The human PSC lines used in this study are listed in Supplementary Table 1 . Fibroblasts from a 50-y-old female ALS patient carrying the D90A SOD1 mutation (ND29149, Coriell Institute, coriell.org), a 3-y-old male SMA patient (GM03813, Coriell Institute) and a 7-m-old SMA patient (GM00232, Coriell Institute) were reprogrammed using the non-integrating Sendai virus as described (Ban et al., 2011) to established iPSC lines ALS-D90A, SMA13 and SMA232. D90D iPSC line was established by correcting the D90A SOD1 mutation in ALS-D90A lines by TALEN technology (Chen et al., 2014). A4V SOD1 mutant ALS iPSC line, established with retrovirus, was obtained from Coriell (ND35671). Human ESC line H9 (WA09 line, NIH registry 0046) and normal iPSC line IMR90-4 were obtained from WiCell. All the PSCs were cultured on irradiated mouse embryonic fibroblasts (MEFs) as described in the standard protocol http://www.wicell.org .
Gm03813
Manufactured by Coriell Institute for Medical Research
GM03813 is a cell line derived from human foreskin fibroblasts. It is one of the cell lines maintained and distributed by the Coriell Institute for Medical Research as a research tool.
Automatically generated - may contain errors
Lab products found in correlation
Gm00232, by Coriell Institute for Medical Research (3 mentions)
Nd29149, by Coriell Institute for Medical Research (2 mentions)
Nd35671, by Coriell Institute for Medical Research (2 mentions)
Imr90 4, by WiCell (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Attractene transfection reagent, by Qiagen (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Equafetal, by Atlas Biologicals (1 mentions)
2 ome ps, by Eurogentec (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Sh sy5y, by American Type Culture Collection (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
7 protocols using gm03813
1
Generation and Characterization of iPSCs
2
Multiplex Transcriptional Activation Assay
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HEK293T (ATCC), U2OS (ATCC), HeLa (ATCC) and the SMA patient-derived fibroblasat line GM03813 (Coriell Institute) cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) with 10% fetal bovine serum (FBS)(Lonza), 4% Glutamax (Gibco), 1% Sodium Pyruvate (Gibco) and penicillin-streptomycin (Gibco). Incubator conditions were 37 °C and 5% CO2. For activation experiments, cells were seeded into 12-well plates at 100,000 cells per well the day before being transfected with 600 ng (the “quota”) of plasmid DNA with 2.25 µL Attractene transfection reagent (Qiagen). Eighteen nanograms of each reporter minigene plasmid was transfected. The remaining quota was then divided equally among the effector and gRNA plasmids. In cases where there were two or more gRNA plasmids, the quota allocated for gRNA plasmids is further subdivided equally. For ESF effectors, an equally split amount (194 ng) of all three ESF plasmids were co-delivered as an ESF mix. For two-peptides effectors (i.e., the FKBP-FRB systems), the effector plasmid quota was divided equally between the plasmids encoding the individual peptides (146 ng). Media was changed 24 h after transfection. Rapamycin (100 nM final concentration) was added during the media change if applicable. Cells were harvested 48 h after transfection for RT-PCR analysis.
3
Generation and Characterization of iPSCs
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The human PSC lines used in this study are listed in Supplementary Table 1 . Fibroblasts from a 50-y-old female ALS patient carrying the D90A SOD1 mutation (ND29149, Coriell Institute, coriell.org), a 3-y-old male SMA patient (GM03813, Coriell Institute) and a 7-m-old SMA patient (GM00232, Coriell Institute) were reprogrammed using the non-integrating Sendai virus as described (Ban et al., 2011) to established iPSC lines ALS-D90A, SMA13 and SMA232. D90D iPSC line was established by correcting the D90A SOD1 mutation in ALS-D90A lines by TALEN technology (Chen et al., 2014). A4V SOD1 mutant ALS iPSC line, established with retrovirus, was obtained from Coriell (ND35671). Human ESC line H9 (WA09 line, NIH registry 0046) and normal iPSC line IMR90-4 were obtained from WiCell. All the PSCs were cultured on irradiated mouse embryonic fibroblasts (MEFs) as described in the standard protocol http://www.wicell.org .
4
Derivation and Characterization of iPSCs from SMA Fibroblasts
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Control fibroblasts (GM03814 and GM03815, Coriell Institute, Supplementary Table S1 ) and type-I SMA fibroblasts (GM03813, GM09677 and GM00232, Coriell Institute, Supplementary Table S1 ) were reprogrammed to iPSCs using retrovirus containing the Yamanaka factors, OCT4, SOX2, KLF-4 and c-MYC, as previous reported28 (link). Pluripotency of the established iPSC lines was characterized by immunostaining for pluripotency markers and by teratoma formation in SCID mice. They were characterized for G banding karyotyping every 10 passages37 (link). In addition, human WA09 ESC line (also known as H9, WiCell institute, NIH registry 0046) were used as an additional control. SMN1 knockdown (SMNi) and luciferase control (Luc) ESC lines were described before25 (link). The PSCs were maintained on irradiated mouse embryonic fibroblasts as previously described38 (link).
5
Comparative Analysis of Spinal Muscular Atrophy Fibroblasts
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Fibroblasts derived from type II SMA (GM03813, GM22592 and AIDHC-SP22) and non-SMA (GM03814, AIDHC-NMC1, AIDHC-SC1 and AIDHC-SC2) individuals were grown in DMEM containing 10% EquaFETAL (Atlas Biologicals, Fort Collins, CO), 2 mM L-glutamine (Life Technologies, Grand Island, NY) and 1% penicillin-streptomycin (Life Technologies). GM03813 [30 (link)], GM22592 and GM03814 [30 (link)] fibroblast lines were obtained from Coriell Cell Repositories (Camden, NJ) while the other fibroblast lines were generated at Nemours/Alfred I. duPont Hospital for Children. All type II SMA fibroblast lines used in this study contain 0 copies of SMN1 and 3 copies of SMN2 [31 (link)]. GM03814 fibroblasts [30 (link)] were derived from the carrier mother of GM03813 and contain 1 copy of SMN1 and 5 copies of SMN2 [31 (link)]. The other non-SMA fibroblast lines contain 2 copies of SMN1 and 2 copies of SMN2 [31 (link)]. The fibroblast lines were authenticated using short tandem repeat profiling and digital PCR as described previously [32 (link)].
The mouse motor neuron cell line NSC-34 [33 (link)] and the NSC-34-based reporter lines [18 (link);34 (link)] were maintained in DMEM, 5% EquaFETAL, 2 mM L-glutamine and 1% penicillin/streptomycin. In all instances, the cells were maintained in a humidified chamber at 37°C and 5% CO2.
The mouse motor neuron cell line NSC-34 [33 (link)] and the NSC-34-based reporter lines [18 (link);34 (link)] were maintained in DMEM, 5% EquaFETAL, 2 mM L-glutamine and 1% penicillin/streptomycin. In all instances, the cells were maintained in a humidified chamber at 37°C and 5% CO2.
6
Fibroblast Transfection of tcDNA AONs for SMA
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Human fibroblasts from a 3-year-old type I SMA patient (GM03813, Coriell Cell Repositories) and WT fibroblasts (GM03814, Coriell Cell Repositories) were grown in DMEM with 20% fetal bovine serum and 1% penicillin-streptomycin (100 U/mL). tcDNA AONs were synthetized by Synthena as previously described,47 , 48 (link) and 2′OMePS was obtained from Eurogentec. The two sequences used were SMN2 TSL (39;55) 5′-(pTTAATTTAAGGAATGTG)-3′ and SMN2 TCI7(10;24) 5′-(pCTTTCATAATGCTGG)-3′.
2′OMePS and tcDNA transfection was performed with oligofectamine (Invitrogen) and incubated for 48 hr without serum and antibiotics.
2′OMePS and tcDNA transfection was performed with oligofectamine (Invitrogen) and incubated for 48 hr without serum and antibiotics.
7
Culturing Various Cell Lines
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All tissue culture media and reagents were purchased from Life Technologies. HeLa, HEK-293 and SH-SY5Y cells were obtained from American Type Culture Collection. Primary fibroblast cells from an SMA type I patient (GM03813) were obtained from Coriell Cell Repositories. Mouse NSC-34 cells were obtained from Dr. Neil Cashman (University of Toronto). HeLa, HEK-293 and mouse NSC-34 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). SH-SY5Y cells were cultured in a 1:1 mixture of minimum essential medium (MEM) and F-12 nutrient mixture supplemented with 10% FBS. GM03813 cells were cultured in MEM supplemented with 15% FBS and 2 mM GlutaMAX.
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