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Using seamless cloning technology, the GRA23 gene was inserted into the pBiFC-VC155 vector, while PEX3 and TRAP1 genes were inserted into the pBiFC-VN173 vector, creating recombinant plasmids pBiFC-GRA23-VC155, pBiFC-PEX3-VN173, and pBiFC-TRAP1-VN173, respectively

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DCE-MRI BBB permeability maps were created using the slope of contrast agent concentration in each voxel over time, calculated using a linear fit model as described previously 70,71,72