Ad lacz

The construction of recombinant adenovirus vector for the RB94 gene was carried out using Gateway™ clone technology (GeneSil, Wuhan, China). Briefly, total RNA was extracted from a human embryo and reversed transcribed to obtain object cDNA, then the hRB94 gene fragment was amplified by polymerase chain reaction (PCR). The attB-flanked PCR primers were designed and used to amplify the hRB94 gene by PCR. An entry clone was performed by a BP recombination reaction with attB-PCR products and donor vector pDONRTM221. Then the entry clone and the target vector Ad/CMV/V5-DEST with attR1 and attR2 sites was recombined in vivo to create the expression clone (Ad-hRB94) using an efficient LR recombination reaction. Next, the expression clone was confirmed by PCR and sequencing. Ad-hRB94 was digested with Pac I and transferred into 293A cells to be packaged into adenovirus stock. Ad-hRB94 was amplified by infection of 293A cells and the titer was measured. Ad-LacZ, a replication-defective control adenovirus not carrying the RB94 gene, was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). The viruses were amplified and plaque-purified. Titers were determined by standard plaque assays.

The adenovirus encoding CFTR (Ad-CFTR) was generated by ViraPower Adenoviral Expression System (Invitrogen, NY, U.S.A.). The wild-type full-length mouse CFTR was cloned into pAd/CMV/V5-DEST Gateway vector (Invitrogen). The vector was linearized by standard cesium chloride/Ethidium Bromide equilibrium density gradient centrifugation. Linear DNA was transfected into QBI-293 cells at 50–70% confluence. The recombinant adenovirus was packaged and amplified in 293A cells and purified by Fast Trap Adenovirus Purification and Concentration Kit (Millipore, MA, U.S.A.). The virus titer was determined by absorbance at 260 nm. The adenovirus expressing LacZ alone (Ad-LacZ) was obtained from Invitrogen and used as a negative control.

The P1 and 3CD genes of the EV71 Neu (pinf7-54A) strain were amplified by PCR and individually inserted into the shuttle vector pENTR4 (Invitrogen). The nucleotide element of the elongation factor-1α (EF-1α) promoter was inserted into the 3’ end of the P1 gene and the 5’ end of the 3CD insert to generate the pENTR4-P1/EF-1α/3CD construct. The 3CD gene alone was inserted into pENTR4 to generate the pENTR4-3CD construct. The pENTR4-P1/EF-1α/3CD and pENTR4-3CD constructs were enzymatically recombined into the ΔE1/ΔE3 (replication-incompetent) Ad5 vector pAd/CMV/V5-DEST [33 (link)] to form recombinant pAd-EVVLP and pAd-3CD, respectively. pAd DNA was transfected into the 293A packaging cell line to produce the recombinant adenoviruses designated Ad-EVVLP and Ad-3CD. Ad-LacZ carrying a luciferase reporter gene as a vector control was obtained from Invitrogen. The recombinant viruses were purified and concentrated using Vivapure adenoPACK 100RT (Satorius Stedin Biotech). The purified virus titers were determined using a modified standard plaque assay. Various Ad virus dilutions were added to each well of 293A cells plated in a 6-well tissue culture plate. After overlaying the cultures with DMEM containing 0.75% methylcellulose, the cultures were incubated at 37 °C for 10 to 12 days and plaques were counted. The typical yield of adenoviruses was approximately 1 × 10⁹ pfu/mL.