The iPSC-SR2 line was derived from MRC-5 fibroblasts (ATCC) by overexpressing Oct4, Sox2, Nanog, and cMyc using retroviral vectors (pMXs-cMyc, pMXs-Klf4, pMXs-hOct3-4, and pMXs-Sox2; Addgene) [33 (link)]. The retroviral vectors were produced by transient transfection of 293 T cells. The fibroblasts were incubated in the viral supernatants containing 5 μg/ml polybrene (Sigma) for 4 h. The transduced cells were then incubated for 3 weeks until development of the pluripotent clones. After isolation the clones were grown in mTeSR1 medium (Stem Cell Technologies) on a Matrigel® substrate (BD Biosciences). The cultures were split mechanically using the StemPro EZ Passage tool (Invitrogen).
Pmxs hoct3 4
Manufactured by Addgene
Sourced in United States, Australia
The PMXs-hOCT3/4 is a plasmid vector that expresses the human OCT3/4 gene. OCT3/4 is a transcription factor that plays a crucial role in the maintenance of pluripotency in embryonic stem cells. This plasmid can be used for various research applications involving the study of stem cell biology and gene regulation.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel substrate, by BD (2 mentions)
Pmxs c myc, by Addgene (2 mentions)
Stempro ezpassage tool, by Thermo Fisher Scientific (2 mentions)
Pmxs sox2, by Addgene (2 mentions)
Polybrene, by Merck Group (2 mentions)
Pmxs hsox2, by Addgene (2 mentions)
Pmxs klf4, by Addgene (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Stempro medium, by Thermo Fisher Scientific (1 mentions)
Mrc 5 fibroblasts, by American Type Culture Collection (1 mentions)
Pmxs nanog, by Addgene (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Pmxs hklf4, by Addgene (1 mentions)
Pmxs hc myc, by Addgene (1 mentions)
Hek293t lentix cells, by Takara Bio (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using pmxs hoct3 4
1
Generation of iPSC-SR2 Line from MRC-5 Fibroblasts
2
Generation of iPSC Lines from Fibroblasts
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The iPSC-WT cell line was derived from MRC-5 fibroblasts (ATCC), and the iPSC-DS clones were derived from AG06872 fibroblasts (Coriell). The fibroblasts were transduced with retroviral vectors (pMXs-cMyc, pMXs-Nanog, pMXs-hOct3-4, and pMXs-Sox2; Addgene) to overexpress Oct4, Sox2, Nanog, and c-Myc transgenes. The retroviral vectors were produced by transient transfection of 293T cells. Following this, the fibroblasts were incubated for 4 h in the viral supernatants containing 5 μg/mL polybrene (Sigma). The transduced cells were then incubated for 3 weeks until development of the pluripotent clones. After isolation, the clones were grown in StemPro medium (Invitrogen) on a Matrigel® substrate (BD Biosciences). The cultures were split mechanically using the StemPro EZ Passage tool (Invitrogen).
3
Lentiviral and Retroviral Transduction of Stem Cells
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Lentiviral Particles harboring shRNA of mouse Oct3/4, Sox2, Nanog and Control shRNA Lentiviral Particles were purchased from Santa Cruz Biotechnology. MLV particles used for Oct4 and Sox2 overexpression were generated using the pCL-Eco packaging plasmid and pMXs-hOCT3/4 or pMXs-hSOX2 (Addgene through Dr. Jacob Hanna). Both plasmids were co-transfected to HEK293T cells using jetPEI™. 24 h later, viruses were filtered and frozen in −80 °C. Viruses were transduced to mESCs or MEFs using Polybrene ® (Santa Cruz) in final concentration of 1 µg/ml. 24 h later supernatant was replaced to a fresh growth media.
4
Derivation of human induced pluripotent stem cells
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Derivation of human iPSCs was performed as described previously [14 (link)]. All plasmids for generating iPSCs were purchased from Addgene (Cambridge, MA, USA); these plasmids included pMXs-hOCT3/4 (Addgene 17217), pMXs-hSOX2 (Addgene 17218), pMXs-hKLF4 (Addgene 17219), and pMXs-hc-MYC (Addgene 17220).
For granulosa cells, four consecutive transductions were performed. Six days after the first transduction, fibroblasts and papilla cells were trypsinized and reseeded at 5 × 104 cells per 100 mm dish on mouse embryonic fibroblast feeders. Granulosa cells were trypsinized and replated at 1 × 105 cells per 100 mm dish on mouse embryonic fibroblast feeders 8 days after the first transduction. On the next day, the media were replaced with hESC media, as described above. Approximately 30 days after transduction, colonies were picked manually and transferred into 0.5 ml hESC media in 24-well plates, before being scaled up.
For granulosa cells, four consecutive transductions were performed. Six days after the first transduction, fibroblasts and papilla cells were trypsinized and reseeded at 5 × 104 cells per 100 mm dish on mouse embryonic fibroblast feeders. Granulosa cells were trypsinized and replated at 1 × 105 cells per 100 mm dish on mouse embryonic fibroblast feeders 8 days after the first transduction. On the next day, the media were replaced with hESC media, as described above. Approximately 30 days after transduction, colonies were picked manually and transferred into 0.5 ml hESC media in 24-well plates, before being scaled up.
5
Lentiviral OCT4-GFP Vector Construction
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POU5F1, the human OCT4 gene, cDNA and enhanced green fluorescent protein (GFP) marker were sub-cloned, using restriction enzyme digests, from the retroviral vectors pMXs-hOCT3/4 (a gift from Shinya Yamanaka, Addgene plasmid # 17217; http://n2t.net/addgene:17217 ; RRID:Addgene_17217) [43 (link)] and pRUF-iG2-Gateway (Stan Gronthos, The University of Adelaide, Australia) respectively, into a third generation lentiviral vector (a gift from Jialiang Wang, Addgene plasmid # 46970; http://n2t.net/addgene:46970 ; RRID:Addgene_4697) [51 (link)] (Fig. S1 ). The control empty vector (EV) lacked the transcription factor cDNA. Plasmids were verified by Sanger sequencing (Australian Genome Research facility, Australia). Lentiviral plasmids were cotransfected with psPAX2 (a gift from Didier Trono, Addgene plasmid # 12260; http://n2t.net/addgene:12260 ; RRID:Addgene_12260) and pCMV-VSV-G (a gift from Bob Weinberg, Addgene plasmid # 8454; http://n2t.net/addgene:8454 ; RRID:Addgene_8454) [52 (link)] packaging plasmids into HEK293T Lenti-X™ Cells (Takara Bio) using Lipofectamine™ 2000 Transfection Reagent (Invitrogen) to initiate production of replication-incompetent, Vesicular Stomatitis Virus-Glycoprotein-coated, recombinant lentiviral particles. Viral supernatants were harvested 48 h after transfection and ultracentrifuged to concentrate the virus.
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