Pcagen sbp dicer1

Plasmids containing the sequence of mouse DICER (pCAGEN-SBP-DICER1, Addgene), MAVS (GE-healthcare, MMM1013-202764911) and DGCR8 (Macias et al., 2012 (link)) were used to amplify the open reading frame using specific primers containing restriction sites (Supplementary file 1). The amplified and digested fragments were ligated in pLenti-GIII-EF1α for MAVS and pEF1α-IRES-dsRED-Express2 for DGCR8 and DICER. Verified plasmids containing the genes of interest were transfected in mESCs using Lipofectamine 2000 and selected with the appropriate antibiotic. After several weeks of selection, colonies were isolated, expanded and tested for expression by qRT-PCR and Western blot.

IUE was performed according to a previous publication.²⁸ Plasmids of pCAGEN‐SBP‐DICER1 (#50558), pCAGGS‐NICD (#26891) and pCAG‐GFP (#11150) were purchased from addgene. Mouse Jag1 was amplified and cloned into HindIII/BamHI sites of p3xFLAG‐CMT‐14 vector. Pregnant mice at E13.5 were anaesthetized with isoflurane (3% for induction and 2% during surgery for maintenance). A 1.5 cm incision was made along the linea alba in the lower abdomen, and the uterine horns were exposed. Desired plasmids (1.5 mg/mL) diluted in 1ul sterile Tris‐EDTA buffer (pH 7.4), which contained Fast Green (Sigma), were injected into the lateral ventricle of embryos at E13.5. Five 50 ms pulses of 33 V with 950 ms intervals were applied with a BTX electroporation system (ECM830). After electroporation, the uterine horns were placed back and the incision was sutured. The embryonic brains were used for further experiments at E15.5.