2.3. Viral vector construction and transduction
This protocol is extracted from research article:
Kir6.1 improves cardiac dysfunction in diabetic cardiomyopathy via the AKT‐FoxO1 signalling pathway
J Cell Mol Med, Feb 6, 2021; DOI: 10.1111/jcmm.16346

A recombinant adeno‐associated virus serotype 9 containing Kir6.1 (AAV‐9) and a recombinant adenovirus encoding Kir6.1 (Ad‐Kir6.1) were packaged by Shanghai HanBio Company (Shanghai, China). The AAV‐9 capsid has previously been reported to show a modest preference for cardiac tissue in vivo. 15 The mice were randomized into two groups and injected with the null control virus (AAV‐C, 2.70 × 1011 GC/mL, 100 μL per mouse) or AAV‐9 (3.97 × 1011 GC/mL, 100 μL per mouse) via the tail vein before being fed standard rodent chow or an HFD. For the in vitro experiments, after 48 hours of cell culture, the medium was changed with fresh DMEM containing serum and NRVMs were transfected by adding adenoviruses expressing green fluorescent protein (Ad‐C, Viral titre 1.58 × 1010 PFU/mL) or GFP‐fused Kir6.1 (Ad‐Kir6.1, Viral titre 1.58 × 1010 PFU/mL). The adenovirus dose is indicated as multiplicity of infection (MOI). After 8 hours of infection, the medium was changed to fresh DMEM containing serum for another 8 hours of culturing, and then, the cells were serum starved for 8 hours. Then, insulin (100 nmol/L, 24 hours; Sigma‐Aldrich) was used to induce insulin resistance. Cells in the control group were treated with 100 nmol/L insulin for 0.5 hours. More details are provided in Figures S4 and S5, Table S2.

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