The recombinant adenovirus vector encoding murine GLEA2 was constructed using the Adeno-XTM Expression System (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, the GLEA2 cDNA was cloned into the shuttle vector pDC315 and sequenced. The desired replication-deficient adenovirus containing the full length cDNA of GLEA2 was generated by homologous recombination through co-transfection of plasmids pDC315-GLEA2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roch, Germany). After several rounds of plaque purification, the adenovirus containing the GLEA2 gene was amplified and purified from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at -80 °C in phosphate buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant, El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (Ad-LacZ), was used as a control vector.
Adeno xtm expression system
Manufactured by Takara Bio
Sourced in United States
The Adeno-X™ Expression System is a tool for the expression of recombinant proteins in mammalian cells. It utilizes adenoviral vectors to efficiently deliver and express the gene of interest in a variety of cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dotap liposome reagent, by Roche (4 mentions)
Pmd18 t, by Takara Bio (1 mentions)
Lipofectamine liposome reagent, by Thermo Fisher Scientific (1 mentions)
Pmd18 t, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Viratrap adenovirus purification kit, by Biomiga (1 mentions)
Cb17 scid mice, by Charles River Laboratories (1 mentions)
12 protocols using adeno xtm expression system
1
Generating Recombinant Adenovirus Encoding GLEA2
2
Generating Sorafenib-Resistant Hepatoma Cells with PD-L1 Overexpression
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The miRNA expression vector pSuper was obtained from OligoEngine (Seattle, WA, USA). Subcloning of hsa–pre-miR-1 fragment into pSuper to form the expression vector pSuper–miR-1 has been described in previous studies17,18 (link). For PD-L1 overexpression in miR-1-expressing sorafenib-resistant hepatoma cells, the recombinant adenovirus vector encoding PD-L1 was constructed using the Adeno-XTM Expression System (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. Briefly, the PD-L1 cDNA was cloned into the shuttle vector pMD-18T (Takara, Dalian, Liaoning, China) and sequenced. Sequences of primers for full-length human PD-L1 cDNA amplification are shown as follows: 5′-TACTGCAGAAGATGAGGATATTTG CTGTC-3′(forward) and 5′-ATTGAATTCTTACGTCTCCTCCAAATGTG-3′ (reverse). The desired replication-deficient adenovirus containing the full-length cDNA of PD-L1 was generated by homologous recombination through cotransfection of plasmids pMD-18T-PD-L1 and pBHG1oXE1, 3Cre in HEK 293 cells using the Lipofectamine liposome reagent (Invitrogen, Carlsbad, CA, USA). After several rounds of plaque purification, the adenovirus containing the PD-L1 gene was amplified and purified from cell lysates. The infectious titer was determined by a standard plaque assay.
3
Determining Adenoviral Titer via Plaque Assay
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PFUs per ml were determined by performing 2-fold serial dilutions of samples in 96-well plates containing 6 x 104 cells/well of HEK293 cells (ATCC CRL1573TM) as described by the manufacturer (Adeno-XTM Expression System User Manual, Clontech Laboratories Inc.) [13 ]. Cells were washed with PBS prior to infection. Infected cells were incubated for 5 days at 37˚C in a 5% CO2 atmosphere. To determine the virus titer, plaques caused by the cytopathic effect of viral proliferation were counted. To minimize experimental error, only plates containing between 10 and 100 plaques were counted, depending on the size of the cell culture plate used. According to statistical analysis, when 100 plaques are counted the sample titer will vary by plus or minus 10%.
4
Constructing Adenoviruses with circRNA
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To construct the adenoviruses, circRNA mm9-circ-008009 (renamed as DICAR) is a vector synthesized by Chenechem Company (Shanghai, China). DICAR was first inserted into pcDNA3.1 with the endogenous flanking sequence (1-kb upstream). The upstream flanking sequence was then partially copied and then inserted in the inverted orientation downstream. Adenovirus-DICAR-shRNA without the downstream reverse sequence served as the negative control. All these vectors were finally cloned into the Adeno-X TM Expression System (Clontech) in accordance with the manufacturer’s instructions.41 (link)
5
Construction of lincRNA-p21 Adenoviruses
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The lincRNA-p21 related adenoviruses were constructed using the Adeno-XTM expression system (Clontech). Briefly, full length of mouse lincRNA-p21 was subcloned into adenoviral shuttle vector pDC315 (Microbix Biosystems, Ontario, Canada) to generate pDC315-lincRNA-p21 (Ad-lincRNA-p21). LincRNA-p21 shRNA harboring lincRNA-p21 siRNA #1 (Ad-lincRNA-p21-shRNA) was designed, synthesized and inserted into pDC315-U6-shRNA. Adenoviruses were then produced by co-transfecting HEK293T cells with each adenoviral construct together with the packaging vectors pBHGloxdelE13cre (Microbix Biosystems, Ontario, Canada) using Lipofectamine 2000 (Life Technologies), according to the instructions of the manufacturer. Ad-lincRNA-p21, Ad-lincRNA-p21-shRNA and Ad-control (used as control) were then amplified and purified using the ViraTrapTM adenovirus purification kit (Biomiga Inc., San Diego, Calif., USA). All the adenoviruses were tagged with HA epitope.
6
Adenoviral Delivery of VEGF in Mice
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Recombinant adenoviruses expressing mouse VEGF164 or human VEGF165 were produced using the Adeno‐XTM Expression System (Clontech, Saint‐Germain‐en‐Laye, France) according to the manufacturer's recommendations. Adenoviral vectors were diluted in physiological solution and injected in TA and GC muscles in the lower hindlimb of immune‐deficient CB.17 SCID mice (Charles River Laboratories) at the titer of 1 × 108 infectious units/injection, with a 30‐gauge needle syringe, as previously described 41 . Information on ischemia experiments is provided below.
7
Mutagenesis and Adenoviral Transduction of miR-128
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The introduction of mutations into the miR-128 binding site in the 3′UTR of prohibitin was performed using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The construct was sequenced to check that only the desired mutations had been introduced.
Preparation of adenoviral miR-128 was synthesized by polymerase chain reaction using rat cardiomyocyte DNA as the template. The upstream primer was 5′-TTTCATTCTTGGGCTCTTTG-3′. The downstream primer was 5′-GAAGAGAAAGCAATAGCTAC-3′. It was cloned into the Adeno-XTM Expression System (Clontech) according to the manufacturer's instructions.
Preparation of adenoviral miR-128 was synthesized by polymerase chain reaction using rat cardiomyocyte DNA as the template. The upstream primer was 5′-TTTCATTCTTGGGCTCTTTG-3′. The downstream primer was 5′-GAAGAGAAAGCAATAGCTAC-3′. It was cloned into the Adeno-XTM Expression System (Clontech) according to the manufacturer's instructions.
8
Constructing TIPE-2 Adenoviral Vector
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The recombinant adenovirus vector encoding TIPE-2 was constructed using the Adeno-XTM Expression System (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. Briefly, the TIPE2 cDNA was cloned into the shuttle vector pDC315 and sequenced. Amplification conditions were: 95 °C for 3 min and then 95 °C for 15 s, 55 °C for 15 s and 72 °C for 30 s for 40 cycles. Primers used for this study were synthesized by Invitrogen Corporation and shown as follows:
5`-TCAGAAACATCCAAGGCCAGAC-3`(sense) and 5`-CGG ACC GA CCAGCCATTTTAC-3` (antisense) for TIPE-2. The desired replication- deficient adenovirus containing the full-length cDNA of TIPE-2 was generated by homologous recombination through cotransfection of plasmids pDC315-TIPE-2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roche, Germany). After several rounds of plaque purification, the adenovirus containing the TIPE-2 gene was amplified and purified from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at − 80 °C in phosphate-buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (rAd-LacZ) was used as a control vector for DC transduction.
5`-TCAGAAACATCCAAGGCCAGAC-3`(sense) and 5`-CGG ACC GA CCAGCCATTTTAC-3` (antisense) for TIPE-2. The desired replication- deficient adenovirus containing the full-length cDNA of TIPE-2 was generated by homologous recombination through cotransfection of plasmids pDC315-TIPE-2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roche, Germany). After several rounds of plaque purification, the adenovirus containing the TIPE-2 gene was amplified and purified from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at − 80 °C in phosphate-buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (rAd-LacZ) was used as a control vector for DC transduction.
9
Recombinant Adenovirus for TIPE-2 Expression
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Construction of recombinant adenovirus encoding TIPE-2
The recombinant adenovirus vector encoding TIPE-2 was constructed using the Adeno-XTM Expression System (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. Brie y, the TIPE2 cDNA was cloned into the shuttle vector pDC315 and sequenced. The desired replication-de cient adenovirus containing the full-length cDNA of TIPE-2 was generated by homologous recombination through cotransfection of plasmids pDC315-TIPE-2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roche, Germany). After several rounds of plaque puri cation, the adenovirus containing the TIPE-2 gene was ampli ed and puri ed from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at -80 •C in phosphate-buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (rAd-LacZ) was used as a control vector for DC transduction.
The recombinant adenovirus vector encoding TIPE-2 was constructed using the Adeno-XTM Expression System (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. Brie y, the TIPE2 cDNA was cloned into the shuttle vector pDC315 and sequenced. The desired replication-de cient adenovirus containing the full-length cDNA of TIPE-2 was generated by homologous recombination through cotransfection of plasmids pDC315-TIPE-2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roche, Germany). After several rounds of plaque puri cation, the adenovirus containing the TIPE-2 gene was ampli ed and puri ed from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at -80 •C in phosphate-buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (rAd-LacZ) was used as a control vector for DC transduction.
10
Adenoviral Vector Encoding TIPE-2 Construction
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The recombinant adenovirus vector encoding TIPE-2 was constructed using the Adeno-XTM Expression System (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. Brie y, the TIPE2 cDNA was cloned into the shuttle vector pDC315 and sequenced. The desired replication-de cient adenovirus containing the full-length cDNA of TIPE-2 was generated by homologous recombination through cotransfection of plasmids pDC315-TIPE-2 and pBHG1oXE1, 3Cre in HEK 293 cells using the DOTAP liposome reagent (Roche, Germany). After several rounds of plaque puri cation, the adenovirus containing the TIPE-2 gene was ampli ed and puri ed from cell lysates by banding twice in CsCl density gradients. Viral products were desalted and stored at -80 •C in phosphate-buffered saline (PBS) containing 10% glycerol (v/v). The infectious titer was determined by a standard plaque assay. A second recombinant El-, E3-deleted adenovirus carrying the LacZ protein under the control of CMV promoter (rAd-LacZ) was used as a control vector for DC transduction.
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