Skin fibroblasts were transfected with pCXLE‐hOct3/4‐shp53, pCXLE‐hSox2‐hKlf4, pCXLE‐hLin28‐hL‐Myc (Addgene, Cambridge, USA) using the Neon Transfection System according to the manufacturer's protocol. After 5 days, the medium was replaced with DMEM/F12 medium supplemented with Glutamax, 20% knockout serum replacer, 1% NAA, 0.1 mmol/l β‐mercaptoethanol (Life Technologies), and 50 ng/ml bFGF (PeproTech, Hamburg, Germany). At day 10, cells were trypsinized and maintained on irradiated mouse embryonic fibroblasts (MEFs) in human iPS cell medium. Colonies were selected based on morphology (Okita et al, 2011 ; Yu et al, 2011 ), further passaged, and expanded. As quality control for iPS clones, we performed alkaline phosphatase (Vector Blue Alkaline Phosphatase Substrate Kit; Vector Laboratories, SK5300) and pluripotent markers staining.
Pcxle hoct3 4 shp53
Manufactured by Addgene
Sourced in United States
The PCXLE-hOCT3/4-shp53 is a plasmid vector that contains the human OCT3/4 gene and a short hairpin RNA (shRNA) targeting the p53 gene. This plasmid is commonly used in research to study the role of OCT3/4 and p53 in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Pcxle hsk, by Addgene (4 mentions)
Pcxle hul, by Addgene (3 mentions)
Pcxwb ebna1, by Addgene (2 mentions)
Matrigel, by Corning (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Vector blue alkaline phosphatase substrate kit, by Vector Laboratories (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Addgene (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Vitronectin coated plates, by Thermo Fisher Scientific (1 mentions)
Minimum essential media, by Thermo Fisher Scientific (1 mentions)
Epi5 episomal ipsc reprogramming kit, by Thermo Fisher Scientific (1 mentions)
Stemflex, by Thermo Fisher Scientific (1 mentions)
Nucleofector iib device, by Lonza (1 mentions)
Stemfit medium, by Ajinomoto (1 mentions)
Four well plates, by SPL Life Sciences (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Y 27632, by MedChemExpress (1 mentions)
5 protocols using pcxle hoct3 4 shp53
1
Generation of Human iPS Cells
2
Fibroblast Reprogramming to iPSCs
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Fibroblasts were isolated from skin biopsy explants from the proband harboring a biallelic ACTL6B p.(Val421_Cys425del) variant and a sex‐matched unaffected familial carrier (biological mother of the affected patients) described in Yüksel et al. (2019 ). Fibroblasts were cultured in Minimum Essential Media (Gibco), supplemented with 20% (v/v) fetal bovine serum (Gemini) and 1% (v/v) antibiotics (Pen/Strep 10,000 U/ml). Low passage fibroblasts were reprogrammed to iPSCs using Epi5 Episomal iPSC Reprogramming Kit (ThermoFisher) with the following modifications: fibroblasts were electroporated and plated onto Matrigel (Corning)‐coated plates. Reprogramming vectors pCXLE‐hOCT3/4‐shp53 (Addgene, 27077, RRID:Addgene_27077), pCXLE‐hSK (Addgene, 27078, RRID: Addgene_27078), pCXLE‐hUL (Addgene, 27080, RRID:Addgene_27080), and pCXWB‐EBNA1 (Addgene, 37624, RRID:Addgene_37624) were gifts from Shinya Yamanaka and prepared in‐house (Okita et al., 2013 ). The medium was changed to StemFlex (ThermoFisher) on day 15. iPSC colonies were manually collected and individual clones were expanded in feeder‐free conditions on vitronectin‐coated plates (ThermoFisher) for karyotyping (Karyostat; Invitrogen) and characterization of pluripotency.
3
Establishing the FF-PB-3AB4 hiPSC Line
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The human induced pluripotent stem cell (hiPSC) line FF-PB-3AB4 was established from a healthy donor’s peripheral blood mononuclear cells (PBMCs), as described previously46 (link),47 (link). In brief, PBMCs were electroporated with the episomal vectors pCXLE-hOCT3/4-shp53 (#27077; Addgene, Cambridge, MA, USA), pCXLE-hSK (#27078; Addgene), pCXLE-hUL (#27080; Addgene), and pCXWB-EBNA1 (Addgene; #37824) using the Nucleofector IIb device (Lonza, Basel, Switzerland) and plated on iMatrix-511-coated cell culture plates (Nippi, Inc., Tokyo, Japan). The iPSCs were induced by changing the medium to StemFit medium (Ajinomoto, Tokyo, Japan). Twenty-nine days after electroporation, colonies were isolated and expanded for validation.
The institutional review board of Kobe University Graduate School of Medicine approved this study (No. 1722), and informed consent was obtained from the donor. FF-PB-3B4 showed typical human embryonic stem cell-like morphology (Fig. S1A ), expressed pluripotent stem cell markers OCT3/4 and NANOG (Fig. S1B ) and had the ability to differentiate into cells comprising all three germ layers in vitro (Fig. S1C ). In addition, we performed a “pluritest” and confirmed that the generated clone was pluripotent and similar to validated normal PSCs (Fig. S1D ). Karyotype of FF-PB-3AB4 was normal (data not shown).
The institutional review board of Kobe University Graduate School of Medicine approved this study (No. 1722), and informed consent was obtained from the donor. FF-PB-3B4 showed typical human embryonic stem cell-like morphology (Fig. S
4
Episomal Vector Reprogramming Protocol
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Three episomal vectors were utilized for reprogramming, as per previous reports of fibroblast transfection success [26 (link)]: pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL (Addgene Plasmid #27077, #27078, and #27080, respectively). Plasmid-containing E. coli were cultured, and colonies picked and expanded in LB Medium with ampicillin. Plasmid extraction was accomplished through the PureYieldTM Midiprep System (Promega) as per the manufacturer’s instructions.
5
Generation of iPSCs from Orbicularis Oculi Muscle Stem Cells
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For the generation of iPSCs, Orbicularis oculi muscle stem cells (OOM-SCs) were procured from Konkuk University Medical Center (KUMC 2019-05-043). OOM-SCs were cultured as sources of iPSCs. OOM-SCs were maintained following the method reported in our previous study [24 (link)]. Reprogramming factors (pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL) were purchased from Addgene and transfected using the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Each plasmid was delivered at a ratio of 1:1:1, for 3 μg in total. Transfected cells were seeded on pre-coated Matrigel (Corning, Bedford, MA, USA) plates and cultured in minimum essential medium (MEM)-α (Gibco™, USA) supplemented with 10% FBS (Gibco™, Waltham, MA, USA) and 1% P/S (Gibco™, Waltham, MA, USA) for three days. After three days, the medium was completely replaced with a hESC proliferation medium containing WT-bFGF (Peprotech, USA) or TS-bFGF with 10 µM Y-27632 (MedChemExpress, USA). The medium was replaced every 2 days until iPSC colony formation. The colonies were transferred into four-well plates (SPL Life Sciences, Pochon, Republic of Korea) coated with Matrigel (Corning, Bedford, MA, USA), then cultured according to the hESC culture method.
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