The full length pre-miR-34c was chemically synthesized by GeneChem and was introduced into the GV217 lentiviral vector (GeneChem, Shanghai, China, Fig. S1) in the unique EcoRI site to construct a lentivirus encoding miR-34c (lenti-miR-34c); this construct was confirmed by using nucleotide sequencing. The specific inhibitor of miR-34c was constructed by cloning the complementary nucleotides of miR-34c into the GV280 lentiviral vector (GeneChem, Fig. S1) between the AgeI and EcoRI sites. The cells were seeded in a 6-well plate at a density of 5 × 104 cells/well and were infected with lenti-miR-34c or its inhibitor when the cells reached 30% confluence. Three days after infection, the efficiency of infection was evaluated by observing the EGFP expression by using a fluorescence microscope (Nikon, 80i).
Gv280 lentiviral vector
Manufactured by Genechem
The GV280 lentiviral vector is a laboratory tool used for gene delivery and expression studies. It is a replication-defective lentiviral vector that can be used to introduce genetic material into target cells. The GV280 vector provides a platform for researchers to study gene function, investigate disease mechanisms, and develop potential therapeutic interventions.
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4 protocols using gv280 lentiviral vector
1
Overexpression and Inhibition of miR-34c
2
Overexpression and Knockdown of miR-34c in Colonic SMCs
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The full length of pre‐miR‐34c was chemically synthesized by GeneChem and introduced into the GV217 lentiviral vector (GeneChem, Shanghai, China) in the unique EcoRI site to construct the lentivirus encoding miR‐34c (GV217‐miR‐34c). The specific inhibitor of miR‐34c was constructed by cloning the complementary nucleotides of miR‐34c into the GV280 lentiviral vector (GV280‐inhibitor, GeneChem) between the AgeI and EcoRI sites. The mouse colonic SMCs were seeded in a 6‐well plate at a density of 5 × 104 cells/well and were infected with lenti‐miR‐34c or its inhibitor when the cells reached 30% confluence. Three days later, the efficiency of infection was evaluated by observing EGFP expression with the fluorescence microscope (Nikon, Ni; Fig. S1 A). Overexpression and knock‐down of miR‐34c were further confirmed by real‐time PCR.
3
Lentiviral Transduction of miR-206-3p and HDAC4 in Neuropathic Pain
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The full-length Hdac4, miR-206-3p mimic, or miRNA-NC was subcloned into
the GV280 lentiviral vector (GeneChem, Shanghai, China) to construct a lentivirus encoding
miR-206-3p (LV-miR-206-3p mimic), lentivirus HDAC4 (LV-HDAC4), and LV-miRNA-NC,
respectively. The LV-miR-206-3p vector was constructed by annealing the sense,
5’-CCGGTGGAATGTAAGGAAGTGTGTGGTTCAAGAGACCACACACTTCCTTACTTCCATTTTTG-3’, and antisense,
5’-AATTCAAAAATGGAAGTAAGGAAGTGTGTGGTCTCTTGAACCACACACTTCCTTACATTCCA-3’, oligonucleotides,
which formed the double-stranded DNA molecule. Then it was subcloned into the AgeI/EcoRI
site of the GV280 lentiviral vector. The sequence of the mature miR-206-3p is as follows:
5’-UGGAAUGUAAGGAAGUGUGUGG-3’. Construction of the LV-miRNA-NC vector was similar to that
of the LV-miR-206-3p vector.
Intrathecal drug administration was accomplished using a microinjection syringe connected
to the intrathecal catheter. LV-miR-206-3p, LV-miRNA-NC (10 µl), or
LV-HDAC4 were delivered at day 5 (d5) after CCI.
the GV280 lentiviral vector (GeneChem, Shanghai, China) to construct a lentivirus encoding
miR-206-3p (LV-miR-206-3p mimic), lentivirus HDAC4 (LV-HDAC4), and LV-miRNA-NC,
respectively. The LV-miR-206-3p vector was constructed by annealing the sense,
5’-CCGGTGGAATGTAAGGAAGTGTGTGGTTCAAGAGACCACACACTTCCTTACTTCCATTTTTG-3’, and antisense,
5’-AATTCAAAAATGGAAGTAAGGAAGTGTGTGGTCTCTTGAACCACACACTTCCTTACATTCCA-3’, oligonucleotides,
which formed the double-stranded DNA molecule. Then it was subcloned into the AgeI/EcoRI
site of the GV280 lentiviral vector. The sequence of the mature miR-206-3p is as follows:
5’-UGGAAUGUAAGGAAGUGUGUGG-3’. Construction of the LV-miRNA-NC vector was similar to that
of the LV-miR-206-3p vector.
Intrathecal drug administration was accomplished using a microinjection syringe connected
to the intrathecal catheter. LV-miR-206-3p, LV-miRNA-NC (10 µl), or
LV-HDAC4 were delivered at day 5 (d5) after CCI.
4
Lentiviral Transduction of miR-144
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The full length miR-144, miR-144-specific inhibitor or their corresponding negative control oligonucleotide were cloned into the GV280 Lentiviral vector (Genechem, Shanghai, China) containing a CMV-driven enhanced green fluorescent protein (EGFP) reporter, to construct lentivirus encoding miR-144 (LV-miR-144), lentivirus inhibiting miR-144 (LV-anti-miR-144), LV-NC and LV-anti-NC. Cells under good culture conditions from each experimental group were seeded into 6-well plates at a density of 5 × 104 cells/well one day prior to viral infection, and were infected with LV-miR-144, LV-NC, LV-anti-miR-144, LV-anti-NC, respectively. Three days after infection, the expression of EGFP was monitored under a fluorescence microscope (Leica, Solms, Germany) and miR-144 levels were detected by real-time PCR to assess infection efficiency.
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