Two different shRNAs against mouse MFN2 were constructed using Broad Institute software (one beginning at base pair 2009 and the other at 3355) and cloned into pSico (Addgene). A scrambled sequence was also constructed. The LV was then produced by cotransfecting the pSico vector harboring the siRNA sequence with the following packing, Res, and Pol plasmids: pRSV-Rev, pMDLg/pRRE, and pMD2.G, respectively. Transfection was achieved using polyethylenimine, and the supernatant containing the virus was harvested at 48 and 72 hours afterward. The effectiveness of the LV was tested on HEK293 cells to determine titer.

The virus was injected into 3-week-old mice by retro-orbital injection. Animals were euthanized 4 weeks after injection, the kidneys were removed for histological analysis, and the cells were isolated for experiments.

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