Generation of pLenti-CMV-YFP and retroviral pBMN-mCherry were previously described11 (link). pCDH-CMV-MCS-EF1a-eGFP-T2A-Puro (System Bioscience #CD510B-1) was a kind gift from Dr. Weilin Jin (Shanghai Jiao Tong University, China). To generate the pLVX-tdTomato-IRES-Hygro construct, tdTomato cDNA was isolated from the pQC-membrane tdTomato IX construct (Addgene #37351) by polymerase chain reaction (PCR) and subcloned into the pLVX-IRES-Hyg vector (Clontech #632185). pLenti-VIIb-EVP-Neo-mVenus-p27K- was generously provided by the Paddison laboratory (FHCRC). This vector encodes a mutant form of p27 that lacks cyclin-dependent kinase inhibitory activity, and an mVenus reporter for quiescence72 . pBABE-YAP1-S127A, S127AS397A and S94A mutant constructs73 were kind gifts from the Vasioukhin lab (FHCRC). YAP mutant cDNA regions were amplified and subcloned into the pCDH-CMV-MCS-EF1a-eGFP-T2A-Puro construct with a FLAG tag at the 5’ end. Stable knockdown of human DAG1 was achieved with shRNAs cloned with a mir30 background in the pGIPZ vector (Dharmacon). To target human ITGA6, ITGB1, YAP1 or murine DAG1, the pLKO.1-blast shRNA plasmid (Addgene#26655) was used. All shRNA sequences are detailed in Supplementary Table 3 .
Plvx ires hyg vector
Manufactured by Takara Bio
Sourced in Canada, China
The PLVX-IRES-Hyg vector is a lentiviral expression vector designed for the expression of genes of interest. It contains an internal ribosome entry site (IRES) sequence to allow for the co-expression of the gene of interest and a hygromycin resistance gene, enabling selection of transduced cells.
Automatically generated - may contain errors
Lab products found in correlation
Pgipz vector, by Horizon Discovery (2 mentions)
Pqc membrane tdtomato 9 construct, by Addgene (2 mentions)
Plko 1 blast shrna plasmid, by Addgene (2 mentions)
Polybrene, by Merck Group (2 mentions)
Lenti ht packaging mix, by Takara Bio (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
In fusion cloning, by Takara Bio (1 mentions)
Plvx puro vector, by Takara Bio (1 mentions)
Mtagbfp, by Takara Bio (1 mentions)
Pfu dna polymerase, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Enzynomics (1 mentions)
Dpn 1, by Enzynomics (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Pgl3 vector, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cmv promoter, by Promega (1 mentions)
Hygromycin, by Roche (1 mentions)
Pwpi lentiviral vector, by Addgene (1 mentions)
7 protocols using plvx ires hyg vector
1
Generating Lentiviral and Retroviral Constructs for Cell Line Engineering
2
Overexpression and Knockdown of ACTL6A and MYC in Cell Lines
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Human ACTL6A cDNA was PCR-amplified and cloned into the pLVX-IRES-Hyg vector (Clontech). Two shRNA against ACTL6A and one shRNA against MYC in pSuper-retro-neo vector were purchased from Sigma-Aldrich. Transfection of plasmids was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. Cells (2 × 105) were seeded and infected by retrovirus generated by pLVX-IRES-Hyg-ACTL6A and pSuper-retro-neo-ACTL6A-shRNA/-MYC-shRNA for 3 days. The stable cell lines expressing ACTL6A, ACTL6A-shRNAs and MYC-shRNA were selected with 200 μg/mL hygromycin and 400 μg/mL G418 for 7 days, respectively. The indicated sequences were provided in the Supplementary Table S1 .
3
Generating Lentiviral and Retroviral Constructs for Cell Line Engineering
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Generation of pLenti-CMV-YFP and retroviral pBMN-mCherry were previously described11 (link). pCDH-CMV-MCS-EF1a-eGFP-T2A-Puro (System Bioscience #CD510B-1) was a kind gift from Dr. Weilin Jin (Shanghai Jiao Tong University, China). To generate the pLVX-tdTomato-IRES-Hygro construct, tdTomato cDNA was isolated from the pQC-membrane tdTomato IX construct (Addgene #37351) by polymerase chain reaction (PCR) and subcloned into the pLVX-IRES-Hyg vector (Clontech #632185). pLenti-VIIb-EVP-Neo-mVenus-p27K- was generously provided by the Paddison laboratory (FHCRC). This vector encodes a mutant form of p27 that lacks cyclin-dependent kinase inhibitory activity, and an mVenus reporter for quiescence72 . pBABE-YAP1-S127A, S127AS397A and S94A mutant constructs73 were kind gifts from the Vasioukhin lab (FHCRC). YAP mutant cDNA regions were amplified and subcloned into the pCDH-CMV-MCS-EF1a-eGFP-T2A-Puro construct with a FLAG tag at the 5’ end. Stable knockdown of human DAG1 was achieved with shRNAs cloned with a mir30 background in the pGIPZ vector (Dharmacon). To target human ITGA6, ITGB1, YAP1 or murine DAG1, the pLKO.1-blast shRNA plasmid (Addgene#26655) was used. All shRNA sequences are detailed in Supplementary Table 3 .
4
Fluorescently-tagged and biotinylatable human γTuRC production
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To generate a fluorescently-tagged and biotinylatable human γTuRC, the coding region for full-length human GCP2 (amino acids 1-902) was amplified by PCR using its cDNA as template (NM_001256617.1, Origene). The mTagBFP (blue fluorescent protein, Evrogen) coding sequence was also amplified by PCR. Both PCR-amplified sequences were cloned into a pLVX-Puro vector (Clonetech) using Gibson assembly (In-Fusion cloning, Takara), to form GCP2_G5A_TEV_G5A_mTagBFP_G5A_BAP, an expression construct for GCP2 which is C-terminally tagged with mTagBFP and biotin acceptor peptide (BAP: GLNDIFEAQKIEWHE), both separated from GCP2 by a TEV protease cleavage site. Glycine linkers (G5A) were placed between sequences. To facilitate the in vivo biotinylation of tagged γTuRC E. coli biotin ligase BirA was cloned into a pLVX-IRES-Hyg vector (Clonetech) using Gibson assembly to form HA_ G5A_BirA; an expression construct of BirA with an HA-tag added to the BirA N-terminus, separated by a G5A-linker. Primers used for cloning are listed in the Key Resources Table .
5
Generating GFP-tagged Sur8 Overexpression Lentiviral Plasmids
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For generating GFP-tagged Sur8-overexpressing lentiviral plasmids, GFP was inserted into pLVX-IRES-Hyg vector (#632185, Clonetech, Palo Alto, CA) at NotI enzyme site prior to cloning Sur8. Human Sur8 complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the following primers: forward 5′-ATAACCGGGATCCACCATGAGTAGTAGTTTAGGA-3′; reverse 5′-TCTAGAGGATCCGACCATGGCACGATATGG-3′. It was inserted into the pLVX-IRES-Hyg vector expressing GFP after digestion with BamHI (Enzynomics, Daejeon, Korea).
For site-directed mutagenesis, point mutations of Sur8 were introduced by PCR using Pfu DNA polymerase (Invitrogen, Carlsbad, CA). The mutagenic oligonucleotides used for mutagenesis are shown inSupplementary Table 1 . PCR reactions were run in the following condition: 15 cycles of 30 seconds at 95°C, then 1 minute at 54°C followed by 10 minutes at 68°C. The PCR products were digested with DpnI (Enzynomics) for 1 hour for removal of the template plasmids. All mutant plasmids were verified by DNA sequencing (Cosmogenetech).
For site-directed mutagenesis, point mutations of Sur8 were introduced by PCR using Pfu DNA polymerase (Invitrogen, Carlsbad, CA). The mutagenic oligonucleotides used for mutagenesis are shown in
6
Overexpression of miR-539 Regulates FSCN1
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Viral vectors containing the miR-539 (GenBank ID: 664612) construct and the green fluorescent protein (GFP) construct were purchased from Takara (Shiga, Japan). The miR-539 sequence was sub-cloned into the pLVX-IRES-Hyg vector (Takara) to generate pLV-miR-539. The viral particles were harvested 48 h after transfection of pLV-miR-539 into HEK293T cells using the Lenti-HT Packaging Mix (Takara). HCCLM3 and SK-HEP1 cells were infected with the harvested recombinant lentivirus in the presence of 6 µg/ml Polybrene (Sigma, St. Louis, MO, USA). The empty pLV-GFP vector encoding GFP was used as the control (Fig. 1 ). The viral particles were harvested as previously described (11 (link)) and used in all subsequent studies.
The full-length 3′-UTR of the FSCN1 gene containing the putative miR-539 binding sites was amplified by PCR and was inserted into the pGL3 vector under the control of the CMV promoter (Promega, Madison, WI, USA). The coding sequences of FSCN1 were amplified by PCR and cloned into the pCDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA) to generate pCDNA-FSCN1. The primer sequences used to amplify the target genes are listed inTable I . The correct insertion of the PCR-amplified sequences was confirmed by sequencing. The plasmid was transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
The full-length 3′-UTR of the FSCN1 gene containing the putative miR-539 binding sites was amplified by PCR and was inserted into the pGL3 vector under the control of the CMV promoter (Promega, Madison, WI, USA). The coding sequences of FSCN1 were amplified by PCR and cloned into the pCDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA) to generate pCDNA-FSCN1. The primer sequences used to amplify the target genes are listed in
7
Overexpression of miR-130b in Colorectal Cancer
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The human primary microRNA 130b gene (pri-miR-130b) was amplified by PCR from human genomic DNA using the following primers: Forward 5′-ATATTCTCGAGGGGGATCTCCC-3′ and Reverse 5′-ATATCGGATCCTCTTACCCCAG-3′ , and then subcloned into the pLVX-IRES-Hyg vector (TaKaRa, Dalian, China) to generate pLVX-miR-130b. The virus particles were harvested 48 h after the transfection of pLVX-miR-130b into HEK-293T cells using the Lenti-HT packaging mix (TaKaRa, Dalian, China). The SW480 cells were infected with the harvested recombinant lentivirus in the presence of 6 µg/ml Polybrene (Sigma, St Louis, USA). The SW480 cells were maintained in complete growth medium in the presence of 1 mg/ml Hygromycin (Roche Applied Science, Mannheim, Germany). The PCR amplicon of pri-miR-130b was also subcloned into the pWPI lentiviral vector (Addgene, Cambridge, MA, USA) to generate pWPI-miR-130b. The empty pWPI vector, encoding green fluorescent protein (GFP), was used as the control. The virus particles were harvested as described earlier. The SW620 cells stably expressing GFP were selected by fluorescent-activated cell-sorting (FACS), with the use of a Vantage SE Diva cell sorter (Becton Dickinson, Franklin Lakes, NJ, USA).
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