Figure 1.

In vitro and in vivo tests of CLCN7^mutant-specific siRNAs. (a) Cartoon depicting the pEGFP-C1 vector used in the study. (b) HEK293 cells stably transfected with the pEGFP-C1 vector carrying the indicated mutations. Expression of the CLCN7 gene was quantified by real-time RT-PCR on RNA extracted from mutant transfectants, against cells transfected with the empty vector, which did not express CLCN7 mRNA (first bar from left). (c–e) HEK293 cells transfected with the indicated vectors, were treated with the CLCN7^mutant-specific siRNA listed in Table 2 as the most effective per each mutation. Concentration-dependent regulation of CLCN7 assessed by real-time RT-PCR, normalized with GAPDH. (f) RT-PCR using primer pairs specific for the Clcn7^G213R mRNA showing transcript amplification only in heterozygous (Clcn7^G213R/WT) and homozygous (Clcn7^G213R/G213R) osteoclasts, while in wild-type osteoclasts (Clcn7^WT/WT) no transcript was amplified. (g) Direct DNA sequencing of the amplified transcript shown in f for the Clcn7^G213R/WT osteoclasts, demonstrating only the mutant sequence. (h) Osteoclasts generated from the bone marrow mononuclear cells of Clcn7^WT/WT and Clcn7^G213R/WT mice were treated with the indicated concentration of scrambled (SCR) or Clcn7^G213R-specific siRNA. Real-time RT-PCR was performed using the primer pairs specific for the mutant transcript validated in (f) and (g). (i) Osteoclasts were generated from the bone marrow mononuclear cells of Clcn7^WT/WT and Clcn7^G213R/WT mice onto bone slices and treated with the indicated concentration of SCR and Clcn7^G213R-specific siRNA. At the end of experiment, cells were removed by sonication and bone resorption evaluated by the pit assay. (j) Three-month-old Clcn7^WT/WT mice were injected once i.p. with 4 mg/kg of Clcn7^G213R-sticky siRNA jetPEI conjugate and sacrificed at the indicated time point. Sera were collected and evaluated for total RNA concentration by Nanodrop. (k) Ten-day-old Clcn7^G21R/WT mice were injected once i.p. with the indicated doses of SCR- or of Clcn7^G213R-sticky siRNA jetPEI conjugate. After 48 hours, mice were sacrificed, RNA was extracted from tibias, and evaluated by real-time RT-PCR using the primer pairs specific for the Clcn7^G213R mRNA validated in (f) and (g). In b–e, h–k data are the mean ± SD of three independent experiments or three animals/group. b–e,h,I,k: Student's t-test. j: one-way analysis of variance (ANOVA). For c–e, statistics was also performed by one way ANOVA (shown in Supplementary Table S3).