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Lentiviral shrna pools

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Lentiviral shRNA pools are a collection of lentiviral particles that contain short hairpin RNA (shRNA) sequences. These shRNA sequences can be used to knock down the expression of target genes in cells through RNA interference (RNAi). The lentiviral particles can be used to transduce a population of cells, resulting in the stable integration of the shRNA sequences into the host cell genome.

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Lab products found in correlation

Pegfp c1, by Takara Bio (1 mentions) Pcmv gag pol, by Cell Biolabs (1 mentions) Xfect, by Takara Bio (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) D luciferin, by GoldBio (1 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions) C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)

3 protocols using lentiviral shrna pools

1

Engineered Retroviral Expression System for Cancer Research

Cited 2 times
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pNGL, pΙκΒαDN, and pCAG.LUC (#74409) have been described elsewhere8 (link),25 (link),33 (link). Lentiviral shRNA pools (Santa Cruz) are described in Supplementary Table 8. A pMIGR1-based (#27490) bicistronic retroviral expression vector was generated by replacing eGFP sequences with puromycin resistance gene (#58250). KrasG12C,Chuk, Ikbkb, Ikbke, and Tbk1 cDNAs were cloned via reverse transcriptase-PCR (RT-PCR) from LLC or MC38 RNA using specific primers (Supplementary Table 6) and were subcloned into peGFP-C1 (Takara, Mountain View, CA). eGFP, eGFP.KrasG12C, eGFP.Chuk, eGFP.Ikbkb, eGFP.Ikbke, and eGFP.Tbk1 cDNAs were subcloned into the new retroviral expression vector (#58249, #64372,# 87033, #58251, #87444, and #87443, respectively). Retroviral particles were obtained by co-transfecting HEK293T cells with retroviral vectors, pMD2.G (#12259), and pCMV-Gag-Pol (Cell Biolabs, San Diego, CA) at 1.5:1:1 stoichiometry using CaCl2/BES. After 2 days, culture media were collected and applied to cancer cells. After 48 h, media were replaced by selection medium containing 2–10 μg/mL puromycin. Stable clones were selected and subcultured11 (link). For stable plasmid/shRNA transfection, 105 tumor cells in six-well culture vessels were transfected with 5 μg DNA using Xfect (Takara), and clones were selected by G418 (400–800 μg/mL) or puromycin (2–10 μg/mL).
Marazioti A., Lilis I., Vreka M., Apostolopoulou H., Kalogeropoulou A., Giopanou I., Giotopoulou G.A., Krontira A.C., Iliopoulou M., Kanellakis N.I., Agalioti T., Giannou A.D., Jones-Paris C., Iwakura Y., Kardamakis D., Blackwell T.S., Taraviras S., Spella M, & Stathopoulos G.T. (2018). Myeloid-derived interleukin-1β drives oncogenic KRAS-NF-κΒ addiction in malignant pleural effusion. Nature Communications, 9, 672.
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2

Urethane and MTT Assay for Cell Viability

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Urethane (CAS#51-79-6) and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay were from Sigma (St. Louis, MO), adenoviruses from the Vector Development Lab of the Baylor College of Medicine (Houston, TX), D-luciferin from Gold Biotechnology (St. Louis, MO), HEK293T cells from ATCC (Wesel, Germany), and Lewis lung carcinoma (LLC) and A549 lung adenocarcinoma cells from the NCI Tumor Repository (Frederick, MD). Primers and antibodies are listed in Supplementary Tables S2 and S3. Lentiviral shRNA pools (Santa Cruz, Dallas, TX) are described in Supplementary Table S4.
Vreka M., Lilis I., Papageorgopoulou M., Giotopoulou G.A., Lianou M., Giopanou I., Kanellakis N.I., Spella M., Agalioti T., Armenis V., Goldmann T., Marwitz S., Yull F.E., Blackwell T.S., Pasparakis M., Marazioti A, & Stathopoulos G.T. (2018). IκB kinase α is required for development and progression of KRAS-mutant lung adenocarcinoma. Cancer research, 78(11), 2939-2951.
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3

Induction of Solid Tumors in Mice

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C57BL/6 mouse Lewis lung carcinoma (LLC) and B16F10 skin melanoma cells were obtained from the NCI Tumor Repository (Frederick). For RNA interference, the following proprietary lentiviral shRNA pools were obtained from Santa Cruz Biotechnology (Palo Alto): random control shRNA (shC, sc-108080), GFP control (sc-108084), anti-EGFR-shRNA (sc-29302-V), and stable transfections of the LLC and B16F10 cells were generated as described previously.[22 (link)] C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor) and were bred at the Center for Animal Models of Disease of the University of Patras, Greece. Experiments were approved a priori by the Veterinary Administration of the Prefecture of Western Greece, and were conducted according to Directive 2010/63/EU. Experimental mice were sex-, weight-, and age-matched. For induction of solid tumors, mice were anesthetized using isoflurane inhalation and received subcutaneous injections of 100 μL PBS containing 0.5 x 106 LLC or B16F10 clones.
Bölükbas D.A., Datz S., Meyer-Schwickerath C., Morrone C., Doryab A., Gößl D., Vreka M., Yang L., Argyo C., van Rijt S.H., Lindner M., Eickelberg O., Stoeger T., Schmid O., Lindstedt S., Stathopoulos G.T., Bein T., Wagner D.E, & Meiners S. (2020). Organ-restricted vascular delivery of nanoparticles for lung cancer therapy. Advanced therapeutics, 3(7), 2000017.
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