Rat glial cell line derived neurotrophic factor

MEFs derived from C57BL6 embryos (isolated at day 13.5 post-coitum) were used as feeders for GSC culture. MEF cells treated with mitomycin-C were plated on 0.1% gelatin-coated dishes before GSC culture. Rosa-CreER^T2 GS cell line was generated in our lab. To establish Rosa-CreER^T2 GSC lines, postnatal mouse testes (day P5) from Rosa-CreER^T2 mice were digested into cell suspension and then seed into 12-well plates for colony formation. GSC colony were moved to feeder cells and cultured with StemPro medium (Thermo) supplement with rat glial cell line-derived neurotrophic factor (20 ng ml⁻¹, R&D systems), mouse EGF (20 ng ml⁻, BD Bioscience) and human FGF2 (10 ng ml⁻, Life Technology). To induce CRE activity in GSCs carrying Rosa-CreER^T2, 4-OHT (1 μM) was added to the GSC medium at the indicated time points. RA (100 nM; Wako) was used to induce mTORC1 signalling and GSC differentiation. CLQ (10 μM; Sigma-Aldrich) was used to inhibit lysosomal degradation. The 293T cell line (CRL-3216) purchased from ATCC was used for lentivirus production.

mSSCs were derived from 5.5 days postpartum (dpp) B6D2F1 male mice. GFP⁺ SSCs were derived from 5.5 dpp male mice generated by mating female EGFP-C57BL/6 mice with male DBA/2 mice. mSSC culture was performed according to a previous study, with culture supplemented with 20 ng/ml mouse epidermal growth factor (EGF) (Thermo Fisher), 10 ng/ml human basic fibroblast growth factor (bFGF) (R&D), 10 ng/ml rat glial cell line-derived neurotrophic factor (R&D) and 10³ U/ml ESGRO (Millipore) 29 (link).
To establish stable Cst3 knockdown mSSC lines, small hairpin RNAs (shRNAs) against Cst3 were constructed using the pLKO.1-puro shRNA system (Sigma). The sequences were as follows: negative control shRNA (shNC): 5'-CAACAAGATGAAGAGCACCAACTCGAGT-TGGTGCTCTTCATCTTGTTGTTTT-3'; Cst3-KD shRNA #1, 5'-CCGGCGTGGCTGGAG-TGAACTATTTCTCGAGAAATAGTTCACTCCAGCCACGTTTTTG-3'; Cst3-KD shRNA #2, 5'-CCGGCCATCTGATGAGGAAGGCACTCTCGAGAGTGCCTTCCTCATCAGATG-GTTTTTG-3'. For lentivirus packaging, plasmids were transiently transfected into HEK293T cells. Viral supernatant containing a final concentration of 4 μg/ml polybrene (Sigma) was used to infect mSSCs. Positively transduced cells were selected using puromycin (Thermo Fisher) at a final concentration of 0.4 μg/ml.