Design of a viral vector for knocking down Yellow Fluorescent Protein expression (and whatever it is attached to).
Virus will be constructed and produced by the UF Vector Core Lab.
To rescue limited brain regions in the FKO mice, we will construct a rAAV virus which will express a short-hairpin RNA (shRNA) that causes degradation of YFP, using previously published shYFP sequences PMID 16676321 PMID 18708075. PMID 15582578 ( Targeted degradation of the 771-nt YFP portion of a YFP-tagged transgene should be sufficient to block expression of the complete transgene PMID 16676321.
The construct will also contain DsRed or mCherry as the reporter gene to allow co-localization with the constitutive YFP reporter in 14-3-3 FKO mice. A rAAV-DsRed vector without shYFP will be constructed as a control virus.
From Michel et al 2005 PMID 15582578:
Oligonucleotides encoding the sense and antisense strands of shRNA against GFP/YFP PMID 15582578:
siGFP sense: 5’-gat ccc gca agc tga ccc tga agt tca ttt caa gag aat gaa ctt cag ggt cag ctt gct ttt ttg gaa a-3’ siGFP anti-sense: 5’-agc ttt tcc aaa aaa gca agc tga ccc tga agt tca ttc tct tga aat gaa ctt cag ggt cag ctt gcg a-3’
used to construct SiRNA vector pAAV9(5)-hSyn-DsRed2N1-EGFP-siRNA gb|AY640634.1 GenBank AY640634.1