Mantle transcriptomes of Crassostrea nippona after Hox4 knockdown

This project investigates the role of the Hox4 gene within the cis-regulatory network underlying shell formation in Crassostrea nippona. A shell repair assay was conducted on two-year-old adult oysters. A hole approximately 2 mm in diameter was drilled into the left valve of each individual. Small interfering RNAs (siRNAs) were diluted to 1 ug/uL in DEPC-treated water containing phenol red (1:20 dilution) to allow visual tracking during injection. A small gap was created near the adductor muscle on the right valve using forceps to provide access for injection. In the experimental group, each oyster received a 20 uL injection of Hox4-siRNA solution into the pericardial cavity using a microsyringe. Control oysters were injected with an equal volume of negative control solution. Injections were administered every 48 hours for a total of three times. During the experiment, oysters were maintained at 18-20 degrees C and fed daily with Chlorella sp. No mortality occurred throughout the duration of the experiment. Mantle tissues surrounding the shell holes were harvested one day after the final injection for RNA-seq analysis.