Short-hairpin RNAs (shRNAs) directed against the α4 GABAAR subunit were designed, cloned and tested, as in (Rewal et al. 2009), and as described below. A 21-nucleotide (nt) small-interfering RNA (siRNA) sequences of the rat α4 subunit (GenBank accession NM080587) was designed using the GenScript web-based program. Specificity of the siRNA sequence was verified by a BLAST search, termed α4-1, and has the following sequence: 5′-AUAACAUGACAGCUCCAAAUA. A non-related 19-nt sequence (5′-AUGAACGUGAAUU GCUCAA) was used as a negative siRNA control. To employ viral delivery of double-strand siRNA, the adenoviral shuttle vector pRNAT-H1.1 (GenScript Corporation) and the adenoviral vector Adeno-X (Clontech) were used. pRNAT-H1.1 is a vector for vector-based siRNA cloning with an H1 promoter to express shRNA, and contains GFP under control of the CMV promoter. For each siRNA sequence, two complementary DNA oligos, containing sense and antisense siRNA sequences with a stem-loop structure, were synthesized, annealed and ligated into BamHI and HindIII sites of pRNAT-H1.1 vector following the vector cloning protocol. The pRNAT-shRNA recombinants were confirmed by sequencing before subcloning into the cloning sites of I-Ceu I and PI-Sce I of Adeno-X vector.
