Transgenesis in Strongyloides and related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing.
Transgenesis in Strongyloides and related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing.
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DOI：
10.1017/s0031182016000391
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发表时间：
2017-03
期刊：
影响因子：
2.4
通讯作者：
Li X
中科院分区：
文献类型：
--
作者：
Lok JB;Shao H;Massey HC;Li X
蛔虫和副蛔虫的转基因于2006年完成，其基础是20多年前秀丽隐杆线虫的转基因技术。这些技术的适应是可能的，因为圆线虫和相关的寄生虫属进行至少一代的自由生活发育，成年雄性和雌性居住在被受感染宿主粪便污染的土壤中。这种寄生虫的转基因是通过将DNA结构体微注射到自由生活的雌性远性腺的合胞体中完成的。在粪类圆线虫中，质粒编码的转基因在基因转移后的F1代中以启动子调控的方式表达，但随后沉默。在粪球菌中，转基因的稳定遗传和表达需要将其整合到基因组中，而使用piggyBac转座子系统创建的整合子已衍生出稳定的系。更直接的基因功能研究涉及突变的转基因构建体的表达，旨在改变细胞内运输和发育调节，揭示了胰岛素调节转录因子Ss-DAF-16的功能。类圆线虫和副类圆线虫的转基因为这类寄生虫的基因组编辑和转录操作开辟了强有力的新方法。本文介绍了其中一种方法CRISPR/Cas9的原理证明。
Transgenesis for Strongyloides and Parastrongyloides was accomplished in 2006 and is based on techniques derived for Caenorhabditis elegans over two decades earlier. Adaptation of these techniques has been possible because Strongyloides and related parasite genera carry out at least one generation of free-living development, with adult males and females residing in soil contaminated by feces from an infected host. Transgenesis in this group of parasites is accomplished by microinjecting DNA constructs into the syncytia of the distal gonads of free-living females. In Strongyloides stercoralis, plasmid-encoded transgenes are expressed in promoter-regulated fashion in the F1 generation following gene transfer but are silenced subsequently. Stable inheritance and expression of transgenes in S. stercoralis requires their integration into the genome, and stable lines have been derived from integrants created using the piggyBac transposon system. More direct investigations of gene function involving expression of mutant transgene constructs designed to alter intracellular trafficking and developmental regulation have shed light on the function of the insulin-regulated transcription factor Ss-DAF-16. Transgenesis in Strongyloides and Parastrongyloides opens the possibility of powerful new methods for genome editing and transcriptional manipulation in this group of parasites. Proof of principle for one of these, CRISPR/Cas9, is presented in this review.