Protocol to investigate the effects of localized gene expression disruption on mouse limb development via direct siRNA injection and explant culture

Here, we present a protocol for localized gene knockdown in embryonic mouse hindlimb explants using direct small interfering RNA (siRNA) injection, combined with a semi-quantitative morphology scoring system to assess developmental outcomes. We describe steps for mating of samples, preparing hindlimb explants for culture and siRNA-Lipofectamine complex, and direct injection of the siRNA-Lipofectamine complex. We then detail procedures for explant culture and maintenance, data analysis, tissue fixation, and cryoprotection. This protocol approach enables spatially precise modulation of gene expression during limb and joint development. • Steps for direct injection of gene expression modulators into embryonic limb explants • Guidance on explant culture and localized gene modulation • Instruction for semi-quantitative scoring of limb and joint morphology Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for localized gene knockdown in embryonic mouse hindlimb explants using direct small interfering RNA (siRNA) injection, combined with a semi-quantitative morphology scoring system to assess developmental outcomes. We describe steps for mating of samples, preparing hindlimb explants for culture and siRNA-Lipofectamine complex, and direct injection of the siRNA-Lipofectamine complex. We then detail procedures for explant culture and maintenance, data analysis, tissue fixation, and cryoprotection. This protocol approach enables spatially precise modulation of gene expression during limb and joint development.