Pb to hngn2

Manufactured by Addgene

Sourced in United Kingdom

The PB-TO-hNGN2 is a plasmid that contains the human Neurogenin-2 (hNGN2) gene under the control of a tetracycline-inducible promoter. This plasmid can be used for the inducible expression of hNGN2 in a variety of cell types.

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3 protocols using pb to hngn2

Plasmid-Based Transcription Factor Differentiation

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The plasmids PB-TO-hNGN2 (Addgene plasmid #172115), PB-TO-hNIL (NGN2, ISL1, and LHX3, Addgene plasmid #172113), EFa1-Transposase (Addgene plasmid #172116) were used for transcription factor-based differentiation experiments. For the p53 reporter assay performed on all sub-lines, the PG13-mCherry p53 reporter plasmid was generated by replacing the luciferase sequence of PG13-Luc reporter plasmid^{61 (link)} (Addgene), which contains 13 copies of a p53-binding site followed by the polyoma promoter, with the mCherry sequence by the In-Fusion HD Cloning Plus kit (Takara). For the transposon-based p53 reporter assay performed on KOLF2.1J, the pCMV-hyPBase plasmid was obtained from Wellcome Trust Sanger Institute.^{62 (link)} The PB-PG13-mCherry-EF1a-PuroR-EGFP plasmid, generated by inserting the PG13-mCherry sequence into the PB-EF1a-PuroR-EGFP plasmid (H Oguro, unpublished), contains the PiggyBac 3’-inverted terminal repeat (PB 3’ITR), 5’-HS4 chicken β-globin (cHS4) insulator, PG13-mCherry reporter, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), bovine growth hormone polyadenylation signal (BGHpA), EF1α promoter, puromycin registrant gene, P2A peptide, EGFP, BGHpA, cHS4 insulator, and PB 5’ITR, in this order.

Pantazis C.B., Yang A., Lara E., McDonough J.A., Blauwendraat C., Peng L., Oguro H., Kanaujiya J., Zou J., Sebesta D., Pratt G., Cross E., Blockwick J., Buxton P., Kinner-Bibeau L., Medura C., Tompkins C., Hughes S., Santiana M., Faghri F., Nalls M.A., Vitale D., Ballard S., Qi Y.A., Ramos D.M., Anderson K.M., Stadler J., Narayan P., Papademetriou J., Reilly L., Nelson M.P., Aggarwal S., Rosen L.U., Kirwan P., Pisupati V., Coon S.L., Scholz S.W., Priebe T., Öttl M., Dong J., Meijer M., Janssen L.J., Lourenco V.S., van der Kant R., Crusius D., Paquet D., Raulin A.C., Bu G., Held A., Wainger B.J., Gabriele R.M., Casey J.M., Wray S., Abu-Bonsrah D., Parish C.L., Beccari M.S., Cleveland D.W., Li E., Rose I.V., Kampmann M., Aristoy C.C., Verstreken P., Heinrich L., Chen M.Y., Schüle B., Dou D., Holzbaur E.L., Zanellati M.C., Basundra R., Deshmukh M., Cohen S., Khanna R., Raman M., Nevin Z.S., Matia M., Van Lent J., Timmerman V., Conklin B.R., Chase K.J., Zhang K., Funes S., Bosco D.A., Erlebach L., Welzer M., Kronenberg-Versteeg D., Lyu G., Arenas E., Coccia E., Sarrafha L., Ahfeldt T., Marioni J.C., Skarnes W.C., Cookson M.R., Ward M.E, & Merkle F.T. (2022). A reference human induced pluripotent stem cell line for large-scale collaborative studies. Cell stem cell, 29(12), 1685-1702.e22.

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Plasmids for Cellular Imaging

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Plasmids used include PGK EGFP-LC3 (Addgene plasmid #200427; subcloned from PGK mCherry-LC3B, gift from Michael Ward, National Institutes of Health, Maryland), PGK 4xMito-mEmerald (Addgene plasmid #200430; subcloned from 4xMito-mScarlet-I, Addgene plasmid #98818), PGK ARF6^Q67L-CFP (Addgene plasmid #200428; subcloned from CMVARF6^Q67L-CFP, Addgene plasmid #11387), PGK CFP (Addgene plasmid #200429; subcloned from CMV ARF6^Q67L-CFP, Addgene plasmid #11387), CMV mScarlet-LC3B (Addgene plasmid #200431; subcloned from EGFP-LC3B, gift from T. Yoshimori, Osaka University, Japan, with mScarlet from Addgene plasmid #85054), PGK mScarlet-LC3B (Addgene plasmid #200083; subcloned from CMV mScarlet-LC3B, Addgene plasmid #200431), CMV EGFP-PPM1H-WT (#DU62939, MRC PPU reagents and services, University of Dundee, Scotland), PGK EGFP-PPM1H-WT (Addgene plasmid #200084, subcloned from #DU62939, MRC PPU reagents and services, University of Dundee, Scotland), CMV EGFP-PPM1H-H153D (subcloned from pLVX HA PPM1H H153D #DU66215, acquired from MRC PPU reagents and services, University of Dundee, Scotland), CMV FLAG-LRRK2-R1441G (#DU13077, MRC PPU reagents and services, University of Dundee, Scotland), PB-TO-hNGN2 (gift from iPSC Neurodegenerative Disease Initiative (iNDI) & Michael Ward, Addgene plasmid #172115), and piggyBac^™ transposase vector (available from Transposagen).

Dou D., Smith E.M., Evans C.S., Boecker C.A, & Holzbaur E.L. (2023). Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell reports, 42(5), 112448.

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Plasmids for Cellular Imaging

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