TY  - GEN
T1  - Two plasmid modules for introducing the auxin-inducible degron into the fission yeast Schizosaccharomyces pombe by PCR-based gene targeting
AU  - Song, Xiuyi
AU  - Xu, Ruoming
AU  - Sugiyama, Tomoyasu
DO  - 10.17912/micropub.biology.000442
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000442/
AB  - Recent application of the plant auxin-inducible degron (AID) system to the fission yeast Schizosaccharomyces pombe added a new option to eliminate a specific protein (Kanke et al. 2011). In this system, a protein fused with AID is subjected to proteasome-dependent protein degradation in the presence of auxin and the auxin receptor F-box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1). However, no plasmid was currently available for AID tagging by the PCR-based method (Bähler et al. 1998). To expedite the construction of AID-tagged yeast fission strains, we added new assortments of AID-tagging plasmids to the PCR-based gene tagging platform described previously (Bähler et al. 1998). When we designed our plasmids for C-terminal AID tagging, we considered the following four criteria. First, the mini-AID (mAID) tag described in the previous study (Nishimura and Kanemaki 2014) rather than the full-length AID (Kanke et al. 2011) should be used to minimize the size of the AID tag. Second, a 5×FLAG tag or GFP should be included for downstream experiments such as western blotting and fluorescence microscopy. Third, primer sequences for module amplification should be the same as those described previously to maximize the universality and utility of the genetic components (Bähler et al. 1998). Finally, the drug-resistant marker for selection is kanMX6, which is one of the most commonly used selection markers for fission yeast. Thus, we constructed two plasmids for use as PCR templates, pFA6a-mAID-GFP-kanMX6 and pFA6a-3×mAID-5×FLAG-kanMX6 (Figure 1A).
PY  - 2021
JO  - microPublication Biology
ER  -