The origin of the HAFTL pre-B cell line, its derivatives C10 (C/EBPαER-GFP) and C11 (C/EBPαER-hCD4), and induction of transdifferentiation (treatment with 100 uM β-est and grown in the presence of 10 nM Il-3 and 10 nM CSF-1) have been described previously (Bussmann et al., 2009; Xie et al., 2004 ). The shC/EBPβ-KD07 directed to the ORF of Cebpb was purchased from Sigma (Mission shRNA System) in a pLKO.1-puro lentiviral backbone. An shRNA against PU.1 cloned into LMP-GFP virus (Open Biosystems) was a gift from Dr. M. Sieweke (Sarrazin et al., 2009 (link)). The 3T3 cell culture conditions and the PU.1-GFP construct have been described previously (Feng et al., 2008 (link)). Phagocytosis of yeast was performed as described previously by Rapino et al. (2013) (link). To test for statistical differences of C/EBPα binding after knockdown of PU.1, we applied the Student’s t test, one-tailed, alpha level (0.05).
Mission shrna system
Manufactured by Merck Group
Sourced in United States
The Mission® shRNA system is a tool that enables the knockdown of target gene expression through the use of short hairpin RNA (shRNA) technology. The core function of this system is to provide a reliable and efficient method for silencing gene expression in cell-based studies.
Automatically generated - may contain errors
10 protocols using mission shrna system
1
Differentiation of HAFTL Pre-B Cells
2
Rab20 Knockdown Impacts EGF Uptake
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Rab20-knockdown RAW264.7 macrophages were produced using a MISSION shRNA system (Sigma-Aldrich, USA) as described previously (Pei et al., 2012 (link)). Cells stably transfected with Rab20 shRNA oligo 2643 (5′-CCTTTACAAGAAGATCCTGA-3′) or empty control shRNA plasmid pLKO.1 were seeded 2 d before experiment in T25 flasks. Then cells were washed twice with PBS and starved in serum-free DMEM for 5 h at 37°C. For EGF uptake, cells were washed twice with ice-cold PBS and incubated for 1 h on ice in uptake medium (2% BSA and 20 mM HEPES, pH 7.5, in serum-free DMEM) containing 2.5 μg/ml recombinant EGF (BioLegend). After incubation, cells were washed three times with ice-cold PBS to remove unbound ligands. PBS was replaced by uptake medium, and flasks were transferred to a 37°C incubator and incubated for the indicated time periods (10, 30, and 60 min). Then cells were washed with PBS, scraped, and processed for Western blot.
3
Inhibition of RECK Expression via Lentiviral shRNA
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For the inhibition of RECK expression, five different specific shRNA sequences (sequentially numbered from shRECK23 through shRECK27) were tested. These vectors and the structural ones belonged to the Mission® shRNA system (Sigma-Aldrich, St. Louis, MO, USA). Viral recombinant particles were generated by co-transfection of the 293FT virus packer cell line with these recombinant lentiviral vectors. Transduced cells were selected by the addition of 2 μg/mL puromycin.
4
Lentiviral Knockdown of CFTR Rescue
5
Lentiviral Knockdown of CFTR Rescue
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Lentiviral particles harboring shRNA sequences specific for the target proteins were generated in HEK293T cells using the Mission® shRNA system with validated shRNAs (Sigma-Aldrich, St.Louis, MO) following standard protocols70 . CFBE41o- cells were infected with lentiviral particles for 16 h and cultivated for additional 48 h prior to harvest. Lentivirus production and infection is covered under TSRI approval #01- 13- 10- 07 and all steps were carried out in a BSL2/3 certified laboratory. Rescue of ΔF508 CFTR was monitored by Western blotting followed by immunodetection of CFTR using rat monoclonal 3G11 antibody. The RNAi Consortium (TRC) identification numbers for the shRNAs used are given in Table S15 .
6
Stable Rab20 Knockdown Cell Lines
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The MISSION® shRNA system (Sigma-Aldrich) was used to obtain stable Rab20-knockdown cell lines. Five Rab20 shRNA oligos were tested, including shRNA2640 (5′-CTGACAGAAACAGCCAACAA-3′), shRNA 2641 (5′-CTATCATCCTTACATACGAT-3′), shRNA 2642 (5′-CTCCTCTTTGAAACCTTGTT-3′), shRNA 2643 (5′-CCTTTACAAGAAGATCCTGA-3′) and shRNA 2644 (5′-AAGATCCTGAAGTACAAGAT-3′). The control shRNA plasmid was pLKO.1-puro empty vector. Macrophages were transfected with the corresponding plasmids by using JetPEI Macrophage as described above. After 16 h of transfection, puromycin (3 µg/ml, MP Biomedicals, Santa Ana, CA) was added as the selective agent and the medium was changed every 2 days with the addition of fresh antibiotic. After 1–2 weeks the colonies were selected and expanded. Rab20 knockdown in the cells from the selected colonies was checked by western blotting.
7
Knockdown of TNF-RI in Mesenchymal Stem Cells
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The knockdown of TNF-RI was accomplished using the MISSION shRNA system from Sigma–Aldrich. shRNA lentiviral plasmids (in pLKO.1-puro) with the clone IDs NM_011609.2-1538s1c1, NM_011609.2-996s1c1, and NM_011609.2-1652s1c1 were employed to generate lentiviral particles, which were used to infect MSC (MSC + TRkd) as described previously [4 (link)]. The efficacy of knockdowns was monitored by Western blot analysis before intravenous injection into recipient mice. The non-target shRNA control SHC002, which does not target a mouse or human mRNA was used as a negative control.
8
Cell Line Maintenance and Manipulation
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HEK293T, SKOV3, ES-2, MEFs were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. A549, EPLC, H292, H1299, H1975, H522, H661, H125, H157, H322, H23, H1650, H358, and H2122 were grown in RPMI 1640 with 10% FBS and 1% penicillin/streptomycin. All cells lines were maintained at 37 °C in a humidified atmosphere of 5% CO2. The pellets of a panel of ovarian cancer cell lines, CAOV3, OVCAR3, TOV21G, CHI, CHI-CisR, M41, PEO1 and DOV, were kindly provided by Dr. Johnathan M. Lancaster and Dr. Douglas Marchion from H. Lee Moffitt Cancer Center. Transient transfections were carried out with Lipofectamine 2000 (Invitrogen) or polyethyleneimine (PEI). The USP10 knockdown cell lines were established either with the MISSION® shRNA system (Sigma) or TRIPZ inducible Lentiviral shRNA (GE Dharmacon). Lentiviruses were produced using a standard protocol (http://www.addgene.org/tools/protocols/plko/ ). USP10 knockout MEFs were generated in Dr. Zhenkun Lou’s laboratory at Mayo Clinic (unpublished data). A549 control and p53KO cells were described in Heyza et al.38 (link). All cell lines were purchased from ATCC. Otherwise they were described as the above.
9
Targeting CD36 in Cancer Stem Cells
10
Targeted Protein Knockdown Using MISSION shRNA
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