Publication date: Apr 19, 2019
After incubation in HBSS with 0. 25% trypsin (Gibco) for 20 min at 37 ^0C, dissociated neurons were resuspended in HBSS and seeded on poly-D-lysine-coated 6-multiwell or 12-multiwell plates, or MatTek glass bottom culture dishes (P35G-1. 0-14-C).
Cells were cultured in maintenance media (Neurobasal-A medium (#12349015, Thermo Fisher Scientific) supplemented with 2 mM GlutaMAX, 200 mM B27 supplement and 1% Penicillin-Streptomycin) at 37 ^0C in a humidified incubator with 5% CO .
Synthetic RNA oligonucleotides (target site: 5′-GTGGTGCATGGTGTGGCAAC-3′, 225 pmol crRNA/tracrRNA, IDT) were annealed by heating to 95 ^0C for 2 min in duplex buffer (IDT) then cooled slowly, followed by addition of 122 pmol recombinant eSpCas9_1. 1 protein (in 10 mM Tris-HCl, pH 7. 4, 300 mM NaCl, 0. 1 mM EDTA, 1 mM DTT), incubation at room temperature for 20 min, and addition of 500 pmol of a 100 nt ssDNA oligonucleotide (IDT Ultramer) as a homology-directed repair template to introduce the desired base change.
After recovery, plating at single cell density and colony picking into 96 well plates, 288 clones were screened for heterozygous and homozygous mutations by high throughput sequencing of amplicons spanning the target site using an Illumina MiSeq instrument using primers A53TintF and A53TintR Final cell lines were expanded and further validated by a second round of sequencing.
Human iPSCs were cultured in TeSR-E8 medium (Stem Cell Technologies) on Vitronectin (Thermo Fisher Scientific) coated plates.
Cells were grown for 11 days in knockout serum replacement media (KSR) supplemented with LDN-193189 (100 nM, Stemgent), SB431542 (10 uM, R&D Systems), SAG (100 nM, Enzo Life Sciences), Purmorphamine (2 uM, Stemgent), FGF8a (100 ng/ml, R&D Systems) and CHIR99021 (3 uM, Stemgent) and 2 mM L-glutamine (Thermo Fisher Scientific).
Media was changed to NB medium on day 12 containing Neurobasal medium, B27 (1cD7) and 2 mM L-glutamine (Thermo Fisher Scientific) supplemented with CHIR99021 (3 uM, Stemgent) (until day 14) and with BDNF (20 ng/ml, Peprotech), GDNF (20 ng/ml, Peprotech), Ascorbic Acid (200 uM, Sigma-Aldrich), TGFβ3, 1 ng/ml, Thermo Fisher Scientific), dibutyryl cAMP (500 uM; Sigma-Aldrich), and DAPT (10 uM, Stemgent).
At day 21 cells were dissociated with StemPro Accutase (Thermo Fisher Scientific) and replated at 300,000 cells/cm2 in dishes pre-coated with Matrigel in final differentiation medium (NB with BDNF, GDNF, TGFb3, DAPT, dbcAMP and Ascorbic Acid).
Maintenance of zebrafish stocks and transgenic lines: Zebrafish were bred and maintained under standard conditions at 28. 5 +/- 0. 5 ^0C on a 14 h light: 10 h dark cycle.
Embryos were collected at 4-5 h post-fertilisation in embryo medium (EM) (5 mM NaCl, 0. 17 mM KCl, 0. 33 mMCaCl , 0. 33 mM Mg SO , 5 mM HEPES) and thereafter kept in a temperature controlled incubator at 28 ^0C and staged according to established criteria29.
Hu α-syn (A53T) mice, also called SNCA(A53T)G2-3 line, or PrPsynA53T Tg mice from the Jackson Laboratory (Bar Harbour, ME, USA) express a 436 bp human α-synuclein cDNA bearing the familial Parkinson’s disease-linked A53T missense mutation driven by the mouse prion protein promoter21.
The primer sequences for mRFP-GFP-LC3 mice genotyping are: Minipigs: Wild-type female Bama pigs, weighing 12-33 kg (Guangzhou Institutes of Biomedic