Adenovirus 5 carrying shRNA against mouse-MUL1 (AAV-shR-MUL1) was generated at the Emory virus core facility. The shR-MUL1 (5′-GAGCTGTGCGGTCTGTTAA -3′) was synthetized and subcloned into a pAAV-U6-GFP expression vector (Cell Biolabs). The eGFP cDNA was used as a control after sub-cloning into pAAV-MCS vector (Cell Biolabs). The full length of mouse VPS35-D620N cDNA was sub-cloned into the pAAV -EGFP-T2A-V5 vector (Vector Biolabs). Infected 293 cells from 25 to 50 15-cm tissue culture plates were collected and centrifuged. Then the cells were lysed by three freeze–thaw cycles. The AAV virus was purified using the iodixanol gradient centrifugation procedure and the virus titers were measured by real-time PCR. For stereotaxic injection, 1.5 μl viral solution (1 × 1012 vg/ml) was injected into the right ventral midbrain (anterior, −2.8 mm; lateral, −1.3mm; dorsal, −4.2 mm relative to bregma) with a 30-gauge microsyringe at a rate of 0.5 μl/min.
Paav mcs vector
Manufactured by Cell Biolabs
Sourced in United States
The PAAV-MCS vector is a plasmid-based vector designed for the expression of recombinant proteins in mammalian cell lines. It features a multiple cloning site (MCS) which allows for the insertion of a gene of interest. The vector also contains an adenovirus-derived promoter (PTV) which drives the expression of the inserted gene.
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Lab products found in correlation
Paav u6 gfp expression vector, by Cell Biolabs (1 mentions)
Phelper, by Cell Biolabs (1 mentions)
Paav dj, by Cell Biolabs (1 mentions)
Aav gfp, by SignaGen (1 mentions)
Pires2 egfp vector, by Takara Bio (1 mentions)
Veriti 96 well fast thermal cycler, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Aavpro purification kit, by Takara Bio (1 mentions)
Aavpro titration kit, by Takara Bio (1 mentions)
Platinum taq high fidelity kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using paav mcs vector
1
Adenoviral Delivery of shRNA against MUL1
2
Promoterless AAV-DJ/8 Vector Construction
3
Circadian Rhythms Monitoring via CRE-Luc2P Assay
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A luciferase reporter driven by a tandem repeat of the Gpr19 CRE sequence (3 × CRE-Luc2P) was inserted between the ITR sequences of pAAV-MCS vector (Cell Biolabs Inc) to obtain pAAV-3 × CRE-Luc2P. HEK293T cells cultured in dish were co-transfected with pAAV-3 × CRE-Luc2P, pAAV-DJ, and pHelper according to the manufacturer's instructions (Cell Biolabs Inc). Three days after transfection, cells were harvested and resuspended in 1 ml of DMEM, followed by four freeze–thaw cycles and centrifugation. The titers of 3 × CREwt-Luc2P and 3 × CREmut-Luc2P virus solutions were ~ 8 × 1012 genome copies/mL. The SCN slices were prepared according to our standard method59 (link). Two days after the preparation of SCN slices, the AAV solution (3 μL per slice) was inoculated on the surface of the SCN slices. Infected slices were further cultured for ~ 14 days. Thereafter, luminescence from the culture was measured with a dish-type luminometer (Kronos Dio, ATTO) at 35 °C using 1 mM luciferin59 (link). The luminescence was monitored for 2 min at 20-min intervals for each slice. The raw data were smoothed using a 1-h moving average and further detrended by subtracting a 24 h running average.
4
Knockdown of Hap1 and c-kit Overexpression
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Adenoviral Hap1-specific and scramble siRNAs were prepared in our previous study [81 (link)]. Viral stocks were adjusted to 1X108 viral particles/μl before use. Neuro2A cells were incubated with adenoviral Hap1 siRNA at a multiplicity of infection of 50. Twenty-four h after infection, the virus-containing medium was removed, and the c-kit plasmid was transfected for another 48 h before performing the protein stability assay. Mouse c-kit cDNA was subcloned into a pAAV-MCS vector (Cell Biolabs). The human synapsin-1 promoter sequence was inserted into the construct to replace the original promoter in the vector. AAV-c-kit (Serotype 9) was packaged and amplified by the viral vector core at Emory University. AAV-GFP (Serotype 9, SignaGen Laboratories) under the same promoter was used as a control.
5
Amplification and Cloning of hKv Genes
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The primer sets used to amplify the hKvs (hKv1.1, hKv1.3, hKv1.4, hKv1.5, hKv3.1, and hKv11.1) gene are provided in Table 2 . The amplification was performed in the Veriti™ 96-Well Fast Thermal Cycler (Applied Biosystems, Waltham, MA, USA) with a thermal cycling program consisting of predenaturation for 3 min at 95 °C, followed by 30 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 58 °C, and extension for 30 s at 72 °C, with a final extension for 3 min at 72 °C. A Human cDNA Clone Set (OriGene Technologies GmbH, Herford, Germany) was used as the template. A MCS (multi-cloning site) digested from the pAAV-MCS vector (Cell Biolabs, San Diego, CA, USA) was ligated into the pIRES2-EGFP vector (Clontech Laboratories, Mountain View, CA, USA) to form pAAV–CMV–IRES2–EGFP. The PCR products were ligated into the pAAV–CMV–IRES2–EGFP vector at the NheI and SalI sites to construct pAAV–CMV–hKv–IRES2–EGFP.
6
Inducible AAV-Mediated MMP-3 Expression
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AAV-2/9 containing the enhanced green fluorescent protein (eGFP) reporter gene (Vector Biolabs) was initially used to assess viral transduction and expression in the anterior chambers of wild type mice (C57/BL6). Murine MMP-3 cDNA was incorporated into Bam HI/Xhol sites of the pAAV-MCS vector (Cell Biolabs Inc) for constitutive expression of MMP-3. A null virus was used as contralateral control using the same capsid and vector. The inducible vector was designed by cloning MMP-3 cDNA into a pSingle-tTS (Clontech) vector. This vector was then digested with BsrBI and BsrGI and the fragment containing the inducible system and MMP-3 cDNA was ligated into the Not1 site of expression vector pAAV-MCS, to incorporate left and right AAV inverted terminal repeats (L-IRT and R-ITR). AAV-2/9 was generated using a triple transfection system in a stable HEK-293 cell line (Vector Biolabs). For animals injected with the inducible virus, after a 3-week incubation period, 0.2% doxycycline (D9891, Sigma) in PBS was administered twice daily to the eye for 10–16 days to induce viral expression. A similar inducible virus expressing eGFP was used as a control in the inducible study.
7
Elf1 cDNA Cloning and AAV Packaging
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