In vivo RNAi Knockdown Protocol

For in vivo RNAi, different target gene fragments were PCR amplified with 2×Hieff®PCR Master Mix (With Dye) (Yeasen Biotech, Shanghai, China). The corresponding gene fragment was cloned into the pMD™18-T Vector (Takara, Dalian, China) for sequencing to ensure the accuracy of the PCR amplification. The T7 promoter sequence was linked to the 5′ end of the corresponding dsRNA primer, and the PCR amplified product was used to synthesize the dsRNA template. The dsRNA was synthesized and purified using the T7 RiboMAX Express RNAi kit (Promega, WI, USA) according to the manufacturer’s instructions. Control dsRNA (CK, a 92 bp noncoding sequence from the pSTBlue-1 vector) was used [78 (link), 79 (link)]. A volume of 2 μl of dsRNA (2 μg/μl) was injected into the abdomen of 1-day-old adult females and 3-day-old adult females with a 10 μl Hamilton microsyringe [25 (link), 76 (link)]. All primers used for dsRNA synthesis in this study are summarized in Table S1 (Additional file 1).