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Shrna targeting

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ShRNA targeting is a laboratory tool used to suppress the expression of specific genes. It utilizes short hairpin RNA (shRNA) molecules to induce RNA interference (RNAi) and inhibit the production of target proteins.

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Lab products found in correlation

Neomycin sulphate, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Rwpe 1, by Thermo Fisher Scientific (1 mentions) Du145, by Merck Group (1 mentions) Lncap, by Merck Group (1 mentions) Oligonucleotide, by Merck Group (1 mentions) K sfm, by Thermo Fisher Scientific (1 mentions) Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Merck Group (1 mentions) Trypsin edta, by Merck Group (1 mentions) Lncap, by American Type Culture Collection (1 mentions) Rwpe 1, by American Type Culture Collection (1 mentions) Du145, by American Type Culture Collection (1 mentions) Puromycin, by InvivoGen (1 mentions) Doxycycline, by Merck Group (1 mentions) Pfastbac, by Thermo Fisher Scientific (1 mentions) Chaetocin, by Selleck Chemicals (1 mentions) Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions) Plko 1 puro 1864, by Addgene (1 mentions) Pmd2 g 12259, by Addgene (1 mentions)

Close spelling variants of this product

Non-targeting shRNA control MISSION shRNA targeting set Control non-targeting shRNA vector Mission Lentiviral Non-Targeting shRNA clone SHC002 ShRNA construct targeting mouse Tcf7l2 RBL2 and non-targeting control shRNA lentiviral transfer plasmids Non-targeting shRNA Lentiviral shRNA construct targeting Glycerol stocks of lentiviral shRNA targeting Lentiviral Mission short hairpin RNA (shRNA) clones targeting

6 protocols using shrna targeting

1

Knockdown of ERK1/2 via Lentiviral shRNA

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shRNA targeting ERK1 and ERK2 (Sigma-Aldrich, St Louis, MO, USA) were encapsulated into lentivirus that were then. Lentivirus were produced in 293T cells using second generation lentiviral system (Invitrogen).
Ricard N., Zhang J., Zhuang Z.W, & Simons M. (2019). Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis. Cells, 9(1), 38.
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2

Lentiviral Knockdown of LIPG

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shRNA targeting LIPG (target sequence: 5′-TTACACGGATGCGGTCAATAA-3′), which was cloned into a lentiviral-based pLKO.1-puro expression vector, was purchased from Sigma-Aldrich. Lentiviral particles were produced in packaging cells (293FT cells) by co-transfection with packaging plasmids. Stable LIPG knockdown (KD) cells were selected using the puromycin selectable marker.
Shirasaki T., Murai K., Ishida A., Kuroki K., Kawaguchi K., Wang Y., Yamanaka S., Yasukawa R., Kawasaki N., Li Y.Y., Shimakami T., Sumiyadorj A., Nio K., Sugimoto S., Orita N., Takayama H., Okada H., Thi Bich P.D., Iwabuchi S., Hashimoto S., Ide M., Tabata N., Ito S., Matsushima K., Yanagawa H., Yamashita T., Kaneko S, & Honda M. (2023). Functional involvement of endothelial lipase in hepatitis B virus infection. Hepatology Communications, 7(9), e0206.
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3

Investigating AKR1C3 Knockdown in Prostate Cell Lines

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ShRNA targeting AKR1C3 oligonucleotides containing the short hairpin sequence (Sigma–Aldrich) were annealed and inserted in a pcDNA3.1 vector (Invitrogen). Stably transfected cells were exposed to 100 mg/ml neomycin sulphate (Sigma–Aldrich) as a selection agent. Human prostate cell lines used RWPE-1, LNCaP, PC-3, DU 145 were purchased from American Type Cell Culture (ATCC) Manassas, Virginia USA. RWPE-1 cells were maintained within keratinocyte serum free medium (K-SFM) (Invitrogen GIBCO) used in combination with the recommended supplements of bovine pituitary extract (0.05 mg/ml) (BPE) and human recombinant epidermal growth factor (5ng/ml) (EGF). LNCaP, PC-3 and DU 145 cells were maintained in RPMI 1640 medium (Sigma–Aldrich) containing 10% fetal bovine serum (Invitrogen), 2 mM l-glutamine and containing 100 units/ml penicillin and 100 μg/ml Streptomycin. Cells were kept at 37 °C in 95% air and 5% CO2. Cells were washed in sterile phosphate buffered saline (PBS) and split using Trypsin-EDTA (Sigma–Aldrich) and seeded into new flasks containing fresh media. All experiments were conducted using cells between passages 15 and 28.
Doig C.L., Battaglia S., Khanim F.L., Bunce C.M, & Campbell M.J. (2015). Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells. The Journal of steroid biochemistry and molecular biology, 155(Pt A), 47-55.
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4

Generation of SOX4-depleted HMLE Cells

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To generate SOX4-depleted HMLE cells and respective control, two independent pLKO.1-puro lentiviral constructs expressing shRNA targeting SOX4 (Sigma) or expressing shRNA control were used, respectively. Lentivirus particles were produced by transfection of HEK293T cells with 3.25 ug of Pax2, 1.8 μg of pMD2.G and 5 μg of shRNA contracts. Twenty four hours after transfection, medium was replaced by MEGM:DMEM/F12 media. Viral supernatants were collected 24 h after and filtered through a 0.22 μm filter and added to the HMLE cells. Cells were selected using 1μg/ml of puromycin (invivoGen). Cells were maintained in 1 μg/ml of puromycin through the cell culture.
Vervoort S.J., Lourenço A.R., Tufegdzic Vidakovic A., Mocholi E., Sandoval J.L., Rueda O.M., Frederiks C., Pals C., Peeters J.G., Caldas C., Bruna A, & Coffer P.J. (2018). SOX4 can redirect TGF-β-mediated SMAD3-transcriptional output in a context-dependent manner to promote tumorigenesis. Nucleic Acids Research, 46(18), 9578-9590.
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5

Histone Mutant Expression and Epigenetic Enzyme Purification

Cited 1 time
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Doxycycline (Sigma; 10,000 × stock solution; 1 mg/ml) was resuspended in 70% ethanol. Biotin (BioShop and Sigma) was prepared as a 1000 × stock solution (50 mM) by dissolving 250 mg in 2 ml 30% v/v NH4OH on ice and neutralized by slowly adding 18 ml HCl (1 N), before sterile filtration through a 0.22 µm filter and storage at 4 °C. Chaetocin (Selleck) and OTS186935 (MedChemExpress) were dissolved in DMSO, aliquoted, and stored at − 80 °C.
H3.3 (H3-3a) or H3.1 (H3C2) cDNA that was WT or had K27M or G34R mutations was amplified from existing cDNA stocks and cloned between the XbaI/BamHI sites of a pCDH-CMV-MCS-EF1α-Hygro (SystemBioscience) previously modified to encode a FLAG/HA tag between the BamHI/NotI sites. K9M mutations were introduced with the Q5 site-directed mutagenesis kit (NEB). pcDNA5-FLAG-BirA* plasmids to create Flp-In T-REx HEK293 cells were generated as previously described [34 (link)]. Bacterial histone expression vectors were created by cloning cDNA into pET3a [17 (link)]. EHMT2 cDNA was amplified from pLenti6-MK1-EHMT2-V5 (Addgene #31113) and cloned between the KpnI and EcoRI sites of pFastBac (Invitrogen) to create pFastBac_EHMT2. SUV39H2 cDNA was amplified from a HEK293T cDNA library and inserted by Gibson assembly into pDest_pACE1 to create pACE1_SUV39H2. All plasmids were sequenced before use. shRNA targeting H3K9 methylases were from Sigma.
Siddaway R., Canty L., Pajovic S., Milos S., Coyaud E., Sbergio S.G., Vadivel Anguraj A.K., Lubanszky E., Yun H.Y., Portante A., Carette S., Zhang C., Moran M.F., Raught B., Campos E.I, & Hawkins C. (2022). Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma. Acta Neuropathologica, 144(5), 1027-1048.
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6

Lentiviral Manipulation of CAMK2A and SOX2

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ShRNA targeting human CAMK2A and EZH2 were from Sigma-Aldrich (St Louis, MO). ShRNA targeting SOX2 a and b (26353, 26352), the negative control vector pLKO.1-puro (1864), the envelope vector pMD2.G (12259) and packaging vector psPAX2 (12260) were from Addgene (Cambrige, MA; http://www.addgene.org). Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression. Lentiviral production and infection were performed as previously described10 . Stable cells were either selected by treating puromycin (2 µg/ml, Sigma-Aldrich) or by FACS using BD Aria (BD Biosciences).
Wang S.Q., Liu J., Qin J., Zhu Y., Tin V.P., Yam J.W., Wong M.P, & Xiao Z.J. (2020). CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation. Cell Death & Disease, 11(6), 410.
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