Figure 1. Functional confirmation of NHEJ-related protein genes by a reporter system.

(a) A NHEJ reporter plasmid was constructed for expression of mCherry under an IE2 promoter, when no I-SceI expressed in cells. (b) The reporter plasmid was co-transfected with helper plasmid into BmN4-SID1 cells, followed by puromycin selection generating NHEJ reporter cells. The cells showed stable mCherry expression. When I-SceI expression vector was transfected into the cells, I-SceI targeting sites were digested. Meanwhile, NHEJ pathway proteins repaired the broken DNA, resulting to construction of a cassette, which expressed EGFP and luciferase proteins. Luciferase assay (Luc assay) was taken after 3 days post-transfection of I-SceI expression vector into NHEJ reporter cells. (c) To test the roles of predicted factors involved in silkworm NHEJ pathway, dsRNAs for the factor gens were added into the culture medium of NHEJ reporter cells, followed by transfection of I-SceI expression vector 3 days later. (d) Another 3 days later, cells were separated to two parts subjected to Luc assay and RT-PCR, respectively. In (a,d), the mean fold was derived from values of luciferase assay normalized by the total protein concentrations of cells in each treatment (the control was set as 1 fold indicated by a dotted line). Differences between the means were evaluated with a two-tailed Student t-test. Significant differences are as follows: *P < 0.05, **P < 0.01.