Constructs

Gene of interest (GOI) with Kozak sequence GCCACC were amplified from cDNA of MA cells and cloned into retroviral vector MLS (LTR-MCS-SV40-GFP) or MLP (LTR-MCS-PGK-Puro-IRES-GFP). Efnb1 had been cloned into MLS in a previous study.²⁴ Stmn1 and Stmn1 S28A were amplified from cDNA and cloned into MLP in this study. MSCV retroviral vectors with a helper plasmid were co-transfected into HEK293T cells to produce retrovirus. GOI-GFP stable cell lines were established by retrovirus infection with polybrene (20 μg/ml). MLP-based stable cells (Stmn1 and Stmn1 S28A) were selected with puromycin (μug/ml). Fluorescence-activated cell sorting (FACS) was performed using FACSAria II (BD) to sort MLS-based stable cells (Efnb1). The single-clone cell strains were established through the dilution method, in which about 30 cells were seeded in 96 wells of a 96-well plate.