The plasmids Flag-tagged TAZ (Flag-TAZ), Flag-TAZ-4SA, and Flag-TAZ-4SA-S51A were gifts from Dr. Guan Kunliang (UCLA) and were described previously [19 (link), 36 (link), 37 (link)]. Full-length TAZ and its mutants TAZ-4SA and TAZ-4SA-S51A were cloned into pcDNA3.0-HA as HindIII-EcoRI fragments (HA-TAZ, HA-4SA, and HA-4SA-S51A). Full-length TAZ was also cloned into the retroviral expression vector pBABEpuro as a BamHI-EcoRI fragment (pBABE-TAZ). PcDNA3.0-Flag-Merlin (pMerlin), the full-length Merlin isoform I, was obtained from Addgene. Retroviruses were produced by transfecting packaging cells (HEK293) with a three-plasmid system consisting of an empty pBABEpuro vector or pBABE-TAZ, the packaging plasmid pUMVC, and the envelope plasmid pCMV-VSV-G. pUMVC and pCMV-VSV-G were obtained from Addgene. Retroviruses were frozen at −20°C or −80°C for long-term storage. Each amplified DNA fragment was verified by sequencing the inserts and flanking regions of the plasmids. Short hairpin RNAs against human TAZ and TEADs were described previously [19 (link), 21 ]. Sh-TAZ-1 and Sh-TAZ-2 were subcloned into a pLKO.1 puro lentiviral plasmid and verified via direct sequencing. Retroviruses were produced by transfecting packaging cells with pCMV-dR8.2 and pCMV-VSVG, which were obtained from Addgene.
Pcmv vsv g
Manufactured by Addgene
Sourced in United States, Japan, Canada, China, United Kingdom
PCMV-VSV-G is a plasmid that contains the gene encoding the vesicular stomatitis virus (VSV) glycoprotein. This glycoprotein can be used to pseudotype lentiviral vectors, which can then be used for gene delivery applications.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (209 mentions)
Pcmv dr8.2 dvpr, by Addgene (94 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (74 mentions)
Prsv rev, by Addgene (68 mentions)
Polybrene, by Merck Group (60 mentions)
Pmdlg prre, by Addgene (55 mentions)
Pcmv dr8, by Addgene (36 mentions)
Opti mem, by Thermo Fisher Scientific (35 mentions)
Puromycin, by Merck Group (34 mentions)
Pumvc, by Addgene (33 mentions)
Lenti x concentrator, by Takara Bio (31 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (24 mentions)
Puromycin, by Thermo Fisher Scientific (23 mentions)
Lenticrispr v2, by Addgene (20 mentions)
Padvantage, by Promega (19 mentions)
Lenti x 293t cells, by Takara Bio (18 mentions)
Pcmv delta r8, by Addgene (15 mentions)
Fugene hd, by Promega (14 mentions)
Pcmvr8, by Addgene (13 mentions)
Pmd2 g, by Addgene (12 mentions)
587 protocols using pcmv vsv g
1
Cloning and production of TAZ plasmids
2
Lentivirus Mediated Knockdown of MOF
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HEK293T cells were plated 1 day before transfection with psPax2 (viral packaging plasmid; Addgene 12260 ), pCMV-VSV-G (viral envelope plasmid; Addgene 8454 ), and a pLKO.1-based plasmid containing either scrambled sequences or human MOF shRNA (Kapoor-Vazirani et al., 2008 (link), 2011 (link); pCMV-VSV-G and pLKO.1 puromycin; Addgene 8454 and 8453 , respectively) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instruction. The pLKO.1-based plasmids were constructed according to Addgene's protocol. Medium containing lentiviral particles collected 1 day after transfection were filtered through a 0.45-μm filter (Advantec), and then 2 μg/ml polybrene (Sigma-Aldrich) was added. Recipient cells were plated 1 day before the infection and the medium was replaced with a lentiviral-containing medium for 18–24 h. Then, the medium was changed to a fresh medium containing 1 μg/ml puromycin (Thermo Fisher Scientific) for 11-4 cells and 2 μg/ml puromycin for AT5BIVA cells. After 1–2 days of puromycin selection, the medium was replaced with fresh medium.
3
Lentivirus and Retrovirus Production
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Lentiviruses were generated by cotransfection of viral vectors (1.5 μg) with packaging plasmids psPAX2 (0.75 μg) and pCMV-VSVG (0.25 μg; Addgene) into 293T cells with 90% confluency in a 6-well plate. Retroviruses were generated by cotransfection of viral vectors (2 μg) with pCMV-VSVG (0.25 μg; Addgene) into Phoenix-gp cells with 90% confluency in a 6-well plate. Polyethyleneimine (PEI) was added during cotransfection with a ratio of total DNA:PEI = 1:3 to facilitate the binding of the plasmid to the cell surface. Viral-containing supernatants were cleared of cellular debris by 0.45-μm filtration. Target cells were exposed to viral supernatants and mixed with 4 μg/mL polybrene overnight before being washed, grown for 24 hours in fresh media, and then subjected to antibiotic selection or fluorescence-based cell sorting.
4
Complex Reprogramming of Mouse Fibroblasts
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MEFs were derived from E13.5 C57BL/6J embryos (the Jackson laboratory: 000664). Retroviral particles were produced by transfecting 293T-17 cells (ATCC: CRL-11268) with the pGCDN-Sam construct containing Hnf4α-t2a-Foxa1/Fos/Yap1, along with packaging construct pCL-Eco (Imgenex). Lentiviral particles were produced with the envelope construct pCMV-VSV-G (Addgene plasmid 8454), the packaging construct pCMV-dR8.2 dvpr (Addgene plasmid 8455), and the shRNA expression vector for the respective candidate TF to be knocked down. For generation of the complex CellTag library, lentiviral particles were produced by transfecting 293T-17 cells (ATCC: CRL-11268) with the pSMAL-CellTag construct, along with packaging constructs pCMV-dR8.2 dvpr (Addgene plasmid 8455) and pCMV-VSVG (Addgene plasmid 8454). For iEP reprogramming, MEFs (< passage 6) were converted to iEPs as in Biddy et al. (2018 (link)), modified from (Sekiya and Suzuki, 2011 (link)). Colony-formation assays were performed as in Biddy et al. (2018 (link)). Perturb-seq was performed as previously described (Adamson et al., 2016 (link)). Single-cell libraries were prepared using the 10x Genomics Chromium platform.
5
Inducible NSD1 Knockdown Cell Lines
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To generate stable cell lines with inducible NSD1 knockdowns, self-complementary single-stranded DNA oligos (Supplementary Table 2 ) were annealed and cloned into AgeI/EcoRI sites of Tet-pLKO-puro vector (Addgene, #21915). Tet-pLKO-puro vectors were packaged into a lentivirus system with pCMV-VSV-G (Addgene, #8454) and psPAX2 (Addgene, #12260). A pBABE-puro mCherry-EGFP-LC3B plasmid was obtained from Addgene (#22418) and packaged into a retrovirus system with packing plasmid pCMV-VSV-G (Addgene, #8454) and pUMVC (Addgene, #8449). HEK293T cell line was used for retroviral and lentiviral system amplification with TransIT-293 Transfection Reagent (Mirus).
6
Inducible NSD1 knockdown cell lines
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To generate stable cell lines with inducible NSD1 knockdowns, self-complementary single-stranded DNA oligos (Supplementary Table 2 ) were annealed and cloned into AgeI/EcoRI sites of Tet-pLKO-puro vector (Addgene, #21915). Tet-pLKO-puro vectors were packaged into a lentivirus system with pCMV-VSV-G (Addgene, #8454) and psPAX2 (Addgene, #12260). A pBABE-puro mCherry-EGFP-LC3B plasmid was obtained from Addgene (#22418) and packaged into a retrovirus system with packing plasmid pCMV-VSV-G (Addgene, #8454) and pUMVC (Addgene, #8449). HEK293T cell line was used for retroviral and lentiviral system amplification with TransIT-293 Transfection Reagent (Mirus).
7
Detailed Virus Propagation and Titration
8
Production and Infection of Viral Particles
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mCherry/iRFP infecting viral particles were produced as described previously [35 (link),36 (link)]. Briefly, HEK 293T cells were transfected with pQC-mCherry [35 (link)] and with the compatible packaging plasmids pCMV-VSV-G (#8454, Addgene, Watertown, MA, USA) and pUMVC (#8449, Addgene, Watertown, MA, USA), or with pLVX-iRFP [37 (link)] together with pCMV-VSV-G (#8454, Addgene, Watertown, MA, USA) and psPAX2 (#12260, Addgene, Watertown, MA, USA). Viral particle-containing media were collected after 48 h. For cell infection, U251 cells were incubated with viral particles and polybrene (8 μg/mL) (hexadimethrine bromide) for 8–16 h prior medium replacement. Following 48 h, mCherry-infected cells were selected by puromycin and iRFP infected cells by hygromycin.
9
Lentivector-mediated YPEL4 expression
10
KRAS G12D Inducible HPDE Cell Engineering
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The packaging plasmids pCMV-VSV-G (#8454), pCMV-VSV-G (#8454), pMDLg/pRRE (#12251) were obtained from Addgene, P442-empty vector and P442-PLI-AIFM2-WT was kindly provided by J. P. Friedmann-Angeli, pLKO.1-empty vector and pLKO.1-shFSP1 were purchased from Merck (NM_153779/TRCN0000112139/pLKO.1). pCW-Puro-KRASG12D to generate doxycycline KRASG12D-inducible HPDE cells was cloned from pCW (addgene #50661 [15 (link)]) by replacing the existing Cas9 gene by human KRASG12D cDNA.
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