Complementary DNAs encoding human SRPK1 were amplified by polymerase chain reaction using a cDNA library prepared from the human fetal brain as the template and then inserted into the pcDNA3.1 FLAG vector (Invitrogen). The vector expressing a SRPK1-targeting small hairpin RNA was constructed as follows. The short hairpin primers were designed around the potential sequence, namely sh1-SRPK1 (5′-GTGCAGCAGAAATTAATT-3′), which corresponds to nucleotides 1171–1189 of the SRPK1 transcript (GI: 47419935), and the sequence was annealed and then ligated into the PstI site of the pSilencer1.0 U6 vector (Ambion). The MCL-1 minigene reporter was constructed by inserting a complete human MCL-1 genomic fragment containing exons 1, 2, and 3 and intra-exon introns. The genomic fragments were amplified by PCR using the genomic DNA prepared from MRC5 fibroblasts as the template and then inserted into the HindIII/SacI sites to replace the β-galactosidase gene of pCH110 (Amersham Pharmacia). Mutant pCH-MCL-1 vectors containing changed nucleotides were constructed using the QuikChange site-directed mutagenesis system (Stratagene).
Psilencer1.0 u6 vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PSilencer1.0 U6 vector is a plasmid designed for the expression of small interfering RNAs (siRNAs) in mammalian cells. It contains the U6 promoter, which drives the expression of the siRNA, and a selectable marker for antibiotic resistance. The core function of this vector is to provide a tool for the study of gene silencing through RNA interference (RNAi) technology.
Automatically generated - may contain errors
2 protocols using psilencer1.0 u6 vector
1
Constructing SRPK1 and MCL-1 Expression Vectors
2
Hypoxia Pathway Knockdown in Gastric Cancer Cells
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Transfection was performed as described earlier.41 (link) The sequences of shRNA targeting the 19-nucleotide segments were as follows: HIF-1α (sense, 5′-CCTATATCCCAATGGATGA-3′ antisense, 5′-CCTATATCCCAATGGATGA-3′, NM_001530); VHL (sense, 5′-CTGCCAGTGTATACTCTGA-3′ antisense, 5′-CTGCCAGTGTATACTCTGA-3′, NM_000551); GRP78 (sense, 5′–GGAGCGCATTGATACTAGA-3′ antisense, 5′-GGAGCGCATTGATACTAGA-3′, NM_005347); and VDAC (sense, 5′-AGATCAGCTTGCACGTGGACTGAAG-3′ antisense, 5′-TCGAAATCCATGTCGCAGCCCA-3′). The double-stranded oligonucleotides were cloned into an ApaI–EcoRI site in the pSilencer 1.0-U6 vector (Ambion, Austin, TX, USA). The plasmids were transfected into SGC-7901 cells using Lipofectamine 2000 (Invitrogen) for 6 h. After transfection, the cells were cultured for 18 h and then exposed to hypoxic atmosphere for the indicated times. The silencing efficiency was verified by western blotting analysis.
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