Adenoviral vectors AdBMP7 and AdBMP9 were constructed using the AdEasy vector system (Agilent Technologies, Santa Clara, CA) according to the methodology by Luo et al. [24 (link)]. AdBMP7 and AdBMP9 are first generation serotype 5 adenoviruses (ΔE1, ΔE3) that are transgene carriers of the human BMP7 and BMP9 proteins, respectively, directed by the cytomegalovirus (CMV) early promoter. AdBMP2 is a previously described [25 (link)] adenoviral vector carrying the BMP2 protein, kindly donated by Dr. Cristopher Evans. Recombinant adenoviruses were amplified in HEK293 cells, purified with cesium chloride gradients and dialyzed in buffer consisting of 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1 mM MgCl2, and 10% glycerol. Viral titration was performed by determining the optical density at 260 nm and by lytic plaques forming units according to the AdEasy vector system manual (Agilent Technologies).
Adeasy vector system
Manufactured by Agilent Technologies
The AdEasy vector system is a collection of plasmids designed for the generation of recombinant adenoviruses. It provides a simplified and efficient approach for the construction and production of adenoviral vectors. The system includes various components necessary for the cloning and packaging of adenoviral constructs.
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3 protocols using adeasy vector system
1
Adenoviral Vectors for BMP Delivery
2
Constructing Recombinant Adenoviral Genome
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The recombinant adenoviral genome was constructed using AdEasy vector system (Agilent). Initially, the spike gene was subcloned to pShuttle-CMV (RRID: Addgene_16403) using HindIII and SalI (ThermoFisher). Both fragments were ligated using T4 DNA ligase (Promega) overnight. The recombinant pShuttle_S was transformed into E. coli DH5⍺. The confirmed pShuttle_S plasmid was linearized with PmeI (ThermoFisher) and transformed into E. coli BJ5183 competent cells carrying AdEasy-1 plasmid (RRID: Addgene_16399) to generate recombinant adenoviral genome containing spike gene (pAdEasy_S). The confirmed pAdEasy_S was transformed into E. coli DH5⍺ for long term storage. The purified pAdEasy_S was characterized by PacI (ThermoFisher) digestion and PCR of the spike gene. Primer pair used to detect spike gene was 5′-CCATGAAACGGGGCCTGTGTTG-3′ and 5′-TTAGGTATAATGCAGCTTCACCCC-3′. PCR was performed using KOD-Plus-Neo DNA polymerase enzyme (Toyobo) and reactions were preliminary denaturation at 94 °C for 2 min followed by 25 cycles 98 °C for 10 s, 65 °C for 30 s and 68 °C for 4 min followed by 5 min final extension.
3
Recombinant Adenovirus Generation
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To generate recombinant adenovirus, the AdEasy vector system (Agilent Technologies) was used according to the manufacturer's instructions. Briefly, the Mustn1 mRNA sequence as described above was cloned into the intermediate pAdTrack-CMV vector (which expresses a GFP tracer), followed by in vivo recombination into virus production vector pAdEasy-1. Ad-293 cells were then used for virus production and amplification. Control adenovirus was generated in the same way using an empty pAdTrack-CMV vector (also expresses the GFP tracer).
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