使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段
Summary
在这里,我们描述了一个协议,使用CRISPR/Cas9系统将基因敲除引入锥虫的细胞外肌瘤。生长表型可以通过细胞计数来追踪轴酸乳腺培养物,或者通过宿主细胞入侵后细胞内乳腺增殖。
Abstract
锥虫病是一种致病原生动物寄生虫,主要在拉丁美洲引起恰加斯病。为了识别针对T.cruzi的新型药物靶点,验证靶向基因在寄生虫的哺乳动物阶段(乳腺)中的重要性是很重要的。T. 克鲁西的阿马斯蒂在宿主细胞内复制;因此,如果不经过其他发展阶段,就很难进行淘汰实验。最近,我们小组报告了一种生长状况,其中乳腺可异种复制长达10天,而不会失去其类似乳腺的特性。通过使用这种时间轴质乳腺培养,我们成功地将gRNA直接引入Cas9表达的乳腺,导致基因敲除,并仅在乳腺阶段分析其表型。在本报告中,我们描述了一个详细的方案,用于产生体外衍生的细胞外乳腺,并在CRISPR/Cas9介导的敲除实验中利用轴培养。敲除的乳腺的生长表型可以通过轴培养的细胞计数或宿主细胞入侵后细胞内乳腺的复制来评估。该方法绕过寄生虫阶段分化通常涉及生产转基因或敲除的乳腺。利用时间轴突培养具有扩大T.cruzi特定阶段研究的实验自由的潜力。
Introduction
锥虫病是恰加斯病的致病剂,主要在拉丁美洲1流行。T. cruzi具有独特的生命周期阶段,因为它在昆虫载体和哺乳动物宿主2之间传播。T. cruzi在吸血三胺虫的中肠道中复制为表皮球菌,在被沉积在人类或动物宿主之前,在后肠中分化成传染性代谢性胰小体球菌。一旦锥虫通过叮咬部位或通过粘膜进入宿主体,寄生虫就会侵入宿主细胞,并转化为称为乳腺的无旗体圆形。乳腺在宿主细胞内复制,并最终分化成锥虫,从宿主细胞中爆发并进入血流,感染另一个宿主细胞。
由于目前可用的化疗药物,苯甲酰氨基苯甲酸和尼富蒂莫克斯,引起不良副作用,并在疾病的慢性阶段无效3,这是极大的兴趣,确定针对T.cruzi的新药物靶点。近年来,CRISPR/Cas9系统已成为一个强大的工具,有效地执行基因敲除在T.cruzi,无论是通过转染单独或单质粒含有gRNA和Cas9 4,通过稳定表达Cas9和随后的引入gRNA 5,6,7或转录模板的gRNA8,或通过电穿孔预成型的gRNA/Cas9 RNP复合物7,9。这一技术进步被高度期待加速恰加斯病的药物靶点研究。
为了继续药物开发,验证靶基因或药物候选化合物在T.cruzi的药效中的重要性至关重要,因为它是寄生虫在哺乳动物宿主中的复制阶段。然而,这是一项具有挑战性的任务,因为由于存在阻塞宿主细胞,因此不能直接操纵乳腺。在利什曼尼亚,一种与T.cruzi密切相关的原生动物寄生虫,开发了一种斧头阿马斯蒂戈特培养方法,并已用于药物筛选测定方法10,11,12, 13.虽然轴突和细胞内乳腺14在化合物的易感性上有一些差异,但维持轴培养的能力为研究利什曼病临床相关阶段的基本生物学15、16。在T.cruzi的情况下,关于自然发生的细胞外乳腺(EA)17和EA 17,18,19的体外生产文献可追溯到几十年前。此外,EA已知具有传染性能力20,虽然小于锥形雄蒂戈特,和马斯蒂戈特宿主入侵的机制已阐明近年来(审查由Bonfim-Melo等人21)。然而,与利什曼尼亚不同,EA在T.cruzi中没有被用作实验工具,主要是因为EA被认为是细胞内寄生虫,因此在实际操作中并未被视为”复制形式”感。
最近,我们小组提议利用T.克鲁兹的EA作为时间轴文化22。T. 克鲁西图拉胡恩菌株的Amastigotes可在37°C下在LIT培养基中无宿主细胞复制长达10天,而不会严重恶化或丧失类似亲子的特性。在无宿主生长期,EA通过常规电穿孔、药物滴定测定与胰蛋白酶化合物和CRISPR/Cas9介导敲除,然后进行生长表型监测,成功地用于外源性基因表达。在本报告中,我们描述了在敲除实验中产生体外衍生EA和利用轴突腺体的详细方案。
Protocol
注:图 1描述了整个实验流程的概述。 图 1:使用 EA 的挖空实验概述。组织培养衍生的胰腺炎被收获并分化成EA.gRNA通过电穿孔转染成Cas9表达的乳腺,并且通过轴仿或通过宿主细胞入侵后的细胞内复制。<a href="https://www.jove.com/files/ftp_upload/59962/599…
Representative Results
通过游出程序隔离胰岛 为了通过游出程序从污染旧的EA中收获新鲜的胰小球,细胞颗粒至少需要孵育1小时。 孵育颗粒超过2小时不会显著增加在溶液中游泳的胰小球的数量(图 2B.在这个特殊的实验中,在初始混合物中,胰小体测定的百分比为38%,在任意给定时间点,游出后百分比超过98%。从两个T-75瓶的汇合培养,我们经常获得3-4 x 107细胞的?…
Discussion
我们证明,在CRISPR/Cas9介导的基因敲除中,通过将gRNA直接电化到Cas9表达的EA中,可以利用T.克鲁兹的乳腺的轴培养。这样,在乳腺发育阶段,可以评估靶基因的本质,而无需经历其他发育阶段。
乳腺转染的另一个有益方面是对大量靶基因的检测方便。一旦Cas9表达T.cruzi和宿主哺乳动物细胞的共同培养建立,它只需要几天时间将组织衍生的胰腺炎转化为EA和转染gRNA,以获得敲?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了JSPS KAKENHI授予编号18K15141到Y.T.的部分支持。
Materials
| 20% formalin solution | FUJIFILM Wako Pure Chemical | 068-03863 | fixing cells |
| 25 cm2 double seal cap culture flask | AGC Techno Glass | 3100-025 | |
| 75 cm2 double seal cap culture flask | AGC Techno Glass | 3110-075 | |
| All-in One Fluorescence Microscope | Keyence | BZ-X710 | |
| Alt-R CRISPR-Cas9 crRNA (for Control) | IDT | custom made | target sequence = GGACGGCACCTTCATCTACAAGG |
| Alt-R CRISPR-Cas9 crRNA (for TcCGM1) | IDT | custom made | target sequence = TAGCCGCGATGGAGAGTTTATGG |
| Alt-R CRISPR-Cas9 crRNA (for TcPAR1) | IDT | custom made | target sequence = CGTGGAGAACGCCATTGCCACGG |
| Alt-R CRISPR-Cas9 tracrRNA | IDT | 1072532 | to anneal with crRNA |
| Amaxa Nucleofector device | LONZA | AAN-1001 | electroporation |
| Basic Parasite Nucleofector Kit 2 | LONZA | VMI-1021 | electroporation |
| BSA | Sigma-Aldrich | A3294 | component of the medium for in-vitro amastigogenesis |
| Burker-Turk disposable hemocytometer | Watson | 177-212C | cell counting |
| Coster 12-well Clear TC-Treated Multiple Well Plates | Corning | 3513 | |
| DMEM | FUJIFILM Wako Pure Chemical | 044-29765 | culture medium |
| Fetal bovine serum, Defined | Hyclone | SH30070.03 | heat-inactivate before use |
| G-418 Sulfate Solution | FUJIFILM Wako Pure Chemical | 077-06433 | selection of transformant |
| Hemin chloride | Sigma-Aldrich | H-5533 | component of LIT medium |
| Hoechst 33342 | Thermo Fisher Scientific | H3570 | staining of nuclei |
| Liver infusion broth, Difco | Becton Dickinson | 226920 | component of LIT medium |
| MES | FUJIFILM Wako Pure Chemical | 349-01623 | component of the medium for in-vitro amastigogenesis |
| PBS (–) | FUJIFILM Wako Pure Chemical | 166-23555 | |
| Propidium Iodide | Sigma-Aldrich | P4864-10ML | staining of dead cells |
| RPMI 1646 | Sigma-Aldrich | R8758 | medium for metacyclogenesis |
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Citazione di questo articolo
Akutsu, Y., Doi, M., Furukawa, K., Takagi, Y. Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System. J. Vis. Exp. (149), e59962, doi:10.3791/59962 (2019).