Lentiviral vector

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Lentiviral Vector is a gene delivery tool used in research applications. It is a viral vector derived from the lentivirus family that can be used to stably integrate genetic material into the genome of target cells. The vector's core function is to serve as a vehicle for the efficient transduction and expression of genes of interest in a variety of cell types.

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PLKO.1 lentiviral vector (22 citations) Lentiviral pGIPZ vectors (4 citations) Lentiviral shRNA pGIPZ vectors; NS (RHS4348) (3 citations) GIPZ lentiviral shRNAmir vectors (3 citations) PGIPZ-lentiviral shRNAmir vectors (3 citations) PLKO lentiviral vectors (3 citations) Control lentiviral pLKO.1 vectors (2 citations) Lentiviral shRNA vectors (2 citations) Shisa2 shRNA lentiviral vectors (2 citations) Lentiviral vectors (pLKO.1) (2 citations)

11 protocols using lentiviral vector

Lentiviral Transduction of ACE2 in uMSCs

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The ACE2 complementary DNA (cDNA) sequence was amplified from total RNA with the following primers: Forward: 5′-AAGCTAGCATAGCCAGGTCCTCCTGGCTCCTTC-3′ and reverse: 5′-AAGTCGACCTAAAAGGAAGTCTGAGCATCATCACTG-3′. The amplification of ACE2 was conducted using the following procedure: Denaturation at 98°C for 30 sec, 35 cycles at 98°C for 10 sec, 55°C for 30 sec, 72°C for 90 sec and a final step at 72°C for 10 min, using the Reverse Transcription System. The sequences were then directionally cloned into the lentiviral vector (Invitrogen Life Technologies). The ACE2 lentiviral vector was co-transfected with packaging plasmid (Invitrogen Life Technologies) and envelope plasmid into 293 T cells (Invitrogen Life Technologies).
During the logarithmic phase, uMSCs were trypsinized and adjusted to a suitable cell density using DMEM/F12. The cells were reseeded into 25-ml culture flasks and then cultured in the incubator until cell density reached 2×10⁵/ml. Cells were subsequently transfected via viral vectors. In addition, green fluorescent protein (GFP) was simultaneously run as a control to label uMSCs and its expression was analyzed by fluorescence microscopy (Nikon Eclipse TE200; Nikon Corporation, Tokyo, Japan).

MIN F., GAO F., LI Q, & LIU Z. (2014). Therapeutic effect of human umbilical cord mesenchymal stem cells modified by angiotensin-converting enzyme 2 gene on bleomycin-induced lung fibrosis injury. Molecular Medicine Reports, 11(4), 2387-2396.

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Generating Meprin α-expressing Cell Lines

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A lentiviral vector coding meprin α with a C-terminal V5 tag was obtained from Thermo Scientific [38 (link)]. HuH7 and Hep3B cells were transduced and stable cell lines were obtained using selection with blasticidin. As a control, cells were transduced with lentiviral particles containing the empty pLX304 plasmid.

Breig O., Yates M., Neaud V., Couchy G., Grigoletto A., Lucchesi C., Prox J., Zucman-Rossi J., Becker-Pauly C, & Rosenbaum J. (2016). Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin. Oncotarget, 8(5), 7839-7851.

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Trophoblast Cell Line Analysis

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The human trophoblast cell line HTR-8/SVneo was purchased from ATCC, USA. RPMI-1640 medium, fetal bovine serum, and penicillin-streptomycin dual antibody were purchased from Gibco, USA. Lipofectamine 2000, Annexin V-FITC/PI apoptosis detection kit, lentiviral vector, pGLO vector, and the dual-luciferase reporter gene detection kit were purchased from ThermoFisher, USA. CCK-8 cell proliferation detection kit, E-cadherin, N-cadherin, Vimentin, and PAK1 and GAPDH protein primary and secondary antibodies were purchased from Abcam, UK. cDNA reverse transcription kit and real-time fluorescence quantitative PCR detection kit were purchased from Takara Company in Japan.

Chen X., Huang J., Peng Y., Han Y., Wang X, & Tu C. (2022). The role of circRNA polyribonucleotide nucleoside transferase 1 on Gestational Diabetes Mellitus. Cellular and molecular biology (Noisy-le-Grand, France), 68(6).

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Lentiviral-mediated CK-α knockdown in breast cancer cells

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The lentiviral vector that carries the transgenes for a CK-α specific shRNA and green fluorescent protein (GFP) was purchased from Thermo Scientific (no. RHS4531). The 293T cells were transfected with a CK-α shRNA lentiviral vector (pLenti-CK-α shRNA), a packing vector (pCMV-dR8.2) and an envelope vector (pCMV-VSVG) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for lentivirus packaging. Forty-eight hours later, the virus-containing supernatant medium was collected, filtered, and concentrated by ultracentrifugation. In brief, 1x10⁵ MCF-7 and MCF-7/TAM cells were seeded in a six-well plate and infected with lentivirus for 6 hours, and after replacing the culture medium, the cells were incubated for an additional 72 hours. The GFP-expressing cells were separated by a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) equipped with a 530-nm filter (bandwidth, ± 15 nm) and a 585-nm filter (bandwidth, ± 21 nm) then analyzed using CellQuest software (BD Biosciences). The downregulation of CK-α in transduced cells was evaluated by RT-PCR and Western blot. The specific CK-α knockdown cells for MCF-7 and MCF-7/TAM were denoted as MCF-7/shCK-α and MCF-7/TAM/shCK-α, respectively.

Kim H.S., Tian L., Kim H, & Moon W.K. (2017). Investigation of discriminant metabolites in tamoxifen-resistant and choline kinase-alpha-downregulated breast cancer cells using 1H-nuclear magnetic resonance spectroscopy. PLoS ONE, 12(6), e0179773.

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Knockdown of lnc-ABCA12-3 Using shRNA

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Short hairpin RNAs (shRNAs) of lnc-ABCA12-3 was constructed into a lentiviral vector (GeneChem, Shanghai, China), and a blank load transfection was sited as the negative control. The sequence against lnc-ABCA12-3 was as follows: 5’‐CCAAGATGCAAAGAAACAT‐3’. The shRNA in the lentiviral vector was transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The transfection efficiency was evaluated by qRT-PCR.

Ma J., Luo Y., Liu Y., Chen C., Chen A., Liang L., Wang W, & Song Y. (2023). Exosome-mediated lnc-ABCA12-3 promotes proliferation and glycolysis but inhibits apoptosis by regulating the toll-like receptor 4/nuclear factor kappa-B signaling pathway in esophageal squamous cell carcinoma. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 27(1), 61-73.

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Knockdown of hsa_circ_0004396 in NSCLC

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For stable knocking down of hsa_circ_0004396, short hairpin RNA against hsa_circ_0004396 was applied. Both sh‐hsa_circ_0004396 and sh‐NC (GenePharma) were constructed into the lentiviral vector (Invitrogen), followed by introduction into A549 cells and cultured in cell medium with 2 μg/ml puromycin.
Meanwhile, based on the producer’s direction of Lipofectamine 3000 (Invitrogen), miR‐615‐5p mimics/inhibitor and its control (miR‐NC/anti‐miR‐NC), empty vector (pcDNA3.1), overexpression vectors of hsa_circ_0004396 (pcDNA3.1‐hsa_circ_0004396), and PAK1 (pcDNA3.1‐PAK1) from Ribobio were transfected into 2 × 10⁵ NSCLC cells in six‐well plates.

Li D., Yan L., Zhang J, & Gu F. (2022). Circular RNA hsa_circ_0004396 acts as a sponge of miR‐615‐5p to promote non‐small cell lung cancer progression and radioresistance through the upregulation of P21‐Activated Kinase 1. Journal of Clinical Laboratory Analysis, 36(6), e24463.

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miR-138-5p Overexpression Lentiviral Protocol

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A lentiviral vector that overexpresses miR-138-5p was purchased from Invitrogen. When T24 cells reached 70% confluence in either 6-well plates or 100-mm dishes, lentivirus at a multiplicity of infection of 10 was added together with Polybrene (final concentration 5 μg/ml) according to the manufacturer’s instructions. Cells were then harvested for animal experiments.

Yang R., Liu M., Liang H., Guo S., Guo X., Yuan M., Lian H., Yan X., Zhang S., Chen X., Fang F., Guo H, & Zhang C. (2016). miR-138-5p contributes to cell proliferation and invasion by targeting Survivin in bladder cancer cells. Molecular Cancer, 15, 82.

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Lentiviral Knockdown and Overexpression of ISLR

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Short hairpin RNA against ISLR (sh-ISLR), negative control shRNA (shRNA-NC), and ISLR plasmids were designed and packaged into lentiviral vector by GenePharma (Shanghai, China). According to the manufacturers’ instructions of Lipofectamine 2000 (Invitrogen, Thermo, USA), the 293 T cells were transfected with lentiviral vector and packaging plasmids. After transfection for 48 h, HGC-27 and AGS cells were inoculated and cultivated in 24-well plates for 24 h. Once to 70–80% confluency, 1 × 10⁵ HGC-27 cells were transfected with lentivirus that carries shISLR-1, shISLR-2, or shRNA-NC to establish ISLR downregulated cell model; meanwhile, 1 × 10⁵ AGS cells were transfected with lentivirus carrying ISLR plasmids or vector to construct ISLR-upregulated cells model. Cells with >80% transfection efficiency were used for the subsequent experiments [21 (link)].

Sun A., Li J., Kong W, & Jiang X. (2022). Silencing of immunoglobulin superfamily containing leucine-rich repeat inhibits gastric cancer cell growth and metastasis by regulating epithelial–mesenchymal transition. Bioengineered, 13(5), 13544-13554.

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Tetracycline-Inducible Lentiviral PPARγ Expression

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Both PPARγ and enhanced green fluorescence protein (EGFP) genes flanked by internal ribosomal entry site were bicistronically constructed under the control of tetracycline-inducible (Tet/On) cytomegalovirus promoter of lentiviral vector (Invitrogen, Carlsbad, CA, USA). Site-directed mutations were introduced into both sumoylation sites (K79R and K367R for PPARγ1; K107R and K395R for PPARγ2) in PPARγ. Lentiviruses were produced and transduced into tumorigenic HBEC-C1 cell lines. Further screening process was performed to select a HBEC-C1-PPARγ clone in which both PPARγ and EGFP expression are tightly regulated upon tetracycline treatment.

Kim J., Sato M., Choi J.W., Kim H.W., Yeh B.I., Larsen J.E., Minna J.D., Cha J.H, & Jeong Y. (2015). Nuclear Receptor Expression and Function in Human Lung Cancer Pathogenesis. PLoS ONE, 10(8), e0134842.

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Lentiviral Vector Construction for hTERT

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For construction of the hTERT-expressing lentiviral vector, a cDNA-coding hTERT (NF-198253.2, 3399-bp cDNA) was synthesized and cloned into the BamHI and XbaI restriction endonuclease sites of the pLenO-DCE vector (Invabio Biotechnology Ltd., Shanghai, China). The pLenO-DCE-hTERT plasmid was then acquired, which also encodes green fluorescent protein (GFP). The products were transformed into competent fibroblasts. The emerged cell colonies were cultured, plasmid DNA was isolated, digested and sequenced to confirm that the correct clone had been made. After the correct sequence was confirmed, lentiviral vector particles were produced in accordance with the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). A control vector driving the expression of GFP (pLenO-DCE-GFP) was also generated. pRsv-REV, Pmd1g-Prre, Pmd2g and pLenO-DCE-hTERT were co-transfected into 293T cells (American Type Culture Collection; ATCC, Manassas, VA, USA) and viral supernatants were harvested 48 and 72 h post-transfection and concentrated using 4 rounds of ultracentrifugation. The cell surpernatant, which contained viral particles, was collected and stored at −80°C. Viral titers were detected by the double dilution method on 293T cells.

Liu Q., Sun Y., Lv Y., Le Z., Xin Y., Zhang P, & Liu Y. (2016). TERT alleviates irradiation-induced late rectal injury by reducing hypoxia-induced ROS levels through the activation of NF-κB and autophagy. International Journal of Molecular Medicine, 38(3), 785-793.

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