Plasmids expressing wild type Rap1 or Rap1F64A were kindly provided by Dr. O. Daumke (22 (link)). Rap1GAP1-expressing plasmids were kindly provided by Dr. P.J. Casey (23 (link)). cDNAs were subcloned into pTRACK CMV and then inserted by homologous recombination into pAdeasy-1 (Agilent Technologies). The viral preparation and purification was carried out as previously described (24 (link)). For the experiments, DRG or hippocampal neurons were plated overnight at 37°C in PLL-coated 24-well plates. Neurons were then transduced with adenoviruses at a final concentration of 1010 plaque-forming units (PFU)/ml. Infected neurons were incubated overnight at 37°C before being trypsinized and used in neurite outgrowth assays.
Padeasy 1
Manufactured by Agilent Technologies
Sourced in United States, China
The PAdEasy-1 is a lab equipment product from Agilent Technologies. It is designed for the construction of recombinant adenoviral vectors. The core function of the PAdEasy-1 is to facilitate the generation of adenoviral DNA constructs for use in various research and development applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
293ad cells, by Cell Biolabs (1 mentions)
Ad293 cells, by Agilent Technologies (1 mentions)
C11995500, by Thermo Fisher Scientific (1 mentions)
Adeasy adenoviral vector system, by Agilent Technologies (1 mentions)
Pmei enzyme, by New England Biolabs (1 mentions)
10 protocols using padeasy 1
1
Adenoviral Expression of Rap1 Mutants
2
Generation of Adenoviral Vectors for miR-26a and MDM2 Modulation
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Vector construction. Anti-miR-26a (5'-cGT GcA AGT AAc cAA GAA TAG GcG TGc AAG TAA ccA AGA ATA GGc GTG cAA GTA Acc AAG AAT AGG-3') and pro-miR-26a (5'-AAG Gcc GTG Gcc TcG TTc AAG TAA Tcc AGG ATA GGc TGT GcA GGT ccc AAG GGG ccT ATT cTT GGT TAc TTG cAc GGG GAc GcG GGc cTG-3'), mdm2-cdNA (5'-Gcc TcT TGc TGc TGA ccA cAc Tcc TGG TA-3') and mdm2-small interfering (si)RNA (5'-cTG cTA ccG TAc AGT cTc AGG cAT GGA cG-3') sequences were individually introduced (each, 0.6 µg/µl) into the pShuttle IRES vector (Agilent Technologies, Inc., Santa clara, cA, USA). Following linearization with PmeI, the pAdEasy-1 (Agilent Technologies, Inc.) and the pShuttle IRES vector were combined to generate a pAdEasy-IRES vector. 293Ad cells (cell Biolabs, Inc., San diego, cA, USA; density, 1x10 6 ) ( 16), were subsequently transfected with the pAdEasy-IRES vector before the liquid supernatant containing viral particles was isolated and collected. The viral particles, including Ad5/anti/miR-26a, Ad5/miR-26a, Ad5/mdm2-cdNA and Ad5/mdm2-siRNA vectors were established individually.
3
Adenoviral Delivery of Flagged FGF11
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Adenoviruses encoding FLAG‐tagged human FGF11 (Ad‐FLAG‐FGF11) and control adenoviruses (Ad‐GFP) were prepared as previously described. Briefly, FLAG‐FGF11 cDNA was inserted into the pAdTrack‐CMV‐expressing GFP vector followed by homologous recombination with pAdEasy‐1, an adenoviral backbone vector (Agilent Technologies, Palo Alto, CA, USA). The Ad‐FLAG‐FGF11 adenoviruses were generated by transfection of a linearized pAd‐FLAG‐FGF11 into AD‐293 cells (Agilent Technologies). The siRNAs against mouse FGF11 (Stealth siRNAs #MSS247199) and the negative control siRNAs (siNS) (AccuTarget™ Negative Control siRNA #SN‐1013) were purchased from Invitrogen (Carlsbad, CA, USA) and Bioneer (Daejeon, Korea), respectively.
4
Adenoviral-mediated overexpression of HDAC4 and HDAC5
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The full-length DNAs of the HDAC4 and HDAC4-mut (S246A, S632A) genes with BglII and XbaI cleavage sites and HDAC5 and HDAC5-mut (S259A, S498A) genes with added KpnI and HindIII sites were synthesized by Genewiz (South Plainfield, NJ, USA) and ligated with the adenovirus shuttle plasmid pAdTrack-CMV (Stratagene, La Jolla, CA.). These constructs were then linearized with PmeI and homologously recombined in AdEasier-1 cells, a derivative of BJ5183 bacteria already containing the adenovirus backbone plasmid pAdEasy-1 (Stratagene/Agilent Technologies, Santa Clara, CA, USA). Positive clones were selected for culture, plasmid extraction, and PacI digestion. The larger fragment was recovered using a gel extraction kit and transfected into HEK293 cells via liposomes (Lipofectamine™ 2000; Invitrogen, Carlsbad, CA). After 8 or 10 days, the recombinant adenovirus was collected by repeated freezing and thawing of the HEK293 cells and used to infect HEK293 cells; after 2 or 3 days, the cells exhibited cytopathic effect, and the virus was collected. The TCID50 method was used to calculate the adenovirus titre. Subsequently, cardiomyocytes were infected using a multiplicity of infection of 50.
5
Plasmid Modifications and Cell Line Generation
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The plasmid pAdEasy-1 was obtained from Agilent Technologies61 (link) and modified as described below. The pC4HSU plasmid was purchased from Microbix and originally developed by Sandig et al.35 (link) The murine cell line EMT6-HER2 was generated as previously described.62 (link) Briefly, murine EMT6 cells were modified for constitutive expression of HER2. The B16-D5-HER2 murine cell line was kindly provided by Louis Weiner.63 (link) A poorly immunogenic subclone of murine B16 cells, D5, was modified to constitutively produce HER2. The human cell line 116 was kindly provided by Philip Ng and cultured as recommended.36 (link) The remaining cell lines were purchased from the vendor ATCC and cultivated according to the provider’s recommendations. Cells were passaged no more than 15 times from the original stocks. C57BL/6 and BALB/c mice were bred in-house at the University Hospital of Basel (Basel, Switzerland). Animals were housed under specific pathogen-free conditions. All animal experiments were performed in accordance with Swiss federal regulations.
6
Adenovirus Rescue and Cultivation
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HEK-293T cells (SC21091507, Hunan Fenghui Biotechnology Co., Ltd, China) were cultured in Dulbecco′s modified Eagle's medium (C11995500, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gemini, 900–108, USA), 100 μg/mL streptomycin, and 100 U/mL penicillin at 37 °C in an atmosphere of 5% CO2. The virus strain rHB2015012 (GeneBank ID: KY612442) was rescued and stored in our laboratory (25 ). The plasmids pShuttle-IRES-hrGFP-2 (240082, Agilent, USA), pshuttle-H1 (BioVector-922362, NTCC, China), pAd-Easy-1 (240005, Agilent, USA), E. coli BJ5183 (200154, Agilent, USA), and E. coli DH5α were maintained in our laboratory. Jingfen No. 1, 4-day-old chicken embryos were purchased from a hatchery in Hubei (China).
7
Generation of Adenoviral Constructs with Mutant Hexon
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The plasmid containing the adenoviral genome, pAdEasy-1, from the AdEasy Adenoviral Vector System (Agilent Technologies) was previously modified to include a mutation to the hypervariable loop 7 (HVR7) of the hexon, which prevents blood factor X binding to virions and thus reduces liver infection (22) . To generate viral constructs, the modified pAdEasy-1_HVR7 plasmid was co-transformed with the pShuttle-MCS variants listed above into recA-proficient E. coli BJ5183 cells, from which the desired recombinants, obtained by homologous recombination, could be isolated for virus production. Packaging and amplification of adenoviral particles was performed by Vector Biolabs and they were purified on two consecutive cesium chloride density gradients and provided directly in PBS with 5% glycerol.
8
Recombinant Adenovirus Production Protocol
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PmeI enzyme (New England BioLabs, Inc., Ipswich, MA, USA) was used to linearly cut pAdTrack-ScFv shuttle plasmid. After purification, it was transformed into competent BJ5183 bacteria with pAdEasy-1 (both from Agilent Technologies, Inc., Santa Clara, CA, USA) for homogenous recombination. After recovery at 37°C for 1 h, bacteria were cultured on a plate with kanamycin and ampicillin. The positive clone was collected for identifying correctly constructed recombinant pAd-ScFv by PCR and PacI enzyme (New England BioLabs, Inc.) restriction digestion.
A total of 5×106/ml HEK293 cells (Cell Bank, Chinese Academy of Sciences, Shanghai, China) were seeded in culture flasks. When the cell density reached 60–70%, pAd-ScFv plasmid linearly cut by PacI endonuclease restriction enzyme was transfected into the cells by using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). After 24 h, the fluorescence in the cells was observed. When 1/3-1/2 of the cells became round and floated, cells were collected, centrifuged and re-suspended. Virus-containing supernatant was obtained by using the Adenovirus Purification and Concentration kit (Merck KGaA, Darmstadt, Germany). Recurrent infection into HEK293 cells was then performed to obtain a large amount of pAd-ScFv recombinant adenovirus.
A total of 5×106/ml HEK293 cells (Cell Bank, Chinese Academy of Sciences, Shanghai, China) were seeded in culture flasks. When the cell density reached 60–70%, pAd-ScFv plasmid linearly cut by PacI endonuclease restriction enzyme was transfected into the cells by using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). After 24 h, the fluorescence in the cells was observed. When 1/3-1/2 of the cells became round and floated, cells were collected, centrifuged and re-suspended. Virus-containing supernatant was obtained by using the Adenovirus Purification and Concentration kit (Merck KGaA, Darmstadt, Germany). Recurrent infection into HEK293 cells was then performed to obtain a large amount of pAd-ScFv recombinant adenovirus.
9
E1A12 Mutant Expression Plasmids and Recombinant Adenoviruses
10
Adenovirus-Mediated Modulation of CircRNA and Lipid Metabolism
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cDNA of circRNA_0001805 and short hairpin RNAs (shRNA) specifically targeting CPT1 and ABCA1 were commercially synthesized (GenePharm, Shanghai, China) and subcloned to the shuttle vector pShuttle and linearized into viral backbone vector pAdEasy-1 (Agilent Technologies, Beijing, China). The selected recombinant construct was amplified using XL10-Gold strain and applied to transfect HEK293 cells using Lipofectamine 2000 (Invitrogen, USA). The adenoviruses were purified and plaque assay was conducted to determine the viral titer (109 PFU/mL). For stable expression or knockdown, primary hepatocytes were transfected with generated adenoviruses above for 24 h and subjected to the subsequent analysis. Empty viruses served as a negative control (NC).
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