A 6.4 kb MluI-ClaI fragment was isolated from pLenti6.2/V5DEST (Invitrogen) and ligated to a linker comprising oligonucleotides 5'-CGATAACTGCAGAACCAATGCATTGGA-3' and 5'-CGCGTCCAATGCATTGGTTCTGCAGTTAT-3'. A library of MluI-PstI linkers was constructed using 24-bp Luminex DNA barcodes9 (link) placed within oligonucleotides 5'-CGCGTXXXXXXXXXXXXXXXXXXXXXXXXCTGCA-3' and 5'-GxxxxxxxxxxxxxxxxxxxxxxxxA-3', where XXX...XXX includes the sense barcode sequence and xxx...xxx includes the antisense barcode sequence, and each of these linkers was individually ligated into the MluI-PstI backbone of the above vector to generate lentiviral barcoding plasmids. Lentivirus was generated from lentivral barcoding plasmids as previously described24 using pCMV-dR8.2 dvpr and pCMV-VSVG packaging vectors in FuGENE6-transfected (Roche Corporation) HEK-293T cells; viral supernatant was collected after 72h, passed through a sterile 0.45μm syringe filter (VWR cat. 28144-007), and stored at −80°C.
Plenti6.2 v5 dest
Manufactured by Thermo Fisher Scientific
The PLenti6.2/V5-DEST is a lentiviral vector designed for the expression of genes of interest in mammalian cells. It provides a gateway-compatible destination vector for the expression of proteins with a C-terminal V5 epitope tag.
Automatically generated - may contain errors
Lab products found in correlation
Hek 293t cells, by Avantor (2 mentions)
Quickchange lightening site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Oligofectamine, by Thermo Fisher Scientific (1 mentions)
Mcf 10a cell line, by American Type Culture Collection (1 mentions)
Psbbi bla, by Addgene (1 mentions)
Lenticrispr v2 blast vector, by Addgene (1 mentions)
Plko 1 blast vector, by Addgene (1 mentions)
6 protocols using plenti6.2 v5 dest
1
Lentiviral Barcoding Plasmid Generation
2
Sema3C Isoform Generation and Knockdown
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Sema3C cDNA (NCBI DQ890847) vector from Genomic and Proteomics Core Facility of German Cancer Research Center was shuttled into pAD/CMV/V5 and pLenti6.2/V5-DEST (Invitrogen) by Gateway cloning for virus production. Sema3CΔ13 and Sema3Cp60 isoforms were generated using the QuickChange Lightening Site-Directed Mutagenesis Kit (Agilent Technologies) to introduce stop codons. PCR primers for the Sema3CΔ13 isoform: (5′-CAAGTTAAAGGCCCTCATCAATAGTTAGTAAAGTAGAAACAGGAGGAATCAGTT-3′), (5′-AACTGATTCCTCCTGTTTCTACTTTACTAACTATTGATGAGGGCCTTTAACTTG-3′); and for Sema3Cp60: (5′-GAAACGGAGGAGCCGAAGATAATAGGTGAGACATGGAAACCCAC-3′) and (5′-GTGGGTTTCCATGTCTCACCTATTATCTTCGGCTCCTCCGTTTC-3′). The translated protein sequences are shown in Fig 1A . Control siRNA (AM4636) and siRNA against human plexinD1 (AM16708, clone ID: 108678) were from LifeTechnologies. Transfection of HUVEC (1.2 × 105 cells) with siRNA duplexes (final concentration, 200 pmol) was performed with Oligofectamine according to the transfection protocol (Invitrogen). shRNA against Nrp-1 (clone ID: 16466, 333723) and Nrp-2 (clone ID: 346687, 176015) were from Thermo Scientific. shRNA against PlexinA2 (clone ID: 389145, 389146) was from Dharmacon.
3
Generating cell lines for fusion gene studies
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The MCF10A cell line was obtained from the American Type Culture Collection and cultured with DMEM medium supplemented with 5% horse serum, 0.5 mg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 µg/mL insulin, and 20 ng/mL EGF. TP53 knockout was performed by CRISPR-Cas9-mediated gene editing, and nine clones with confirmed homozygous knockout were pooled to obtain the TP53-deficient MCF10A cell line. The H6c7 cell line was obtained from Kerafast and cultured with Keratinocyte serum-free medium supplemented with 50 ng/mL bovine pituitary extract and 5 ng/mL EGF.
The fusion genes were synthesized by Trenzyme GmbH and cloned into the lentiviral expression vector pLenti6.2/V5-DEST (Invitrogen). Production of lentiviral particles and transduction of MCF10A and H6c7 cells was performed as previously described (Stolze et al. 2015 (link)). Transduced cells were selected with 10 µg/mL blasticidin to obtain cell lines with stable expression of the fusion genes or empty vector control.
The fusion genes were synthesized by Trenzyme GmbH and cloned into the lentiviral expression vector pLenti6.2/V5-DEST (Invitrogen). Production of lentiviral particles and transduction of MCF10A and H6c7 cells was performed as previously described (Stolze et al. 2015 (link)). Transduced cells were selected with 10 µg/mL blasticidin to obtain cell lines with stable expression of the fusion genes or empty vector control.
4
Lentiviral Barcoding Plasmid Generation
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A 6.4 kb MluI-ClaI fragment was isolated from pLenti6.2/V5DEST (Invitrogen) and ligated to a linker comprising oligonucleotides 5'-CGATAACTGCAGAACCAATGCATTGGA-3' and 5'-CGCGTCCAATGCATTGGTTCTGCAGTTAT-3'. A library of MluI-PstI linkers was constructed using 24-bp Luminex DNA barcodes9 (link) placed within oligonucleotides 5'-CGCGTXXXXXXXXXXXXXXXXXXXXXXXXCTGCA-3' and 5'-GxxxxxxxxxxxxxxxxxxxxxxxxA-3', where XXX...XXX includes the sense barcode sequence and xxx...xxx includes the antisense barcode sequence, and each of these linkers was individually ligated into the MluI-PstI backbone of the above vector to generate lentiviral barcoding plasmids. Lentivirus was generated from lentivral barcoding plasmids as previously described24 using pCMV-dR8.2 dvpr and pCMV-VSVG packaging vectors in FuGENE6-transfected (Roche Corporation) HEK-293T cells; viral supernatant was collected after 72h, passed through a sterile 0.45μm syringe filter (VWR cat. 28144-007), and stored at −80°C.
5
Overexpression and Knockdown of RHOQ
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For overexpression, RHOQ and GFP-RHOQ were cloned into pLenti 6.2/V5-DEST (Invitrogen). For stable knockdown, MISSION shRHOQ plasmids were purchased (Invitrogen; clone numbers TRCN0000289568 and TRCN0000047588). All plasmid inserts were packaged into virus using HEK293 cells and the ViraPower™ packaging mix (Invitrogen), according to manufacturer’s instructions.
6
Optimizing CRISPR/Cas9 Plasmid Systems
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We utilized the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, obtained from Feng Zhang (Addgene #42230), as the basis for constructing CRISPR/Cas vectors. To generate the mAID donor plasmids, we modified constructs of the Kanemaki lab (Addgene #72827 and #121180). In order to incorporate mRuby2, we replaced mCherry2 in the donor plasmid (Addgene #121180).
For the rescue experiments, wild-type (WT) UHRF1 and each of the point mutants (M8R/F46V, Y188A, DAEA, G448D, and H741A) were cloned into pLenti6.2/V5-DEST (invitrogen). Likewise, WT DNMT1 and each of the point mutants (H170V, D381A/E382A/S392A, W464A/W465A, C1226W) were cloned into pSBbi-Bla (Addgene: #60526). To target DNMT3A and DNMT3B, we cloned the oligonucleotide sequences for gRNA into the lentiCRISPR v2-Blast vector (Addgene #83480). Additionally, we cloned the shRNA targeting TET2 into the pLKO.1-blast vector (Addgene #26655). Plasmids were generated using PCR, restriction enzymes, or Gibson Assembly Cloning techniques. All plasmids underwent sequencing prior to their utilization. The oligonucleotide sequences inserted into the lentiCRISPR v2-Blast vector and pLKO.1-blast vector are available in Supplementary Table1 .
For the rescue experiments, wild-type (WT) UHRF1 and each of the point mutants (M8R/F46V, Y188A, DAEA, G448D, and H741A) were cloned into pLenti6.2/V5-DEST (invitrogen). Likewise, WT DNMT1 and each of the point mutants (H170V, D381A/E382A/S392A, W464A/W465A, C1226W) were cloned into pSBbi-Bla (Addgene: #60526). To target DNMT3A and DNMT3B, we cloned the oligonucleotide sequences for gRNA into the lentiCRISPR v2-Blast vector (Addgene #83480). Additionally, we cloned the shRNA targeting TET2 into the pLKO.1-blast vector (Addgene #26655). Plasmids were generated using PCR, restriction enzymes, or Gibson Assembly Cloning techniques. All plasmids underwent sequencing prior to their utilization. The oligonucleotide sequences inserted into the lentiCRISPR v2-Blast vector and pLKO.1-blast vector are available in Supplementary Table
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