A shRNA sequence complementarily binding to rat Smad4 mRNA (NM_019275.2) was selected. The target sequence of shRNA (5’- GCTGTCCTATTGTAACTGT-3’) is homologous to nt 1031–1049 of Smad4 mRNA. The oligonucleotide templates of this shRNA were chemically synthesized and cloned into the linear pSIH1-H1-copGFP shRNA vector (System Biosciences, CA, USA), which was obtained through digestion by BamH I and EcoR I and purified by agarose gel electrophoresis. A scrambled sequence (5’-GAAGCCAGATCCAGCTTCC-3’) was used as a negative control (NC). Sequencing was used to confirm the vectors that were constructed (pSIH1-shRNA-Smad4 and pSIH1-NC).
Psih1 h1 copgfp shrna vector
Manufactured by System Biosciences
Sourced in United States
The PSIH1-H1-copGFP shRNA vector is a plasmid-based system designed for the expression of short hairpin RNA (shRNA) and the simultaneous expression of copGFP (Copepod Green Fluorescent Protein) as a reporter. The vector contains an H1 promoter for shRNA expression and a copGFP expression cassette.
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3 protocols using psih1 h1 copgfp shrna vector
1
Knockdown of rat Smad4 using shRNA
2
Lentiviral Knockdown of IL1RAP in Leukemia Cells
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For knockdown of IL1RAP, we cloned shRNA template oligonucleotides into the pSIH1-H1-copGFP shRNA vector (System Biosciences) and produced lentiviral particles as previously described (Barreyro et al., 2012 (link)). THP-1, HEL, or TF-1 cells were transduced with a nonsilencing control (luciferase) shRNA- or IL1RAP-specific shRNAs (MOI = 1), and transduced (GFP+) cells were FACS sorted before transplantation. For all shRNA transduction experiments, 8 µg/ml polybrene was added to cells before incubation with virus, and spin infection was performed after addition of virus (1,000 rcf for 1 h at 32°C) to facilitate transduction. Knockdown efficiency was measured by flow cytometry using anti–human IL-1RAcP/IL-1R3 APC or PE (FAB676A and FAB676P; R&D) and mouse IgG1 APC (17-4714; eBioscience) or PE (IC002P; R&D) as controls.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP+ cells were FACS sorted and plated between 1.5 × 105 and 2.5 × 105 cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.
Primary human MDS and AML MNCs were transduced with IL1RAP shRNA lentiviruses (MOI = 1). 24 h after shRNA infection, GFP+ cells were FACS sorted and plated between 1.5 × 105 and 2.5 × 105 cells/ml in human complete methylcellulose medium (HSC003; R&D) supplemented with 40 µg/ml low-density lipoproteins (L7914; Sigma-Aldrich). Colonies were scored after 10–14 d.
3
Silencing and Overexpression of Key Regulatory RNAs
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The following steps were involved in the construction of a silencing vector of lncRNA NONRATT013819.2. First, we designed a specific siRNA sequence that complementarily binds to NONRATT013819.2. Then, a complementary shRNA-DNA sequence with a hairpin structure was obtained according to the siRNA sequence. Next, the shRNA-DNA sequences were cloned into the linear pSIH1-H1-copGFP shRNA vector (System Biosciences, Palo Alto, CA, USA) to obtain lncRNA NONRATT013819.2 silencing vector (tagged as pSIH1-shRNA-NONR). Meanwhile, an invalid siRNA sequence was used as a negative control (NC) and tagged as pSIH1-NC. Finally, DNA sequencing was used to confirm the constructed vectors (pSIH1-shRNA-NONR and pSIH1-NC). All the primers are shown in Table S1.
Construction of the overexpression vector of lox. The coding sequence of rat lox (NM_017061.2, 1808 bp) was amplified using PCR, which contained an EcoRI cutting site, Kozak sequence (5′-GCCACC-3′], and a BamHI cutting site. The product of PCR amplification was digested and cloned into a pcDH1-GFP lentiviral expression vector (System Biosciences). The recombinant vector was tagged pcDH1-lox and confirmed by DNA sequencing. Empty vectors were used as controls. All the primers are listed in Table S1.
Construction of the overexpression vector of lox. The coding sequence of rat lox (NM_017061.2, 1808 bp) was amplified using PCR, which contained an EcoRI cutting site, Kozak sequence (5′-GCCACC-3′], and a BamHI cutting site. The product of PCR amplification was digested and cloned into a pcDH1-GFP lentiviral expression vector (System Biosciences). The recombinant vector was tagged pcDH1-lox and confirmed by DNA sequencing. Empty vectors were used as controls. All the primers are listed in Table S1.
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