Pcdh mscvmcs ef1α gfp puro

The WTIP coding sequence with a 3×FLAG-tagged sequence was amplified from HEK293 cells, and then cloned into the pLVX-tre3G vector (Clontech, CA, USA) or pCDH-MSCVMCS-EF1α-GFP+Puro (System Biosciences, Palo Alto, CA, USA) using Hieff Clone^TM One Step Cloning Kit (Yeasen, Shanghai, China) as previously described [21 (link)]. The FOXO3a coding sequence with a 3×FLAG-tagged sequence was amplified from HEK293 cells, and cloned into the pCDH-MSCVMCS-EF1α-GFP+Puro (System Biosciences, Palo Alto, CA, USA) using Hieff Clone^TM One Step Cloning Kit as previously described [23 (link)]. The sequence of shRNAs for WTIP was designed (shWTIP target sequence: 5′-CCGGCAGCGTGTGTGGACATCTCATCTCGAGATGAGATGTCCACACACGCTGTTTTTG-3′).

For stable transfection experiments in ESCs, the sequences coding for E2F6 WT, as well as E68 and MB4 mutants, were inserted into the pCAG-EGFP-IB vector^{60 (link)}. To generate the E68 DNA-binding mutant, the amino acids in positions 68 and 69 were changed from Leu-Val to Glu-Ser^{18 (link)}. To generate the MB4 mutant, the E2F6 marked box domain (aa 180–240) was replaced by the marked box domain of E2F4 (aa 138–198). These constructs were transfected into ESCs by electroporation with the Amaxa Nucleofector II device using the Amaxa Cell Line Nucleofector kit R and stable transfectants were selected with blasticidin (7.5 μg/ml). For the DNA-binding domain swap experiment, the sequences coding for E2F6 WT, DBD1, and MB4 mutants were inserted in the lentiviral expression vector pCDH-MSCV-MCS-EF1α-GFP + Puro (System Biosciences). The DBD1 mutant was generated by replacing the DNA-binding domain of E2F6 (aa 61–129) with the DNA-binding domain of E2F1 (aa 122–187). Viral particles were produced in HEK293T cells and ESCs were infected with 8 μg/ml polybrene. Twenty-four hours after transduction, puromycin (2 μg/ml) was added for 72 h before collecting the RNA samples.

The human CaMK2G coding sequence (NM_172171.2) with a 3×FLAG sequence and a kozak sequence was cloned into the vector pCDH-MSCV-MCS-EF1α-GFP+Puro (System Biosciences, USA) using Hieff Clone TM One Step Cloning Kit (Yeasen, China) [26 (link)].
CRISPR/Cas9 system was applied to construct cells with CaMK2G knockout. Lentivirus vector pCW-Cas9 (doxycycline inducible; #5066) and pLX-sgRNA (lentiviral vector, #50662) were purchased from Addgene (USA). Two seperate sgRNAs against CaMK2G were designed and cloned into pLX-sgRNA following the the CRISPR protocol [37 (link)]. The sequence of two seperate sgRNA against CaMK2G as follows: 5’-CACCGTGCTTTCTCTGTGGTCCGC-3’; 5’-AAACGCGGACCACAGAGAAAGCAC-3’.