The pSilencer 2.1-U6 expression vector was purchased from Ambion (Ambion, AM5762). The RNU6-1 RNA polymerase III promoter and the polylinker region were subcloned into the adenoviral shuttle vector pDC311 (Microbix, PD-01-25). The rat FYN shRNA targeting sequence was 5ʹ-AGGATAAAGAAGCAGCGAAACTGAC-3ʹ. The rat FGF18 shRNA targeting sequence was 5ʹ-CCTGCACTTGCCTGTGTTT-3ʹ. For Scrambled shRNA, an in-house-generated shRNA adenovirus that encodes a scrambled sequence was used as a control. Recombinant adenoviruses were generated by homologous recombination in HEK293T cells as described above. For adenovirus-mediated gene knockdown, these constructed adenovirus vectors were transfected into NRCMs at an MOI of 20× PFU/cell for 24 h. After 24 h, the knockdown efficiency was evaluated.
Psilencer 2.1 u6 expression vector
Manufactured by Thermo Fisher Scientific
The PSilencer 2.1-U6 expression vector is a tool used for the expression of short hairpin RNA (shRNA) sequences in mammalian cells. It provides a convenient platform for the stable or transient expression of shRNA, which can be used for gene silencing experiments.
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4 protocols using psilencer 2.1 u6 expression vector
1
Adenoviral Knockdown of FYN and FGF18 in NRCMs
2
Adenoviral Knockdown of eNOS, iNOS, and Trx in HUVECs
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The pSilencer 2.1-U6 expression vector was purchased from Ambion (Ambion, AM5762). The RNU6-1 RNA polymerase III promoter and the polylinker region were subcloned into the adenoviral shuttle vector pDC311 (Microbix, PD-01-25). The human eNOS-shRNA targeting sequence was 5ʹ-GTGGCCAACGCCGTGAAGATC-3ʹ. The human iNOS-shRNA targeting sequence was 5ʹ-GGTAACAAAGGAGATAGAAAC-3ʹ. The human Trx-shRNA targeting sequence was 5ʹ-GCAGGTGATAAACTTGTAGTA-3ʹ. For Scrambled shRNA, an in-house-generated shRNA adenovirus that encodes a scrambled sequence was used as control. For adenovirus-mediated gene knockdown, these constructed adenovirus vectors were transfected into HUVECs at a MOI of 20 × PFU/cell for 24 h.
3
Adenoviral Knockdown of Murine HXK2
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The pSilencer 2.1-U6 expression vector was purchased from Ambion (Ambion, AM5762). The U6 RNA polymerase III promoter and the polylinker region were subcloned into the adenoviral shuttle vector pDC311 (Microbix, PD-01-25). The HXK2 shRNA targeting sequence was 5′- GCTGCTGTTCCAAGGGAAA -3'. shScramble, an in-house generated shRNA adenovirus that encodes a scramble sequence (5′- CAGTTTGGCACAATCAATA -3′), was used as control. Recombinant adenoviruses carrying the short hairpin RNA against murine HXK2 mRNA under control of the murine vascular Cadherin 5 core promoter were generated by homologous recombination in HEK293 cells as described previously [29 (link)].
4
Recombinant Adenovirus Knockdown of Nrf2
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Recombinant adenovirus vectors were constructed, propagated and titered as previously described (Maejima et al. 2013) (link). Briefly, pBHGloxΔE1, 3 Cre plasmid (Microbix, PD-01-40), including the ΔE1 adenoviral genome, was co-transfected with the pDC316 shuttle vector containing the gene of interest into HEK293 cells using Lipofectamine 2000 (Life Technologies, 11668019). Through homologous recombination, the test genes were integrated into the E1-deleted adenoviral genome. The viruses were propagated in HEK293 cells.
The pSilencer 2.1-U6 expression vector was purchased from Ambion (Ambion, AM5762). The U6 RNA polymerase III promoter and the polylinker region were subcloned into the adenoviral shuttle vector pDC311 (Microbix, PD-01-25). The Nrf2 shRNA targeting sequence was 5′-GCAGTTCAATGAAGCTCAACT-3′. Sh-Scramble, an in-house generated shRNA adenovirus that encodes a scramble sequence 5′-TTCTCCGAACGTGTCACGT-3′, was used as control. Recombinant adenoviruses were generated by homologous recombination in HEK293 cells as described above.
To knockdown Nrf2 expression in late HUVECs, HUVECs were infected with the adenoviral containing shRNA against Nrf2 or nonsense shRNA overnight and the medium was replaced with fresh growth medium 24 h after infection. After transfection for 48 h, the levels of Nrf2 expression were detected by Western blot.
The pSilencer 2.1-U6 expression vector was purchased from Ambion (Ambion, AM5762). The U6 RNA polymerase III promoter and the polylinker region were subcloned into the adenoviral shuttle vector pDC311 (Microbix, PD-01-25). The Nrf2 shRNA targeting sequence was 5′-GCAGTTCAATGAAGCTCAACT-3′. Sh-Scramble, an in-house generated shRNA adenovirus that encodes a scramble sequence 5′-TTCTCCGAACGTGTCACGT-3′, was used as control. Recombinant adenoviruses were generated by homologous recombination in HEK293 cells as described above.
To knockdown Nrf2 expression in late HUVECs, HUVECs were infected with the adenoviral containing shRNA against Nrf2 or nonsense shRNA overnight and the medium was replaced with fresh growth medium 24 h after infection. After transfection for 48 h, the levels of Nrf2 expression were detected by Western blot.
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