Pdr274

The Cas9 nuclease expression vector was generated as follows. The hygromycin B resistance gene cassette was prepared by PCR amplification using primer pair Hyg SS and Hyg AS (Table S1). pGloSensor™-22F cAMP Plasmid (Promega corporation, Madison, Germany) was used as the template. The PCR product was phosphorylated and inserted into pCS2+hSpCas9 (Addgene Plasmid 51815), which had been digested with Mfe I, filled in by Klenow fragment, and dephosphorylated. The resultant vector, pCS2+hSpCas9/Hyg, was used for knockout experiments. The sgRNA expression vector was generated according to the previous method described in [18 (link)]. Briefly, a pair of oligonucleotides was annealed and ligated into pDR274 (Addgene Plasmid 42250), which had been digested with Bsa I. The resultant vector, pDR274-CDK9, was used for KO experiments. A combination of pDR274-CDK9 and pCS2+hSpCas9/Hyg, or pDR274-Mock and pCS2+hSpCas9/Hyg, was co-transfected into OLHNI-2 cells stably expressing medaka Pgr using ScreenFect A (Wako) according to the manufacturer’s instructions. After culture for 48 h, the medium was changed into fresh medium containing 100 μg/mL hygromycin (Wako), and cells were cultured for another 48 h. The cells were harvested and used for the immunoprecipitation/Phos-tag SDS-PAGE analysis.