcare</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium iodide (PI)</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7638-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-lysine&#160;</td>
			<td>Sigma</td>
			<td>P-4832</td>
			<td>Filter sterilize and store at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector&#160; pWPXLd</td>
			<td>Addgene</td>
			<td>12258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vector psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I (RNase-free)</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Oligo(dT)18 Primer</td>
			<td>Thermo Scientific</td>
			<td>SO131&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse secondary antibody (Anti-mouse IgG, HRP-linked Antibody)</td>
			<td>Cell Signaling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering artificial factors to specifically manipulate alternative splicing in human cells

대부분의 진핵 생물 RNA의 프로세싱은 pre-mRNA target과 effector domain을 특이 적으로 결합시키는 RNA 인식 모듈을 포함하여 모듈 형 배열을 갖는 RNA Binding Protein (RBP)에 의해 매개된다. 이전에 우리는 인간 Pumilio 1의 PUF 도메인의 고유 한 RNA 결합 모드를 이용하여 RNA 신진 대사를 조작하기 위해 다양한 인공 RBP를 설계하는 프로그래밍 가능한 RNA 바인딩 스캐 폴드를 생성했습니다. 여기에는 세부적인 프로토콜이 구체적으로 표적 유전자의 선택적인 접합을 조절하도록 설계된 공학적 접합 인자 (ESF)를 구축하기 위해 기술되어있다. 이 프로토콜에는 특정 RNA 표적을위한 맞춤화 된 PUF 스캐 폴드를 설계하고 구성하는 방법, 디자이너 PUF 도메인과 이펙터 도메인을 융합시켜 ESF 발현 플라스미드를 구축하는 방법 및 ESF를 사용하여 표적 유전자의 접합을 조작하는 방법이 포함됩니다. 이 방법의 대표 결과에서, 우리는 또한 일반적인 assays를 설명했다스 플라이 싱 리포터를 사용한 ESF 활동, 배양 된 인간 세포에서의 ESF의 적용 및 스 플라이 싱 변화의 후속 효과. 이 보고서의 상세한 프로토콜을 따르면, 다른 유형의 Alternative Splicing (AS)의 조절을위한 ESF를 설계하고 생성 할 수 있으며, 접합 조절 및 다른 접합 이형의 기능을 연구하기위한 새로운 전략을 제공합니다. 또한, 설계된 PUF 영역과 다른 기능 영역을 융합시킴으로써 연구원은 RNA 공정의 다양한 단계를 조작하기 위해 특정 RNA를 표적으로하는 인공 인자를 설계 할 수있다.

이 보고서는 ESF 및 접합 기자의 설계 및 구성을위한 전체 프로토콜을 설명합니다. 또한 내인성 유전자의 AS 조작에있어 ESF의 추가 적용에 대해 설명한다. ESF 매개 스 플라이 싱 변경의 전형적인 결과를 설명하기 위해 이전 연구의 데이터를 예로 사용합니다. 상이한 기능성 영역을 갖는 ESF는 표적 카세트 엑손 ( 도 1D 및 E )의 ?...

Watch this Scientific Journal Video about Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells at JoVE.com

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

이 보고서는 특히 포유 동물 세포에서 표적 유전자의 스 플라이 싱을 조절하는 신규 한 인공 접합 인자 (ASFs)를 설계하고 구성하는 생명 공학 방법을 설명한다. 이 방법은 RNA 대사의 다른 측면을 조작하는 다양한 인공 요소를 엔지니어에게 확장 할 수 있습니다.

인공 요소 엔지니어 것은 특히 인간 세포에서의 스 플라이 싱을 조작 할

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

engineering-artificial-factors-to-specifically-manipulate-alternative

Research

JoVE Journal

Genetics

8.9K 조회수.  Shanghai Institutes for Biological Sciences (SIBS).  이 보고서는 특히 포유 동물 세포에서 표적 유전자의 스 플라이 싱을 조절하는 신규 한 인공 접합 인자 (ASFs)를 설계하고 구성하는 생명 공학 방법을 설명한다. 이 방법은 RNA 대사의 다른 측면을 조작하는 다양한 인공 요소를 엔지니어에게 확장 할 수 있습니다. 

비디오: Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

갓 고립 된 인간의 산모 Cytotrophoblasts에 siRNA를 형질 및 EMSA 분석

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

연구

유전학

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

생리 산소 조건에 노출 된 인간 세포의 캡 결합 단백질의 분석

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9에 의해 Murine 인간과 조 혈 조상 세포의 고효율 유전자 장애

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

대체 사전 mRNA 접합 교 잡 시험 현장에서 RNA를 사용 하 여 마우스 뇌 섹션에서의 정량 분석

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

인간에서 태그 생 단백질 유도 만능 줄기 세포를 사용 하 여 CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

셀 기반 비 코딩 RNAs 특히 mRNA 번역을 향상 시킬 수 있는 SINEUP의 분석 실험

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Dictyostelium discoideum 셀 선택에 따라 성장과 박테리아에서의 유전 공학

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

렌티 바이러스는 낮은 부착 플레이트에 준비 인간의 의사에 유전자 침묵 중재

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

포유류 세포에 있는 풀린 CRISPR 기지를 둔 유전 스크린

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

인간 다능성 줄기 세포에서 CRISPR-CAS9를 사용하여 정의된 게놈 수정 생성

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...