Abstract:

Objective To establish an approach for evaluating the development of Schistosoma japonicum receiving RNA interference, after being transferred to mice, in order to provide a tool for studying functional genes in the post-genomics era. Methods The primers of S. japonicum cathepsin B1(SjCB1) and green fluorescent protein(GFP) genes were designed to amplify  dsRNA. The 14-day-old schistosomula were collected from infected mice, and cultured by in vitro soaking with 6 mg/L SjCB1 dsRNA to specifically knock down the SjCB1 gene (SjCB1 interference group). In the control group, 6 mg/L green fluorescent protein (GFP) dsRNA was used instead (GFP interference group). The schistosomula after interference were surgically transferred back to the mouse mesenteric vein (3 mice in each group, 40 schistosomula/mouse). The adult worms were then collected after growing to sexual maturity. The numbers of male and female worms recovered, the pairing rate, the recovery rate, and the body length were analyzed. The growth and development of the adult worms were assessed. The relative expression level of SjCB1 gene in 14-day schistosomula and in recovered adult worms was detected by real-time PCR.  Results The recovered numbers of female and male worms, the pairing rate and the recovery rate in the SjCB1 interference group were (9.0 ± 1.4), (13.0 ± 2.9), 100% and 58.60%， and those in the GFP interference group were (9.0 ± 2.1), (11.3 ± 2.5), 100% and 54.67%, respectively. None was significantly different between the two groups (P > 0.05). The relative expression levels of SjCB1 in male and female schistosomula before adoptively transferred were 1.022 ± 0.019 and 0.643 ± 0.105, respectively, in the SjCB1 interference group, and both were significantly lower than those in the GFP interference group (2.880 ± 0.297 and 2.753 ± 0.164) (t = 0.000, 0.000; P < 0.01). The relative expression levels of SjCB1 in female and male worms recovered from the SjCB1 interference group were 1.286 ± 0.211 and 1.182 ± 0.287, respectively, which were significantly lower than those in the GFP interference group (5.508 ± 0.326 and 4.896 ± 1.230) (t = 0.013, 0.000; P < 0.01). The lengths of females in the SjCB1 interference group and the GFP interference group were (7.059 ± 1.255) and (8.433 ± 0.749) cm (t = 0.003, P < 0.01), and the lengths of males were (6.993 ± 2.734) and (10.561 ± 1.375) cm, respectively (t = 0.001, P < 0.01). There were no differences in the appearance, the internal reproductive system and the digestive system between the SjCB1 interference group and the GFP interference group. Females in both groups had eggs with a clear shape in the egg molds and the uterus.  Conclusion A standard procedure has been established to observe the developmental phenotypes in vivo of S. japonicum with RNA interference, and the SjCB1 gene has been knocked down by a soaking method. This procedure can be used to study the phenotypes of development-related genes.

Key words: Schistosoma japonicum, RNA interference, Adoptive transfer