The target sequence for rat Arl13b was identified using BLOCK-iT™ RNAi Designer (Thermo Fisher Scientific): 5′-TGTGATGGTCGGACTTGATAA –3′.
shRNA target sequence with a microRNA stem (shRNAmir) for knockdown of Arl13b was generated by ligating oligonucleotides encoding the target sequences into the pcDNA6.2-GW/EmGFP-miR vector. The shRNA/miR/EmGFP sequence was then cloned via attB/attP site recombination (using BP clonase) into the pDONR221 backbone (ThermoFisher, 12536-017), and via attL/attR site recombination (using LR clonase) into a custom expression vector derived from Gateway pcDNA-DEST47 (ThermoFisher Scientific, 12281-010). This custom expression vector replaced the promoter and polyA regions of Gateway pcDNA-DEST47 with the promoter and polyA from the pCAGGS vector (Stühmer et al., 2002 (link)) to improve its expression in mammalian neurons. The efficiency and specificity of this shRNAs was evaluated in vitro by immunofluorescence.
shRNA target sequence with a microRNA stem (shRNAmir) for knockdown of Arl13b was generated by ligating oligonucleotides encoding the target sequences into the pcDNA6.2-GW/EmGFP-miR vector. The shRNA/miR/EmGFP sequence was then cloned via attB/attP site recombination (using BP clonase) into the pDONR221 backbone (ThermoFisher, 12536-017), and via attL/attR site recombination (using LR clonase) into a custom expression vector derived from Gateway pcDNA-DEST47 (ThermoFisher Scientific, 12281-010). This custom expression vector replaced the promoter and polyA regions of Gateway pcDNA-DEST47 with the promoter and polyA from the pCAGGS vector (Stühmer et al., 2002 (link)) to improve its expression in mammalian neurons. The efficiency and specificity of this shRNAs was evaluated in vitro by immunofluorescence.