Pretrosuper myc shrna

Manufactured by Addgene

Sourced in United States

The PRetrosuper Myc shRNA is a plasmid vector used for the expression of short hairpin RNA (shRNA) targeting the Myc gene. It provides a tool for researchers to study the role of Myc in various cellular processes.

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Lab products found in correlation

Hiperfect transfection reagent, by Qiagen (1 mentions) X tremegene hp reagent, by Roche (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Control non specific scrambled sirna, by Qiagen (1 mentions) 4 hydroxytamoxifen 4 oht, by Merck Group (1 mentions) Pbabepuro myc er, by Addgene (1 mentions) Polybrene, by Merck Group (1 mentions) Puromycin, by Fujifilm (1 mentions) Pmko 1 puro gfp shrna, by Addgene (1 mentions)

3 protocols using pretrosuper myc shrna

Overexpression and Knockdown Strategies

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In order to overexpress BRD4, its coding sequence was subcloned into the lentiviral pLVX-Puro plasmid. BRD4 was knocked down by transfection with the plasmid containing a pSUPER-shBRD4 (C terminus). B7-H6 knockdown was performed using the lentiviral pLKO.1-Puro plasmid containing an shRNA for B7-H6 (Sigma-Aldrich). pRetrosuper MYC shRNA (#15662, Addgene) was used for MYC knockdown. For experiments, 2 × 10⁴ HEL-R cells were plated in 48-well plates, and plasmids were transfected using XtremeGene HP reagent (Roche), following the manufacturer’s instructions. In all assays, transfected clones were selected with 0.5 µg/ml of puromycin (Invitrogen).
For transient silencing assays, JMJD6 and control nonspecific scrambled siRNA were purchased from Qiagen and Dharmacon, respectively. For this purpose, 2 × 10⁴ cells were plated in 48-well plates and transfected with control or specific siRNA (200 nM) using HiPerFect Transfection Reagent (Qiagen).

Baragaño Raneros A., Rodriguez R.M., Bernardo Flórez A., Palomo P., Colado E., Minguela A., Suárez Álvarez B, & López-Larrea C. (2021). Bromodomain protein BRD4 is an epigenetic activator of B7-H6 expression in acute myeloid leukemia. Oncoimmunology, 10(1), 1897294.

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Modulating c-Myc in Ba/F3-ITD cells

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Retroviral packaging and infection of Ba/F3-ITD cells were performed as previously described [48 (link)].
Ba/F3-ITD cells were infected with pMSCVpuro-Flag-cMyc-T58A retroviral plasmid (Addgene plasmid #20076; https://www.addgene.org/20076) [49 (link)]) from Addgene (Cambridge, MA, USA), containing c-Myc with a point mutation at position 58, changing threonine to alanine and thereby inhibiting phosphorylation, or pMSCVpuro empty vector control (Takara Bio USA, Mountain View, CA, USA).
To induce c-Myc overexpression, Ba/F3-ITD cells were infected with the c-Myc expression vector pBABEpuro-myc-ER (estrogen receptor) (plasmid #19128; https://www.addgene.org/19128) [50 (link)] or pBABE-puro empty vector control from Addgene. Myc-ER expression was confirmed by immunoblotting. Ba/F3-ITD cells expressing Myc-ER were cultured with 300 nM 4-hydroxytamoxifen (4-OHT) (Sigma) to activate the myc-ER fusion protein via nuclear translocation of c-Myc [48 (link)].
To accomplish c-Myc knockdown, cells were infected with pRetrosuper Myc shRNA (plasmid #15662; https://www.addgene.org/15662) [51 (link)] or pSUPER retro puro Scr shRNA control (plasmid #30520; https://www.addgene.org/30520) from Addgene. c-Myc knockdown was confirmed by immunoblotting.

Scarpa M., Kapoor S., Tvedte E.S., Doshi K.A., Zou Y.S., Singh P., Lee J.K., Chatterjee A., Ali M.K., Bromley R.E., Hotopp J.C., Rassool F.V, & Baer M.R. (2021). Pim kinase inhibitor co-treatment decreases alternative non-homologous end-joining DNA repair and genomic instability induced by topoisomerase 2 inhibitors in cells with FLT3 internal tandem duplication. Oncotarget, 12(18), 1763-1779.

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Stable Knockdown of MYC in AMU-ML2 Cells

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The pRetrosuper Myc shRNA and pMKO.1‐puro GFP shRNA were gifts from Addgene (plasmids #15662 and #10675, respectively; Cambridge, MA, USA) 33, 34. To obtain cells stably expressing decreased levels of MYC, the MYC shRNA vector (AMU‐ML2/MYCsh) or the control GFP shRNA vector (AMU‐ML2/GFPsh) was introduced into AMU‐ML2 cells. The retroviral plasmids were packaged into 293T cells using the pCL10A vector. Viral supernatants were harvested 96 h after transfection and filtered before infection. The cells were infected with retroviruses in the presence of 8 μg·mL⁻¹ Polybrene (Sigma‐Aldrich). Antibiotic selection (puromycin; 0.3 μg·mL⁻¹; Wako) was begun 48 h after infection and continued for at least 3 days. Following infection and antibiotic selection, the cells were examined for MYC protein levels using western blotting.

Mizuno S., Hanamura I., Ota A., Karnan S., Kanasugi J., Nakamura A., Takasugi S., Uchino K., Horio T., Goto M., Murakami S., Gotou M., Yamamoto H., Watarai M., Shikami M., Hosokawa Y., Miwa H., Taniwaki M., Ueda R., Nitta M, & Takami A. (2018). Establishment and characterization of a novel vincristine‐resistant diffuse large B‐cell lymphoma cell line containing the 8q24 homogeneously staining region. FEBS Open Bio, 8(12), 1977-1991.

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