Virapower lentiviral packaging mix

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ViraPower Lentiviral Packaging Mix is a laboratory product designed to facilitate the production of lentiviral particles. It contains the necessary components for the packaging of lentiviral vectors, enabling the generation of high-titer lentiviral stocks for various research applications.

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ViraPower Packaging Mix (73 citations) ViraPower (42 citations) Lentiviral packaging mix (11 citations) ViraPower Mix (11 citations) ViraPower™ Lentiviral Packaging Mix kit (2 citations)

119 protocols using virapower lentiviral packaging mix

Optimized Multiplexed CRISPR Screening

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The Fred Hutchinson Co-operative Center for Excellence in Hematology Vector Production core produced all virus for the multiplexed enhancer-gene pair screening experiments. For the singleton CRISPRi recapitulation, virus was made in-house by co-transfecting (Lipofectamine 3000, ThermoFisher, L300015) HEK293Ts with the small pools of CRISPRi-optimized CROP-seq with the ViraPower Lentiviral Packaging Mix (ThermoFisher). After 3 days, supernatant was syringe filtered with a 0.45 uM filter (cellulose acetate, VWR) to prepare virus for transduction.
Cells were transduced (8 μg/mL polybrene) with varying titers and amounts of virus to achieve differing MOI. 400,000 and ~2.5 million original cells were transduced for the pilot and at-scale experiments, respectively. At 24 hours post-transduction, cells were spun and resuspended with virus- and polybrene- free media. At a total 48 hours post-transduction, 2 μg/mL puromycin was added to the culture, and changed to 1 μg/mL puromycin at the next passage for maintenance. A total of 10 days post transduction, cells were collected for scRNA-seq or bulkRNA-seq.

Gasperini M., Hill A.J., McFaline-Figueroa J.L., Martin B., Kim S., Zhang M.D., Jackson D., Leith A., Schreiber J., Noble W.S., Trapnell C., Ahituv N, & Shendure J. (2019). A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens. Cell, 176(1-2), 377-390.e19.

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HA-CADM1 Lentiviral Expression in HCC827 Cells

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HA‐CADM1 was cloned in a pENTR/D‐TOPO vector (Thermo Fisher Scientific). The lentiviral expression vector was then obtained by Gateway recombination with pLenti6‐V5/DEST (Thermo Fisher Scientific). The vector obtained and ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) were co–transfected into 293FT cells using Polyethylenimine Max (Polysciences, Warrington, UK). After 48 hours, HCC827 cells were infected with the lentivirus by incubating with the culture supernatant of 293FT cells containing 5 μg/mL Polybrene (Nacalai Tesque). HCC827 cells expressing HA‐CADM1 were selected by 20 μg/mL blasticidin.

Ito T., Nakamura A., Tanaka I., Tsuboi Y., Morikawa T., Nakajima J., Takai D., Fukayama M., Sekido Y., Niki T., Matsubara D, & Murakami Y. (2019). CADM1 associates with Hippo pathway core kinases; membranous co–expression of CADM1 and LATS2 in lung tumors predicts good prognosis. Cancer Science, 110(7), 2284-2295.

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Cloning and Lentiviral Expression of MERIT40 Variants

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The cDNAs for wild-type MERIT40, MERIT40ΔTBM1, MERIT40ΔTBM2 and MERIT40ΔTBM1/2 were amplified by PCR and subcloned into the pBabe-Myc-puro vector. MERIT40G33A vector was generated from pBabe-Myc-MERIT40 using QuikChange II XL Site-Directed Mutagenesis Kit (#200522, Agilent Technologies, Santa Clara, CA). To produce retroviruses, GP2-293 cells were transfected with these vectors and pVSV-G using polyethylenimine (PEI) MAX (#24765-1, Polysciences, Inc., Warrington, PA). A549 or HeLa cells were infected with the retroviruses and selected with 1 μg/mL puromycin for 3 days. To produce lentivirus, the MERIT40 cDNAs were inserted into the pLenti6/V5-Dest vector (V49610, Thermo Fisher Scientific) according to manufacturer's instructions. Lentiviruses were produced by co-transfection of HEK293FT cells with ViraPower Lentiviral Packaging Mix (K497500, Thermo Fisher Scientific) using PEI MAX. After 48 h, A549 or HeLa cells were infected with the lentiviruses and selected with 10 μg/mL blasticidin for 3 days.

Okamoto K., Ohishi T., Kuroiwa M., Iemura S.I., Natsume T, & Seimiya H. (2018). MERIT40-dependent recruitment of tankyrase to damaged DNA and its implication for cell sensitivity to DNA-damaging anticancer drugs. Oncotarget, 9(88), 35844-35855.

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Lentiviral Expression of Gaussia Luciferase and CFP

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Gaussia luciferase (Gluc) was cloned in CSCW, a self-inactivating lentivirus vector, under the control of the cytomegalovirus (CMV) immediate early promoter as previously described (Sena-Esteves 2004); this promoter also controls the expression of CFP separated from Gluc by an internal ribosome entry site (IRES). Lentiviral vectors were constructed by co-transfecting 293-FT cells with this plasmid and ViraPower™ Lentiviral Packaging Mix using Lipofectamine™ 2000 (Thermo Fisher Scientific). The supernatant containing the lentivirus was harvested after 72 hours, concentrated (Amicon® centrifugal filters) and the titer quantified as transducing units/mL. MDA-MB-231 cells were then infected with lentivirus in the presence of 8 µg/mL Polybrene® (Sigma-Aldrich) and transduction efficiency confirmed by counting CFP-positive cells 48 h post-infection. The construction of cDNA constructs in pCDNA-DEST40 vectors (Thermo Fisher Scientific) for expressing C-terminally expressed V5-tagged proteins in mammalian cells has been described previously (22 (link)). EGFR, HER2, HER3, HER4 and a HER3 signal peptide mutant (HER3 G11L/S15L) were transiently expressed in HEK293T cells using jetPRIME™ transfection reagent (Polyplus transfection, New York) according to manufacturer’s protocol.

Kazemi S., Kawaguchi S., Badr C.E., Mattos D.R., Ruiz-Saenz A., Serrill J.D., Moasser M.M., Dolan B.P., Paavilainen V.O., Oishi S., McPhail K.L, & Ishmael J.E. (2020). Targeting of HER/ErbB family proteins using broad spectrum Sec61 inhibitors coibamide A and apratoxin A. Biochemical pharmacology, 183, 114317.

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Establishment of ATF5-Knockdown Cell Lines

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Vectors encoding shRNA-directed against mouse ATF5 (Genecopoeia, Rockville, MD, USA) were transfected into 293FT cells, along with ViraPower Lentiviral Packaging Mix (ThermoFisher Scientific, Waltham, MA, USA), using Lipofectamine^® 2000 Transfection Reagent (ThermoFisher Scientific) according to the manufacturer’s recommendations. Medium was refreshed after 18 h. Virus-containing medium was collected after additional 24 h, filtered through 0.45 µm Filter Unit (Merck Millipore, Billerica, MA, USA), and placed on adherent Mvt1 or Met1 cells in the presence of 8 µg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA). A stable ATF5-KD was achieved by selection of the infected Mvt1 and Met1 cells (obtained from a pool of clones) with 2 µg/ml puromycin (Sigma-Aldrich). Cells infected with a vector containing a scrambled shRNA sequence (pool of clones) were used as control cells (scrambled).

Ben-Shmuel S., Rashed R., Rostoker R., Isakov E., Shen-Orr Z, & LeRoith D. (2017). Activating Transcription Factor-5 Knockdown Reduces Aggressiveness of Mammary Tumor Cells and Attenuates Mammary Tumor Growth. Frontiers in Endocrinology, 8, 173.

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Lentiviral Overexpression of MEIS1 and MEIS2

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To derive cell lines overexpressing lentiviral constructs of MEIS1 or MEIS2, all lentiviral vectors used in this study contain the gene-of-interest in the pReceiver-LV105 backbone (GeneCopoeia). High titer lentivirus was made by separately co-transfecting the LV105 constructs with ViraPower Lentiviral packaging mix (#K497500, Thermo Fisher Scientific) in HEK-293T cells using Lipofectamine 2000 (#11668019, Thermo Fisher Scientific) according to manufacturer's instructions. After 48 and 72 hr, media containing the lentivirus was collected, spun down, and filtered using a 0.45 mm filter and used to infect target CWR-22rv1 or LAPC-4 cells with 5 mg/mL polybrene for 48 hr. Complete media were then replaced followed by selection and maintenance with puromycin (1 mg/mL, Invitrogen). Confirmation of MEIS1 or MEIS2 expression was confirmed using both qRT-PCR and Western blotting (anti-MEIS1 #ab19867, abcam); anti-MEIS2 (#TA337288, OriGene).

VanOpstall C., Perike S., Brechka H., Gillard M., Lamperis S., Zhu B., Brown R., Bhanvadia R, & Vander Griend D.J. (2020). MEIS-mediated suppression of human prostate cancer growth and metastasis through HOXB13-dependent regulation of proteoglycans. eLife, 9, e53600.

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ROR2 Knockdown in LNCaP Cells

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Flag-ROR2 construct (Gao et al, 2011 (link)) was given from Dr. Yingzi Yang (Harvard School of Dental Medicine, Boston, MA, USA) as a gift. ROR2 MISSION shRNA was purchased from Sigma-Aldrich (St. Louis, MO, USA). ROR2 shRNA lentiviral supernatant was generated with ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific, Waltham, MA, USA), and this was used to infect LNCaP cells. The expression of ROR2 was analysed using quantitative RT-PCR and immunoblot.

Lee G.T., Kwon S.J., Kim J., Kwon Y.S., Lee N., Hong J.H., Jamieson C., Kim W.J, & Kim I.Y. (2018). WNT5A induces castration-resistant prostate cancer via CCL2 and tumour-infiltrating macrophages. British Journal of Cancer, 118(5), 670-678.

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Lentivirus-Mediated TP53TG1 Overexpression

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The full-length sequence of TP53TG1 was amplified by PCR and cloned into pLV-EF1α-MCS-IRES-Puro vector (Biosettia, San Diego, CA, USA) to generate TP53TG1 overexpression lentivirus vector (lenti-TP53TG1). The lenti-TP53TG1 vector or empty vector was transfected into 293FT cells together with ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific). After transfection 96 h, lenti-TP53TG1 or lenti-control virus supernatant was harvested to further infect A549/DDP cells. Lastly, stable lentivirus-infected cells were screened with puromycin for at least 1 week.

Xiao H., Liu Y., Liang P., Wang B., Tan H., Zhang Y., Gao X, & Gao J. (2018). TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis. Cell & Bioscience, 8, 23.

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Generating Inducible LNCaP-AI Cell Lines

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For co-immunoprecipitation studies and luciferase reporter assays, above plasmids, as described, were transiently co-transfected into PCa or 293FT cells using lipofectamine 3000 (Thermo Fisher). siRNA targeting AR-FL (AR silencer select, Thermo Fisher, S1539) was transfected into PCa cells using Lipofectamine RNAiMax (Thermo Fisher) by reverse transfection. For generation of doxycycline-inducible LNCaP-AI-myc-Gli2_{628–897_3xNLS}, two steps of lentiviral transduction were performed as previously described [31 (link)]. Briefly, stable LNCaP-AI cells expressing the Tet-Repressor (TR) were generated by transducing lentivirus (pLenti6/TR vector, Thermo Fisher) and ViraPower™ Lentiviral Packaging Mix (Thermo Fisher). Cells expressing TR were selected in 10 µg/ml Blasticidin and subsequently transduced with lentivirus made from either pLenti4-TO (empty Control) or pLenti4-TO_myc-Gli2_{628–897_3xNLS} and were selected under 100 µg/ml Zeocin.

Li N., Truong S., Nouri M., Moore J., Al Nakouzi N., Lubik A.A, & Buttyan R. (2018). Non-canonical activation of hedgehog in prostate cancer cells mediated by the interaction of transcriptionally active androgen receptor proteins with Gli3. Oncogene, 37(17), 2313-2325.

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Lentiviral Vector Production and Titration

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The lentiviral vectors and ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) were co-transfected into 293T cells by Trans IT LT-1 (Mirus, Madison, WI), and the supernatants were recovered at 48 hr post-transfection. The lentivirus titer was determined by using a Lenti XTM qRT-PCR Titration Kit (Clontech, Mountain View, CA), and the expression levels and GFP were determined at 48 hr post-inoculation.

Kurihara T., Fukuhara T., Ono C., Yamamoto S., Uemura K., Okamoto T., Sugiyama M., Motooka D., Nakamura S., Ikawa M., Mizokami M., Maehara Y, & Matsuura Y. (2017). Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9. Scientific Reports, 7, 6122.

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