Generating Transgenics and Knockouts in Strongyloides Species by Microinjection.
Generating Transgenics and Knockouts in Strongyloides Species by Microinjection.
复制标题
通过显微注射在类圆线虫物种中产生转基因和敲除。
DOI：
10.3791/63023
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发表时间：
2021-10-07
影响因子：
1.2
通讯作者：
Hallem, Elissa A.
中科院分区：
文献类型：
--
作者：
Castelletto, Michelle L.;Hallem, Elissa A.
类圆线虫属由多种具有不同宿主范围的皮肤穿透线虫组成，包括粪类圆线虫（Strongyloides stercoralis）和拉氏类圆线虫（Strongyloides ratti）。S.粪寄生虫是一种人体寄生的皮肤穿透线虫，感染了大约6.1亿人，而鼠寄生虫S。ratti与S. stercoralis，并经常被用作S.粪虫两个S。stercoralis和S.通过性腺内显微注射的外源核酸递送技术，响尾蛇易于产生转基因和基因敲除，因此，已经成为尚不适于该技术的其它寄生蠕虫的模型系统。寄生类圆线虫成虫栖息于宿主的小肠，并通过粪便将后代释放到环境中。一旦进入环境，幼虫发育成自由生活的成虫，生活在粪便中并产生后代，必须找到并入侵新的宿主。这种环境世代是圆线虫属物种所特有的，并且在形态上与为C.秀丽线虫可适于与这些寄生线虫一起使用，包括性腺内显微注射。利用性腺内显微注射，可以将多种转基因导入类圆线虫。CRISPR/Cas9组分也可以显微注射以产生突变的类圆线虫幼虫。在这里，性腺内显微注射到类圆线虫的技术，包括准备自由生活的成年人，注射程序，和转基因后代的选择，进行了描述。包括使用CRISPR/Cas9诱变产生的转基因类圆线虫幼虫的图像。本文的目的是使其他研究人员能够使用显微注射来创建转基因和突变的类圆线虫。寄生线虫Strongyloides stercoralis和Strongyloides ratti的功能基因组工具包现在包括转基因、CRISPR/Cas9介导的诱变和RNAi。该方案将展示如何使用性腺内显微注射将转基因和CRISPR组分引入S。stercoralis和S.拉蒂。
The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S. stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S. ratti is closely related to S. stercoralis and is often used as a laboratory model for S. stercoralis. Both S. stercoralis and S. ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique. Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides. The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti now includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.
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影响因子：
64.5
作者：
BARGMANN, CI;HARTWIEG, E;HORVITZ, HR
通讯作者：
HORVITZ, HR
DOI：
10.1073/pnas.1909710117
发表时间：
2020-07-28
影响因子：
11.1
作者：
Gang, Spencer S.;Castelletto, Michelle L.;Hallem, Elissa A.
通讯作者：
Hallem, Elissa A.
影响因子：
2.1
作者：
Dulovic, Alex;Puller, Vadim;Streit, Adrian
通讯作者：
Streit, Adrian
影响因子：
4.6
作者：
Adams, Sally;Pathak, Prachi;Pires-daSilva, Andre
通讯作者：
Pires-daSilva, Andre
影响因子：
--
作者：
Schafer TW;Skopic A
通讯作者：
Skopic A