Tenlen Jennifer R
Department of Biology, Seattle Pacific University, Seattle, Washington 98119
Cold Spring Harb Protoc. 2018 Nov 1;2018(11):2018/11/pdb.prot102368. doi: 10.1101/pdb.prot102368.
Classical genetic analysis in the tardigrade is a challenge because these animals are parthenogens. The publication of the genome has facilitated the study of targeted genes by RNA interference (RNAi), a robust mechanism to disrupt gene function. This protocol describes microinjection of double-stranded RNA (dsRNA) in tardigrades using techniques adapted from protocols originally developed in A DNA template (either genomic or cDNA) is used to prepare dsRNA, to which T7 polymerase binding sites are added at the 5' end of each strand. The dsRNA is injected into adult tardigrades, preferably targeting the gonad or intestine. Injected adults are allowed to recover in spring water and then transferred to culture dishes or individual wells of a 96-well plate.
对缓步动物进行经典的遗传分析具有挑战性，因为这些动物是孤雌生殖的。基因组的公布促进了通过RNA干扰（RNAi）对目标基因的研究，RNAi是一种破坏基因功能的强大机制。本方案描述了利用最初在秀丽隐杆线虫中开发的方案改编的技术，对缓步动物进行双链RNA（dsRNA）显微注射。使用DNA模板（基因组或cDNA）制备dsRNA，在每条链的5'端添加T7聚合酶结合位点。将dsRNA注射到成年缓步动物体内，最好靶向性腺或肠道。注射后的成虫在泉水中恢复，然后转移到培养皿或96孔板的单个孔中。
