Adeasy xl adenoviral vector system

Replication-deficient E1/E3-deleted adenovirus vectors Ad5-CMV-oIL-2 (Ad-oIL-2) and Ad5-CMV-Null (Ad-Null) were constructed using the AdEasy XL Adenoviral Vector System (Agilent). Mouse orthogonal IL-2 clone 3A10 cDNA was synthesized (GenScript) with 5′ KpnI and 3′ HindIII restriction sites and subcloned into the multiple cloning site of pShuttle-CMV (Addgene). pAdEasy-1-containing BJ5183-AD-1 Escherichia coli cells were transformed with PmeI-linearized pShuttle-CMV-oIL-2 for homologous recombination. The resulting recombinant Ad plasmid was sequence-verified and expanded in XL10-Gold Ultracompetent cells before PacI linearization and transfection into HEK293T cells. High-titre adenoviruses were purified by caesium chloride (CsCl₂) gradient centrifugation after multiple rounds of amplification. CsCl₂ was exchanged to A195 buffer^{53 (link)} with Amicon Ultra-15 centrifugal filter units (Millipore). Viral titre (VP per ml) was determined spectrophotometrically (Nanodrop, Thermo Fisher Scientific).

Plasmids were constructed using the pcDNA3.1 (Life Technologies) mammalian expression vector. The Epac2-camps FRET-based cAMP biosensor was modified by site-directed mutagenesis to add targeting sequences in order to record changes in cAMP levels associated with specific membrane microdomains (figure 1A). Epac2-MyrPalm was designed to report cAMP changes in membrane raft domains. This plasmid contains an N-terminal acylation sequence (GCINSKRKD) from Lyn kinase, which results in myristoylation and palmitoylation that targets proteins to lipid raft/caveolar membrane fractions [31] (link). Epac2-CAAX was designed to report cAMP changes in non-raft membrane microdomains. This plasmid contains the CAAX box sequence (KKKKSKTKCVIM) from Rho GTPase attached to carboxyl terminus of Epac2-camps (figure 1A), which results in specific targeting of proteins to non-lipid raft domains of the plasma membrane [31] (link). The DNA fragments encoding Epac2-CAAX and Epac2-MyrPalm were amplified from pcDNA constructs using Platinum PCR Supermix High Fidelity (Agilent Technologies, CA). Amplified sequences were then directionally cloned into pShuttle-CMV vector in MCS using HindIII(5′) and EcoRV(3′) restriction sites. Adenovirus constructs of these plasmids were generated using AdEasy XL adenoviral vector system (Agilent Technologies, CA) following the manufacturer’s protocol.

AdEasy XL Adenoviral Vector System (Agilent) was used for production of adenovirus expressing GFP-fused MeCP2 according to the manufacturer protocol. Cortical primary neurons infected by this MeCP2 fluorescent virus were used in the FRAP experiment.