Nanoparticle-Mediated Gene Silencing in Zebrafish

siRNAs were encapsulated in polyethylene glycol–polylactic acid nanoparticles using a double emulsion-solvent evaporation technique and then injected into the pericardial sac40 (link)65 66 (link). Briefly, zebrafish were allowed to recover for 1 day after ventricular resection. To evaluate the effect of siRNA on its target gene expression, the hearts were collected at 2 d.p.a., and total RNA was isolated to assess the expression of the respective genes by quantitative RT–PCR. To evaluate the effect of genes on cardiomyocyte proliferation, 50 μl polyethylene glycol–polylactic acid nanoparticle-encapsulated siRNAs was injected first, and ∼1 h later, 50 μl 2.5 mg ml⁻¹ BrdU (B5002; Sigma) was injected into the thoracic cavity daily from 7 to 14 d.p.a. The hearts at 14 d.p.a. were collected for subsequent experiments. siRNA sequences for cdkn1a, cdkn1c and dnmt3ab are shown in Supplementary Table 2.