For construction of the expression plasmid for ISG12a, the entire open reading frame of human ISG12a gene was polymerase chain reaction (PCR) amplified from p3XFLAG-CMV-14-ISG12a (kindly provided by Dr. Douglas W Leaman, The University of Toledo), and inserted into pcDNA3.1/V5-His (Invitrogen). The primers for amplification of ISG12a are 5'-CGCGCGGATCCATGG AGGCCTCTGCTCTC-3'(F), and 5'-CTGCAGGAATTCGTAGAACCTCGCAATGA-3'(R). The oligonucleotides encode 19-mer hairpin sequence specific to the ISG12a mRNA were incorporated into the pSilencer-neo plasmid (Ambion). The sequences of ISG12a shRNAs targeting two regions of ISG12a were 5'-AAGTTCATCCTGGGCTCCATT-3' and 5'-AATTAACCCGAGCAGGCATGG-3'. Precursor of miR-942 was amplified from Huh7 cells and inserted into pcDNA3.1/V5-His. The primers for miR-942 are 5'-GCATGGATCCGCTTTAACA ATGGTTCCTCCG-3'(F) and 5'-GCCGGTCTAGAAGCACCTTTTGTTTCTATTAT CACG-3'(R). All constructs were confirmed, and transfected into cells using Lipofectamine 2000 reagents (Invitrogen).
Psilencer neo plasmid
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PSilencer-neo plasmid is a vector used for the expression of short hairpin RNA (shRNA) in mammalian cells. It contains a neomycin resistance gene for selection of transfected cells. The plasmid allows for the stable expression of shRNA to achieve gene silencing.
Automatically generated - may contain errors
5 protocols using psilencer neo plasmid
1
Construction and Transfection of ISG12a Expression Plasmid
2
EPSTI1 Overexpression and Knockdown
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EPSTI1 cDNA was amplified from total cellular RNA isolated from Huh7.5 cells using standard RT-PCR and subsequently subcloned into the p3×FLAG-CMV. The primers for amplification of EPSTI1 are 5′-GGAATTCCATGAACACCCGCAATAGAGT-3′ (forward EcoR I) and 5′-GCTCTAGAGCAGAAAAATAATGTAGCATTTCCC-3′ (reverse Xba I). The shRNAs targeting EPSTI1 were constructed into pSilencer-neo plasmid (Ambion). The sequences of EPSTI1 shRNAs targeting three regions of EPSTI1 were 5′-AACAACAACTCCAGCTGATGC-3′, 5′-AACCGCTGAGTTCTTGAGCAA-3′, and 5′-AAGATGAAGGATGAACAACAT-3′. The negative control shRNA plasmid was purchased from Ambion.
3
Plasmid Construction for NTCP and KDM7A
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The NTCP was constructed in pCDH-CMV-MCS-EF1 plasmid, the primers of NTCP were shown in Table 1 . The short hairpin RNA targeting KDM7A was constructed in pSilencer-neo plasmid (Ambion). The target sequences of KDM7A shRNAs were shown in Table 1 . The primers of HBV promoters to construct luciferase plasmids were listed in Table 1 . pAAV/HBV1.2 was gifted by Professor Pei-Jer Chen (National Taiwan University). pGAS-luc (GAS luciferase plasmid) was obtained from Professor Zhenghong Yuan (Fudan University).
4
Plasmid Construction for NTCP and KDM7A
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The NTCP was constructed in pCDH-CMV-MCS-EF1 plasmid, the primers of NTCP were shown in Table 1 . The short hairpin RNA targeting KDM7A was constructed in pSilencer-neo plasmid (Ambion). The target sequences of KDM7A shRNAs were shown in Table 1 . The primers of HBV promoters to construct luciferase plasmids were listed in Table 1 . pAAV/HBV1.2 was gifted by Professor Pei-Jer Chen (National Taiwan University). pGAS-luc (GAS luciferase plasmid) was obtained from Professor Zhenghong Yuan (Fudan University).
5
Generation of ISG15 Expression Plasmid and Knockdown
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The ISG15 expression plasmid was constructed by amplifying the entire open reading frame of the human ISG15 gene with the polymerase chain reaction (PCR) using p3XFLAG-CMV-14-ISG15 plasmid (kindly provided by Dr Douglas W. Leaman of the University of Toledo) as a template and inserted into the pcDNA3.1/V5-His expression vector (Invitrogen, Carlsbad, CA, USA). The primers for amplification of ISG15 were 5′-GATCACCCAGAAGA TCGGCG-3′ (forward), and 5′-GGATGCTCAGAGGTTC GTCG-3′ (reverse). The oligonucleotides, which encode a 19-mer hairpin sequence specific to the ISG15 mRNA, were incorporated into the pSilencer-neo plasmid (Ambion, Naugatuck, CT, USA). The sequence of ISG12a shRNAs targeting regions of ISG15 was 5′-TTCGTCGCATT TGTCCACCA-3′. miR-138 mimics (5′-AGCUGGUGUUG UGAAUACAGGCCG-3′) were purchased from Genepharma (Suzhou, China). All constructs were confirmed by DNA sequencing and transfected into cells using Lipofectamine 2000 reagent (Invitrogen). The transfected cells were cultured in DMEM with 10% fetal bovine serum for 24-72 h before analysis.
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