Lentiviral constructs expressing CDH11 shRNA and JUN shRNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The EPHB6-wild type, EPHB6-Q926R, EPHB6-del915-917 cDNA constructs were cloned into pCDH-CMV-MCS-EF1-Puro, a lentiviral vector for cDNA expression (System Biosciences, Mountain View, CA, USA). All lentiviral vectors were transfected into 293TN cells (System Biosciences) with Lipofectamine 3000 transfection reagent (Invitrogen, Waltham, MA, USA). Particles were collected 2 days after the transfection of the lentiviral plasmids and used to infect cancer cells. Lentivirus-infected cancer cells were puromycin-selected for 1 week.
Lentiviral constructs
Manufactured by Merck Group
Sourced in United States
Lentiviral constructs are a type of viral vector used in molecular biology and genetics research. They are designed to efficiently deliver genetic material into target cells, including non-dividing and hard-to-transfect cell types. These constructs incorporate the necessary components for virus production and cellular transduction, providing a versatile tool for gene delivery and expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Site directed mutagenesis kit, by Agilent Technologies (2 mentions)
293tn cells, by System Biosciences (2 mentions)
Pcdh cmv mcs ef1 puro, by System Biosciences (2 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Transfection reagent, by Qiagen (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Lentiviral construct, by Hanbio Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Mir 29a mimic, by RiboBio (1 mentions)
Puromycin, by Wisent (1 mentions)
Arpc3, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
11 protocols using lentiviral constructs
1
Lentiviral Transduction of Cancer Cells
2
Generating Stable Alkbh5 and Fto Knockdown Cell Lines
3
Lentiviral Silencing of TG2 in CRC
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In order to ectopically express or silence TG2 in CRCs, lentiviral constructs containing wild type (wt) TG2 or different shRNA's (Sigma-Aldrich, Dorset, UK) that target human TG2 were used to transduce the CRCs and allow optimisation of TG2 knock down by Wang et al. [53 (link)]. Comparison of wt cells with empty vector transduced control cells indicated no differences in the expression of TG2 or EMT markers Supplementary Figure 4 .
shRNA sequences: 5′-CCACCCACCATATTGTTT GAT- 3′
5′-ACAGCAACCTTCTCATC GAGT-3′
shRNA sequences: 5′-CCACCCACCATATTGTTT GAT- 3′
5′-ACAGCAACCTTCTCATC GAGT-3′
4
Overexpression and Knockdown of MUC15 in Cell Lines
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To develop an overexpression vector for MUC15, the pMSCV puro (lentivirus) vector was used. MUC15 gene was isolated from MUC15-expressing colon cancer cells and was used for complementary DNA (cDNA) synthesis. MUC15 cDNA was cloned into pMSCV puro vector and transfected into Nthy-ori 3-1 cells, followed by puromycin treatment for selection.
Lentiviral constructs containing MUC15-specific shRNA conjugated with puromycin resistance genes and tGFP was obtained from Sigma Aldrich (MO, USA). To generate the lentivirus, tGFP-shRNA of Lenti vector and packaging vectors were used to co-transfect with HEK293T cells in accordance with the manufacturer’s instructions. Viral supernatants were harvested and used to transduce FTC-238 cells. Transduction efficiency was analyzed via qPCR and western blotting analyses.
Lentiviral constructs containing MUC15-specific shRNA conjugated with puromycin resistance genes and tGFP was obtained from Sigma Aldrich (MO, USA). To generate the lentivirus, tGFP-shRNA of Lenti vector and packaging vectors were used to co-transfect with HEK293T cells in accordance with the manufacturer’s instructions. Viral supernatants were harvested and used to transduce FTC-238 cells. Transduction efficiency was analyzed via qPCR and western blotting analyses.
5
Lentiviral Manipulation of SMARCAD1 in PANC-1 Cells
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Lentiviral constructs expressing SMARCAD1 shRNAs and negative control were obtained from Sigma. The sequences of target shRNAs were as follows:
Sh1 5'-CCAGCACCTTATGACAATTAA-3'.
Sh2 5'-GCCTTATTTGACAATGCTTAT-3'.
Lentivirus was produced in 293T cells using the transfection reagent (QIAGEN) following the manufacturer's instructions. Supernatant containing virus was collected after 48h transfection. PANC-1 cells were infected with collected virus containing polybrene (10µg/ml), followed by selection with puromycin.
For over-expression of SMARCAD1, pLX304-V5-Blast was constructed and used. PANC-1 cells are transfected at the confluence between 30% and 50% and were selected with Blasticidin S (10µg/mL) after 48 hours of transfection.
Sh1 5'-CCAGCACCTTATGACAATTAA-3'.
Sh2 5'-GCCTTATTTGACAATGCTTAT-3'.
Lentivirus was produced in 293T cells using the transfection reagent (QIAGEN) following the manufacturer's instructions. Supernatant containing virus was collected after 48h transfection. PANC-1 cells were infected with collected virus containing polybrene (10µg/ml), followed by selection with puromycin.
For over-expression of SMARCAD1, pLX304-V5-Blast was constructed and used. PANC-1 cells are transfected at the confluence between 30% and 50% and were selected with Blasticidin S (10µg/mL) after 48 hours of transfection.
6
Lentiviral Knockdown in Schwann Cells
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Lentivirus was produced by transfection of lentiviral constructs purchased from Sigma-Aldrich (table S1) into human embryonic kidney 293T cells with the packaging vectors pMDL, pREV, and pVSV-G using Turbofect transfection reagent (Thermo Fisher Scientific, no. R0531), according to the manufacturer’s instructions. The supernatant was collected at 24 and 48 hours after transfection, concentrated with Lenti-X concentrator (Takara, no. 631231). For knockdown, Schwann cells were treated with short-hairpin lentiviral particles in the presence of hexadimethrine bromide (8 μg/ml). Twenty-four hours after transduction, the cells were selected using puromycin (0.5 μg/ml) (Sigma-Aldrich, P7255-250MG), and puromycin-resistant cell clones were grown, analyzed, and frozen for future use.
7
PARK2 and FASN Knockdown in DLBCL
8
Genetic Manipulation of PARK2 and FASN
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PARK2-HA [wild type (WT) and catalytically inactive (CS)] and PARK2-GFP constructs were a generous gift from Drs. Toshio Kitamura, Ted Dawson, and Edward Fon (Addgene plasmids #38248, 17613, 45875, 45877). pMX-IP-PARK2-HA (S127A, C431S) mutants were generated by using a site-directed mutagenesis kit (Agilent Technologies) according to the manufacturer's recommendations. Retroviral constructs for overexpression of encoding plasmids were transfected in Platinum-A cells, and the particles were concentrated as reported earlier. Lentiviral constructs for knockdown of PARK2 and FASN were purchased from Sigma. For lentiviral packaging, HEK293T/17 cells were seeded at 50% confluence and transfected using packing plasmids psPAX2 and pMD2.G (a generous gift from Dr. Didier Trono; Addgene # 12260 and #12259). Lentiviral particles were concentrated as defined earlier. DLBCLs were treated with polybrene (4 mg/mL, American Bioanalytical) and centrifuged (35 minutes, 300 × g, 25°C). Cell pellets were resuspended in the virus-containing medium for 48 hours, then selected and maintained with puromycin (1 mg/mL).
9
Modulating circHIPK3 in Endothelial Cells
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CMs or CMVECs were transfected at approximately 80% confluence using a lentiviral construct (HANBIO, China) according to the manufacturer's protocol. Some of those lentiviral constructs were empty (LV), and some carried synthesized circHIPK3 (LV-circHIPK3), small interfering RNAs (Sigma) targeting circHIPK3 (LV-sicircHIPK3), or linear HIPK3 (LV-silineHIPK3). In addition, an miR-29a inhibitor, miR-29a mimics, and negative controls were all synthesized by RiboBio (Guangzhou, China) and transfected into CMVECs by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. CMVEC functions and gene expression were evaluated 48 h after transfection. The sequence information is listed in Table 1 .
10
Lentiviral SIRT1 Gene Silencing
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Five lentiviral constructs targeting SIRT1 mRNA were purchased from Sigma as bacterial stocks. Stocks were grown and plasmid isolated as per the manufacturer instructions. shGFP plasmid was purchased from Addgene (plasmid 30323 from David Sabattini) [43 (link)]. 5μg of shRNA plasmid was cotransfected into 293T cells with 3.33μg Gag-Pol-Tat-Rev and 1.37μg VSVG packaging plasmids. After 72 hours, lentiviral cell supernatants were removed, spun briefly to remove cellular debris, concentrated using centrifugal filters (Millpore) and stored at -80°C. 50μL of concentrated lentivirus was used to transduce target cells.
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