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Pmx klf4

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The PMX-KLF4 is a lentiviral expression vector that contains the KLF4 (Kruppel-like factor 4) gene. The vector is designed for the overexpression of the KLF4 transcription factor in mammalian cells.

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Lab products found in correlation

Pmx sox2, by Addgene (6 mentions) Pmx cmyc, by Addgene (6 mentions) Pmx oct4, by Addgene (5 mentions) Plko 1 vector, by Thermo Fisher Scientific (2 mentions) Lipofectamine, by Thermo Fisher Scientific (2 mentions) Src y527f, by Addgene (1 mentions) In fusion system, by Takara Bio (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) L ascorbic acid, by Merck Group (1 mentions) Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PMXs-Klf4

6 protocols using pmx klf4

1

Retroviral and Lentiviral Vector Production

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pMX-Oct4, pMX-SOX2, pMX-KLF4, pMX-cMyc were obtained from Addgene (plasmids 17217, 17218, 17219, 17220 respectively). Retroviral vectors were co-transfected with packaging plasmids (pCMV-gag-pol-PA and pCMV-VSVg, kindly provided by Gerald Pao, Laboratory of Genetics, The Salk Institute, La Jolla, CA) in 293T cells using Lipofectamine (Invitrogen) in accordance with manufacturer’s recommendations.
pBABE-U3-hTR was kindly provided by Kathleen Collins60 (link). U3-hTR was cloned into third generation lentiviral vector kindly provided by Raul Alvarez Rodriguez (Laboratory of Genetics, The Salk Institute, La Jolla, CA). pBABE-TERT and TERT-DN61 (link) were subcloned into pLV-FU-TetO vector. Lentiviral vectors were co-transfected with packaging plasmids (pMDL, Rev and VSVg, kindly provided by Oded Singer, Laboratory of Genetics, The Salk Institute, La Jolla, CA) in 293T cells using Lipofectamine (Invitrogen) in accordance with manufacturer’s recommendations.
Lentiviral supernatants were collected 36 hours after transfection, concentrated by ultracentrifugation at 19400 rpm for 2 hours and resuspended in hES cell media.
The shRNA-expressing lentiviral pLKO.1 vectors were purchased from OpenBiosystems (ThermoFisher) and correspond to the following clones:
Rivera T., Haggblom C., Cosconati S, & Karlseder J. (2016). A balance between elongation and trimming regulates telomere stability in stem cells. Nature structural & molecular biology, 24(1), 30-39.
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2

Lentiviral Oncogene Expression Vectors

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pMX-OCT4, pMX-SOX2, pMX-KLF4 and pMX-cMYC, were obtained from Addgene (plasmids 17217, 17218, 17219 and 17220, respectively). The shRNA p53 construct was previously described and validated49 (link). Constructs for the expression of mutant oncogenes were obtained from Addgene (RasV12 Neo (w108-1) #22259; EGFR D770-N771 insNPG #11016; EGFR (del3) #11015; src Y527F #13660). Coding sequence for the mutant oncogenes were removed by restriction enzymes and subcloned into a lentiviral backbone with the In-Fusion system according to manufacturer's instructions (Takara/Clontech).
Sancho-Martinez I., Nivet E., Xia Y., Hishida T., Aguirre A., Ocampo A., Ma L., Morey R., Krause M.N., Zembrzycki A., Ansorge O., Vazquez-Ferrer E., Dubova I., Reddy P., Lam D., Hishida Y., Wu M.Z., Esteban C.R., O'Leary D., Wahl G.M., Verma I.M., Laurent L.C, & Izpisua Belmonte J.C. (2016). Establishment of human iPSC-based models for the study and targeting of glioma initiating cells. Nature Communications, 7, 10743.
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3

Generation of induced pluripotent stem cells

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iPSC derivation was performed by utilizing retroviral vectors (pMX-OCT3/4, pMX-SOX2, pMX-KLF4, and pMX-cMYC, obtained from Addgene)42 (link). After the transduction of the reprogramming factors, sarcoma cells and ear tip fibroblasts were cultured in ESC media supplemented with human recombinant leukemia inhibitory factor (LIF) (Wako), 2-mercaptoethanol (Invitrogen) and 50 µg/ml l-ascorbic acid (Sigma). The established iPSCs were maintained with ESC media supplemented with LIF, 1 μM PD0325901 (Stemgent), and 3 μM CHIR99021 (Stemgent).
Komura S., Ito K., Ohta S., Ukai T., Kabata M., Itakura F., Semi K., Matsuda Y., Hashimoto K., Shibata H., Sone M., Jo N., Sekiguchi K., Ohno T., Akiyama H., Shimizu K., Woltjen K., Ozawa M., Toguchida J., Yamamoto T, & Yamada Y. (2019). Cell-type dependent enhancer binding of the EWS/ATF1 fusion gene in clear cell sarcomas. Nature Communications, 10, 3999.
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4

Retroviral and Lentiviral Vector Production

Cited 1 time
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pMX-Oct4, pMX-SOX2, pMX-KLF4, pMX-cMyc were obtained from Addgene (plasmids 17217, 17218, 17219, 17220 respectively). Retroviral vectors were co-transfected with packaging plasmids (pCMV-gag-pol-PA and pCMV-VSVg, kindly provided by Gerald Pao, Laboratory of Genetics, The Salk Institute, La Jolla, CA) in 293T cells using Lipofectamine (Invitrogen) in accordance with manufacturer’s recommendations.
pBABE-U3-hTR was kindly provided by Kathleen Collins60 (link). U3-hTR was cloned into third generation lentiviral vector kindly provided by Raul Alvarez Rodriguez (Laboratory of Genetics, The Salk Institute, La Jolla, CA). pBABE-TERT and TERT-DN61 (link) were subcloned into pLV-FU-TetO vector. Lentiviral vectors were co-transfected with packaging plasmids (pMDL, Rev and VSVg, kindly provided by Oded Singer, Laboratory of Genetics, The Salk Institute, La Jolla, CA) in 293T cells using Lipofectamine (Invitrogen) in accordance with manufacturer’s recommendations.
Lentiviral supernatants were collected 36 hours after transfection, concentrated by ultracentrifugation at 19400 rpm for 2 hours and resuspended in hES cell media.
The shRNA-expressing lentiviral pLKO.1 vectors were purchased from OpenBiosystems (ThermoFisher) and correspond to the following clones:
Rivera T., Haggblom C., Cosconati S, & Karlseder J. (2016). A balance between elongation and trimming regulates telomere stability in stem cells. Nature structural & molecular biology, 24(1), 30-39.
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5

Induced Pluripotent Stem Cell Generation

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105 sorted cells were infected with retrovirus generated from Plat-E cells, which were transfected with pMXOct4 (id: 13366), pMXSox2 (id: 13367), pMXKlf4 (id: 13370), and pMXc-Myc (id: 13375) (Addgene) in 6-well dishes with 0.5 ml of each viral supernatant (total 2 ml per well), and spun at 2500 rpm at 20°C for 90 min. The reprogramming factor-infected osteoblasts were cultured in osteoblast medium for 5 days before replating 8 × 105 cells per 10 cm dish precoated with mitomycin C-inactivated mouse embryonic fibroblast in ES maintenance media. Media were changed daily until ES-like colonies were observed. F-iPS cells were generated from dermal fibroblasts of newborn mice. 106 fibroblast cells were plated onto all wells of a 6-well plate and spin-infected with the four viral supernatants. Cells were cultured further in DMEM media supplemented with 15% FBS, 1 × penicillin/streptomycin/glutamine (Invitrogen). On day 5, the cultured cells were trypsinized and replated in four 10 cm dishes on mitomycin C-inactivated mouse embryonic fibroblast with ES maintenance media. Media were changed every day until ES-like colonies were observed.
Wang Y., Liu J.A., Leung K.K., Sham M.H., Chan D., Cheah K.S, & Cheung M. (2018). Reprogramming of Mouse Calvarial Osteoblasts into Induced Pluripotent Stem Cells. Stem Cells International, 2018, 5280793.
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6

Induced Pluripotency Vector Acquisition

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DNA vectors pMX-GFP, pMX-OCT4, pMX-SOX2, pMX-KLF4, and pMX-cMYC (h.sapiens) were purchased from Addgene (Watertown, MA, USA).
Lopez-Caraballo L., Martorell-Marugan J., Carmona-Sáez P, & Gonzalez-Munoz E. (2020). iPS-Derived Early Oligodendrocyte Progenitor Cells from SPMS Patients Reveal Deficient In Vitro Cell Migration Stimulation. Cells, 9(8), 1803.
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