Construction of Vectors

The sequence of rat nestin short hairpin RNA (shRNA) was TCTGAGGCTCCCTGTCCTCCA. Non-targeting control shRNA (MISSION shRNA: SHC002) was purchased from Sigma (St. Louis, MO, USA). The shRNA and the U6-promotor were ligated into adeno-associated virus (AAV) vectors (Takara, Kyoto, Japan). AAV shH10 (addgene, Watertown, MA, USA) and pHelper (Takara) were transformed into HEK293T (Takara) and AAV was purified using AAVpro Purification Kit Maxi/Midi (Takara). The AAV titration protocol²⁹ was used to determine the number of genome-containing particles of an AAV prep using real-time PCR. About 1 × 10¹¹ Vg/µL of AAV was titrated for injection in 4 µL volume in each eye.