Adeno x rapid titer kit

The adeno-associated virus 9 (AAV9) construction compassing cDNA (AAV9-TIE-cDNA) was generated by according to the manufacturers’ recommendations from Shanghai Genechem Co., Ltd. (Shanghai, China). The endothelial cell specific promoter is “pAAV-TIEp-EGFP-MCS-3Flag-SV40 PolyA”. Viruses were packaged and amplified in HEK293A cells and purified using CsCl₂ banding followed by dialysis against 10 mM Tris-buffered saline with 10% glycerol. Titering was performed on HEK293 cells using the adeno-X Rapid Titer kit (BD Biosciences Clontech, PaloAlto, CA, USA) according to the manufacturer’s instructions.

The cDNA encoding rat clusterin was inserted into the pAd-Track-CMV shuttle vector. To produce the recombinant adenoviral plasmid, the resultant shuttle vector was electroporated into BJ5138 cells containing the AdEasy adenoviral vector. The recombinant adenoviral plasmids were transfected, and adenoviruses expressing clusterin were amplified in human embryonic kidney-293 cells and purified using CsCl density centrifugation (Sigma-Aldrich). The viruses were collected and desalted, and the titers were determined using the Adeno-X Rapid Titer Kit (BD Bioscience, San Jose, CA, USA).

We constructed the multiple shRNA expressing vectors against human VEGF, CCR1, and EpCAM as described previously [10 (link)]. The chosen siRNA target sequences are as follows: VEGFR-2, 5′-GGT CCA TTT CAA ATC TCA ACG-3′; CCR1, 5′-GCT TCC ATG CCA GGC TTA TAC-3′; EpCAM, 5′-GCT GGC CGT AAA CTG CTT TGT-3′; and negative silencing control sequence, 5′-GAG ACC CTA TCC GTG ATT A-3′. The shRNA expressing cassettes, including the H1.1 promoter and corresponding oligonucleotides, were amplified from the single shRNA-expressing vector, using the primers listed in Supplementary Figure 1. Further ligation was performed via the same restriction enzyme site to construct the multiple shRNA-expressing vectors. The viruses were propagated in HEK293 cells and purified by CsCl discontinued density gradient centrifugation. Viral titers were determined with Adeno-X Rapid Titer Kit (BD Biosciences Clontech).

pacCMV shuttle vectors encoding the structural proteins core, membrane and envelope (C, M, E), and the nonstructural protein 3 (NS3) from YF-17D virus (GenBank: X03700.1) were obtained from GenScript (Piscataway, NJ, USA). pacCMV vectors encoding the truncated form of NS3 (aa. 201–325) were also obtained from Gen Script (Piscataway, NJ, USA).
From all shuttle plasmids, human type 5 recombinant adenovirus (Ad5) vectors were produced through homologous recombination by standard methods [24 (link)]. After purification on a cesium chloride (CsCl) density gradient, adenoviral stocks were immediately frozen as single use aliquots in 10% glycerol at -80°C, and the infectivity of the stocks was determined using the Adeno-X Rapid Titer kit (BD Clontech). The presence of the inserted transgene was validated in all the recombinant Ad5 vectors by sequence analysis prior to their use for vaccination in mice. The genome of YF-17D virus with the position of the two known class I-restricted epitopes in B6 (H-2^b) mice and the derived Ad-YF vectors, are depicted in Fig 1.