A TGF-β type I receptor-like molecule with a key functional role in Haemonchus contortus development

A serine/threonine‐specific protein kinase ofHaemonchus contortuswith a role in the development

“…The partial sequence was PCR‐amplified using primer pairs using the same cycling conditions as described above and cloned into the pTOPO‐Blunt vector. Bt‐cry1Ac was used as an irrelevant control in this RNAi assay . The partial sequence of Bt‐cry1Ac was PCR‐amplified using primer pairs Bt‐cry1Ac‐sF1/Bt‐cry1Ac‐R1 and Bt‐cry1Ac‐sF2/Bt‐cry1Ac‐R2 (Table S1) and cloned into the pTOPO‐Blunt‐T vector.…”

“…RNAi was carried out as previously described . For RNAi treatment, dsRNA was added to plates to a final concentration of 1 μg/μL after encapsulation in a liposome preparation.…”

“…RNAi was carried out as previously described. 18 For RNAi treatment, dsRNA was added to plates to a final concentration of 1 μg/μL after encapsulation in a liposome preparation. The liposome formulation of dsRNA was prepared as follows: 1 μL of lipofectamine 2000 (Invitrogen, Waltham, USA), 1 μL of RNase Inhibitor (Thermo Fisher Scientific, Waltham, USA) to a final concentration of 0.5 U/ mL, an amount of dsRNA (approximately 10 μL), and 8 μL of dH 2 O were mixed gently, allowed to stand for 10 minutes, then added to worms in the in vitro culture.…”

“…The liposome formulation of dsRNA was prepared as follows: 1 μL of lipofectamine 2000 (Invitrogen, Waltham, USA), 1 μL of RNase Inhibitor (Thermo Fisher Scientific, Waltham, USA) to a final concentration of 0.5 U/ mL, an amount of dsRNA (approximately 10 μL), and 8 μL of dH 2 O were mixed gently, allowed to stand for 10 minutes, then added to worms in the in vitro culture. 19 Using an established method, 18 L3s of H. contortus were exsheathed (called xL3), washed five times in sterile physiological saline and suspended in Earle's Balanced Salt Solution (EBSS) (pH 5.2) medium; xL3s were then treated separately (in triplicate) with dsRNA from Hc-akt-1, dsRNA from an irrelevant gene (Bt-cry1Ac), or remained untreated (control) at 38°C and 20% v/v CO 2 for 3 days using an established method. 18 Subsequently, 30% of worms werer removed and washed thoroughly for subsequent RNA extraction.…”

“…Bt-cry1Ac was used as an irrelevant control in this RNAi assay. 18 The partial sequence of Bt-cry1Ac was PCRamplified using primer pairs Bt-cry1Ac-sF1/Bt-cry1Ac-R1 and Bt-cry1Ac-sF2/Bt-cry1Ac-R2 (Table S1) and cloned into the pTOPO-Blunt-T vector. Each of the six plasmids was linearized and examined on an agarose gel to confirm that cleavage was complete; then, DNA free from contaminating proteins and RNA was obtained by column purification (AxyPrep PCR Cleanup Kit, Axygen, San Francisco, USA) following the manufacturer's instructions.…”