Name: virus delivery of crispr guides to the murine prostate for gene alteration
Uploaded: 2018-04-27T15:30:00.000+00:00
Duration: 6 min 21 s
Description: Watch this Scientific Journal Video about Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration at JoVE.com

MR 是由丹麦癌症协会 (R146-A9394-16-S2) 的一支奖学金资助的。MFB 和 MKT 由 AUFF 新星 (AUFF-E-2015-FLS-9-8) 资助。MR 和 MFB 由研究生院, 健康, 非盟共同出资。E.F.W. 实验室得到西班牙经济部 (SAF2015-70857, 由 ERDF-欧盟共同出资) 的赠款和一个紧急救济高级补助金 (741888-CSI 乐趣) 的支持。
我们要感谢莉莉法哈多梅勒 (基因、发育和疾病;国家癌症研究中心) 对手稿的批判性阅读。

该协议涉及实验室小鼠的手术方法。所有动物实验必须由一个机构动物护理和使用委员会 (IACUC) 单独审查和批准。由于该方法是基于动物的恢复和生存, 确保适当的麻醉, 疼痛管理, 并在任何时候无菌的外科环境。在手术期间, 使用加热垫防止体温过低, 直到麻醉恢复。
1. 开始考虑事项
<ol><li>根据使用的病毒, 不同的效价要求可能适用;相应地稀释病毒。 注: 在本例中, 在 PBS 中稀释的腺相关病毒注射在 1 x 1012的。该病毒具有9型, 并包含在泛素助剂 (图 1) 下的Pten和蛋白表达的指南 RNA。Pten的丢失增加了前列腺上皮的 pAKT 和增生 (13)。</li>
	<li>在手术时使用雄性小鼠在8周的年龄, 以确保充分的前列腺发育。 注意: 本实验中显示的小鼠是在 C57BL/6J 背景12 (图 1) 上 Rosa26-LSL-Cas9-EGFP 敲老鼠。</li>
	<li>新鲜的准备一个一般的麻醉混合, 如果可能的话, 保持它不育。 注意: 为了更好的动物恢复手术后, 强烈建议麻醉解毒剂。<ol>
<li>要准备5毫升麻醉剂的总容量, 混合0.15 毫升 medetomidine 盐酸盐 (1 毫克/毫升), 0.4 毫升咪达唑仑 (5 毫克/毫升), 和0.25 毫升布托啡诺 (10 毫克/毫升) 与4.2 毫升无菌盐水水。 注: 替代麻醉方法, 如可吸入异氟醚, 可以根据机构动物护理指南使用。</li>
		<li>为了逆转盐酸 medetomidine 术后的效果, 将0.1 毫升 atipamezole (5 毫克/毫升) 与4.9 毫升无菌盐水混合使用。</li>
	</ol>
</li>
	<li>通过适当的消毒和灭菌, 准备无菌的外科手术环境。使用前用高压釜手术器械。</li>
</ol>2. 向小鼠前列腺提供病毒
<ol><li>麻醉用无菌1毫升注射器和 27G x ½针腹腔注射动物。使用麻醉剂量的0.01 毫升/克体重。如果手术持续时间超过30分钟, 则每30分钟注射1/3 的起始剂量即可维持麻醉。</li>
	<li>检查麻醉深度, 评估肌肉松弛, 踏板撤退, 和眼睑反射。当观察到反射丢失时, 剃掉动物的下腹部。</li>
	<li>用不育棉拭子小心地用兽医眼膏遮盖动物的眼睛, 以防止因角膜反射不足而失明。</li>
	<li>用70% 乙醇和10% 聚维酮碘擦拭剃腹, 对手术部位进行消毒。将手术区域从手术部位的中心擦洗到外围。</li>
	<li>使用无菌手术剪刀在低腹中线进行垂直皮肤切口, 约1厘米。</li>
	<li>用细点钳抬起腹膜, 以防止损坏下面铺设的器官。小心做一个 < 8 毫米切口使用手术剪刀通过腹膜。 注意: 与人类相比, 啮齿动物的前列腺包括四个单独的裂片。前叶附着于精囊。</li>
	<li>轻轻地将脂肪组织移开, 以发现精囊。使用环钳, 小心地抬起精囊, 直到前前列腺可以识别 (图 1)。</li>
	<li>使用0.5 毫升胰岛素注射器, 用 30G x 8 毫米针在前前列腺上皮细胞中注入30µL 病毒 (3 x 1010病毒微粒) 溶液。尽量减少渗漏, 确保液体在组织内吸收, 形成小气泡。把精囊放回腹腔。</li>
	<li>缝合腹膜与2-3 简单中断针使用6-0 可吸收缝线与13毫米, 3/8 圆, 和锥形点针。</li>
	<li>用3不育 4.8 x 6.5 毫米夹子将皮肤与镊子一起抬起, 以避免损伤腹膜。</li>
	<li>为了更好的恢复, 应用麻醉解毒剂在剂量0.01 毫升/克体重的腹腔注射使用无菌1毫升注射器和 27G x ½ "针。小心地把动物放回笼子里。</li>
</ol>3. 术后程序
<ol><li>在手术后, 保持笼子里的小鼠在加热垫上进行了1小时的治疗, 以防止体温过低直到完全恢复。如果使用解毒剂, 动物应在注射后几分钟内清醒。</li>
	<li>每天监视动物以得到适当的伤口愈合和疼痛征兆。如有必要, 根据机构动物护理指南, 管理额外的止痛药。</li>
	<li>一旦伤口闭合, 4-5 天后取下皮肤夹子。</li>
</ol>

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作者没有什么可透露的。

在本协议中, 我们描述了一种通过病毒注射改变小鼠前列腺前叶基因表达的方法, 为前列腺癌创建了一个强大的新的老鼠模型 (图 2)。在2005年的8中, 首次用利奥et描述了腺病毒的成功管理。我们以前已经展示了如何用腺病毒编码的 recombinase 蛋白可以取代耗时的杂交育种的一个培养的等位基因的组织特定删除9 (图 2D)。由于病毒感染了几个单元格9, 此模型模拟了克隆扩展1415的人类方案, 并且是最佳的癌症研究 (图 2B)。
CRISPR/Cas9 技术的发现为体内基因编辑开辟了新的契机。现在有可能同时改变多个基因, 为这种异癌症提供了一种既节省成本又有时间效率的高价值技术。使用动物模型的研究得益于不仅在生殖, 而且在成人组织中产生基因改变的好处。此外, Cas9 表达小鼠的发展允许病毒传递的创可和 sgRNAs (图 1)。由于指南对基因组没有 Cas9 表达的影响, 这种病毒的工作是非危险的。
腺相关病毒诱发极低的免疫应答, 即使存在长达一年的时间, 病毒也不会集成到宿主基因组中, 这使得它更适合体内挖空研究16。在这方面, 必须指出, 不同的血清型可能会影响不同组织类型的转导效率。因此, 对靶向组织进行优化是实现高效率的关键。使用基因组集成慢病毒北疆可以另一方面允许长期癌基因表达10。
人前列腺不分离在不同的裂片, 并讨论了哪些小鼠前列腺叶最好代表人前列腺。当侧叶被提出时, 不同的裂片在肿瘤起动方面没有显著差异, 如9,17,18,19。病毒传递的另一个关键方面是体内其他细胞可能感染。我们观察到了转基因的前列腺基质细胞。在原位分娩过程中, 可以最大限度地减少风险, 避免血管的发生, 同时防止注射时出现渗漏。进一步的病毒设计, 包括前列腺特异启动子, 如 Probasin 启动子和血清型, 可能会增加细胞特异性。

在过去的十年中, 前列腺癌的检测和治疗有了显著的改善。然而, 随着预期寿命的延长, 前列腺癌的发病率也在增加。在全世界估计有110万例新病例, 这是男性癌症相关死亡的最常见原因之一1。前列腺癌的发展缓慢, 但当癌症进展到晚期转移状态时, 由于治疗选择有限, 预后较差。到目前为止, 只有少数基因被确定为常见的驱动程序在这个癌症, 其异质性和 multifocality 阻碍检测生物标志物和多弹头疾病驱动程序2,3。
传统的生成 GEMMs 的技术往往因其复杂性、及时的费用和成本而受到损害。条件敲除模型已被广泛用于研究前列腺癌候选基因, 这导致胚胎致死时, 灭活在生殖4。大多数常见的模型涉及前列腺特定的 recombinase 驱动由修改 Probasin5或 PSA6启动子集成在 GEMM 通过额外的杂交育种。在这些模型中, 感兴趣的基因将被瞄准大多数前列腺上皮细胞, 产生增生的整个器官, 这可能会损害动物的泌尿道功能7。
通过注射到小鼠前列腺前叶的病毒传递, 可以解决这个问题, 只针对几个细胞8。考虑到实验室的技术先决条件、专门知识和目标, 该方法从各种可能的变化中受益。成功的方法使用了针对 JunB和Pten9或慢病毒北疆目标的腺病毒的目标Pten和Trp53 10 已显示在其他人中。添加转基因, 如荧光素酶, 到病毒结构或 GEMM 将进一步通过生物发光成像的11, 使非侵入性监测疾病进展。
基于 CRISPR/Cas9 技术的基因组编辑揭示了通过快速生成体细胞挖空12来研究癌症的新的快速机会。单导 rna (sgRNAs) 定向到小鼠前列腺前叶的病毒传递建立了一个生理上更相关的前列腺癌模型。通过这种方法, 携带所选突变的单个细胞可以形成能够扩张和侵入的克隆。此外, 使用引导 rna 的多目标基因将产生细胞克隆与改变的不同基因。这将允许肿瘤的异质性和自然选择压力的癌症进展, 这可以揭示每个基因改变或上位机制的重要性。
在这里, 我们提出了一种方法, 提供病毒微粒的小鼠前列腺改变基因表达。小的腹部切口, 鼠前前列腺叶被暴露和病毒微粒被注射入裂片。手术后五天, 手术夹可从皮肤上取出, 前列腺癌可从8周后进行分析。总的来说, 这是一个快速和成本效益高的程序, 它对鼠标没有什么影响, 并允许较大的肿瘤发展, 而不损害鼠标。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B6J.129(B6N)-Gt(ROSA)26Sor&lt;sup&gt;tm1(CAG-cas9*,-EGFP)Fezh&lt;/sup&gt;/J mice</td><td>The Jackson Laboratory</td><td>26175</td><td></td></tr><tr><td>0,5 ml U-100 insulin syringe</td><td>BD</td><td>324825</td><td>for virus injection</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>300013</td><td>for anesthesia injection</td></tr><tr><td>30 G 1/2&#039;&#039; needle</td><td>BD</td><td>304000</td><td>for anesthesia injection</td></tr><tr><td>6-0 Polysorb Suture</td><td>Medtronic</td><td>GL889</td><td>to close the peritoneum</td></tr><tr><td>Disposable sterilized surgery drape</td><td>multiple suppliers</td><td></td><td></td></tr><tr><td>Heating pad</td><td>multiple suppliers</td><td></td><td></td></tr><tr><td>Povidone-Iodine Prep Pad</td><td>Fisher Scientific</td><td>06-669-70</td><td></td></tr><tr><td>Trimming machine</td><td>Aesculap</td><td>GT415</td><td>to shave the abdomen</td></tr><tr><td>Dumont Forceps</td><td>F.S.T</td><td>11252-00</td><td></td></tr><tr><td>Halsey Micro Needle Holder</td><td>F.S.T</td><td>12500-12</td><td></td></tr><tr><td>Iris Scissors</td><td>F.S.T</td><td>14094-11</td><td></td></tr><tr><td>Narrow Pattern Forceps</td><td>F.S.T</td><td>11002-12</td><td></td></tr><tr><td>Ring Forceps</td><td>F.S.T</td><td>11106-09</td><td></td></tr><tr><td>Wound Clip System Handle incl. Clips</td><td>F.S.T</td><td>12030-01</td><td>to close the skin</td></tr><tr><td>Wound Clip System Remover</td><td>F.S.T</td><td>12030-04</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>different models have been used</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Antisedan</td><td></td><td></td><td>obtained from the animal facility</td></tr><tr><td>Butorphanol</td><td></td><td></td><td>obtained from the animal facility</td></tr><tr><td>Medetomidinhydrochlorid</td><td></td><td></td><td>obtained from the animal facility</td></tr><tr><td>Midazolam</td><td></td><td></td><td>obtained from the animal facility</td></tr><tr><td>100% Ethanol</td><td>Fisher Scientific</td><td>22-032-103</td><td></td></tr><tr><td>Eye ointment</td><td>Takeda</td><td>7242</td><td></td></tr><tr><td>Virus (AAV, AV)</td><td>multiple suppliers</td><td></td><td>virus used in this protocol is an in-house production</td></tr><tr><td>pAKT antibody</td><td>CST</td><td>4060</td><td>used 1:200 dillution</td></tr><tr><td>GFP anitbody</td><td>CST</td><td>2956</td><td>used 1:100 dillution</td></tr></tbody></table>

virus delivery of crispr guides to the murine prostate for gene alteration

随着前列腺癌发病率的增加, 新的肿瘤驱动剂或调节剂的鉴定是至关重要的。基因工程鼠模型 (GEMM) 为前列腺癌的肿瘤异质性和其复杂的微进化动力学的阻碍。传统的前列腺癌小鼠模型包括生殖和条件性挖空、癌基因的基因表达和异种移植模型。在这些模型中生成 "从头" 突变是复杂、耗时且成本高昂的。此外, 大多数传统的模型都以前列腺上皮为目标, 而人前列腺癌则是众所周知的, 只在小的细胞子集中进化为孤立事件。有价值的模型不仅需要模拟前列腺癌的启动, 还要对晚期疾病进行进化。
在这里, 我们描述了一种方法来靶向几个细胞在前列腺上皮中的传感细胞的病毒粒子。对小鼠前列腺进行工程化病毒的传递, 可以改变前列腺上皮细胞中的基因表达。病毒类型和数量将据此定义基因改变的靶向细胞数量, 传感几个细胞用于癌症的启动和许多细胞进行基因治疗。通过手术为主的注射在前叶, 远端从泌尿道, 该模型的肿瘤可以扩大而不损害的泌尿功能的动物。此外, 通过仅针对前列腺上皮细胞的一个子集, 该技术能够使肿瘤的克隆扩张, 从而模仿人类肿瘤的启动, 进展, 以及通过基底膜的侵袭。
这项新技术提供了一个强大的前列腺癌模型, 改善生理相关性。动物的痛苦是有限的, 因为没有额外的育种是需要的, 整体动物计数减少。同时, 对新候选基因和通路的分析也加快了, 从而提高了成本效益。

为了评估病毒对小鼠前列腺的传递, 手术后三月分析了样本。Rosa26-LSL-Cas9-EGFP 小鼠12在暴露于病毒表达的培养蛋白的细胞中表达 GFP。用荧光显微镜检查前列腺样品, 以确定带有 GFP 信号的区域 (图 2A)。GFP 的信号表明前列腺上皮细胞的活性, 而不是 CRISPR 引导的基因编辑是否被诱导。免疫组化部分显示高表达 pAKT 的细胞的焦点区域 (图 2B), 表示Pten13的丢失。pAKT 和 GFP 的免疫荧光联合染色识别了双阳性细胞 (图 2C)。这证实了腺相关病毒对前列腺细胞的转化。总的来说, 这些结果表明,在体内CRISPR/Cas9 基因编辑可以在前列腺上皮中使用腺相关病毒和 Rosa26-LSL-Cas9-EGFP 小鼠进行。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57525/57525fig1.jpg"> 图 1: 过程的说明.该过程是在普拉特et . 生成的 Rosa26-LSL-Cas9-EGFP 鼠标中进行的。12前前列腺 (AP) 附着于精囊 (SV)。病毒粒子表达引导 RNA 和一个创可蛋白被注入到前前列腺, 以改变基因表达。<a href="/files/ftp_upload/57525/57525fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57525/57525fig2.jpg"> 图 2: 通过原位病毒传递对小鼠前列腺进行基因改变.7周大的雄性 Rosa26-LSL-Cas9-EGFP 小鼠被注射与腺相关的病毒, 其中含有一个引导 RNA 的Pten和编码的一个创能蛋白。样本分析3月后, 病毒注射。(A) GFP 荧光成像的前前列腺。(B) 染色为 pAKT (褐色) 的组织学切片 (4 µm) 标记克隆区域, 并丢失Pten表达式。虚线框标志着高放大率的提交。(C) 荧光蛋白染色 (绿色) 和 pAKT (红色)。核酸被 DAPI 染色。左: 染色的未注射前叶显示没有信号的 GFP 或 pAKT。右: 染色的注射叶显示荧光蛋白 (细胞质染色) 和 pAKT (细胞膜定位) 的共同定位。(D) Ptenflox/flox小鼠注射了表达的腺病毒。在病毒传递6月后, 4 µm 前叶组织学切片的 H & E 染色。点状椭圆标志着高等级前列腺上皮内瘤样病变的面积。有关详细信息, 请参见引用9。<a href="/files/ftp_upload/57525/57525fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration at JoVE.com

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

该协议描述了一种新建立的方法, 病毒传递到小鼠前列腺。使用 CRISPR/Cas9 技术, 基因过度表达, 或 recombinase 分娩, 该技术允许基因表达的原位改变, 并实施了新的小鼠模型前列腺癌。

CRISPR 指南对小鼠前列腺病毒传递的基因改变

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

virus-delivery-crispr-guides-to-murine-prostate-for-gene

Nanyang Technological University

Research

JoVE Journal

Cancer Research

8.6K 次观看.  Aarhus University. 该协议描述了一种新建立的方法, 病毒传递到小鼠前列腺。使用 CRISPR/Cas9 技术, 基因过度表达, 或 recombinase 分娩, 该技术允许基因表达的原位改变, 并实施了新的小鼠模型前列腺癌。

视频: Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct&#160;in vivo&#160;CRISPR&#160;screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

在Vivo CRISPR/Cas9筛查中同时评估小鼠皮肤和口腔中的基因功能

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

科研

遗传学

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

发育生物学

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

神经科学

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

免疫与感染

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

医学

<meta charset="utf8"></head><body>向导 RNA 的逆转录病毒转导用于 Th17 分化中的基因编辑

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

<meta charset="utf8"></head><body>逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

生物学

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells.
To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named &#34;Nanoblades&#34;) were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for &#34;transfection-free&#34; homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

通过病毒样颗粒（"纳米刀片"）在永生化和原代细胞中递送Cas9 / sgRNA核糖核蛋白复合物

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the ...

CRISPR 指南对小鼠前列腺病毒传递的基因改变

摘要

探索更多视频

此视频中的章节

在Vivo CRISPR/Cas9筛查中同时评估小鼠皮肤和口腔中的基因功能

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

逆转录病毒 CRISPR/Cas9 介导的基因靶向在体外研究 Th17 分化

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

通过病毒样颗粒（"纳米刀片"）在永生化和原代细胞中递送Cas9 / sgRNA核糖核蛋白复合物