The ORF of mouse JMJD3 constructed into lentivirus vector was obtained from Addgene. The ORF of mouse Hes1 was constructed into pCDH-CMV-MCS-EF1-GFP-Puro vector (System Biosciences; catalog no.: CD513B-1) by BamHI and EcoRI double enzyme digestion. The guide RNA targeting mouse JMJD3 or Hes1 was constructed into CRISPR–Caspase9 vector, respectively. The packaging vectors VSVG and Δ8.9 for lentivirus were purchased from Addgene. The primers used for vector construction and guide RNA information have been shown in Table 1 .
To produce lentivirus, human embryonic kidney 293T cells were transfected with a lentiviral plasmid expressing sgRNA/complementary DNA together with the packaging plasmids VSVG and Δ8.9 using calcium phosphate transfection reagent. Lentivirus packaging with nonspecific sgRNA or empty vector was used as control. Viral supernatants were collected at 24 and 48 h after transfection, respectively. Viral supernatants were further concentrated by ∼200-fold using ultracentrifugation at 25,000 rpm for 2 h at 4 °C.
To produce lentivirus, human embryonic kidney 293T cells were transfected with a lentiviral plasmid expressing sgRNA/complementary DNA together with the packaging plasmids VSVG and Δ8.9 using calcium phosphate transfection reagent. Lentivirus packaging with nonspecific sgRNA or empty vector was used as control. Viral supernatants were collected at 24 and 48 h after transfection, respectively. Viral supernatants were further concentrated by ∼200-fold using ultracentrifugation at 25,000 rpm for 2 h at 4 °C.