Psp72 vector

For generation of RNA in vitro, the HIV 5′-UTR (nucleotides 1–497) was cloned into pSP72 vector (Promega) using BglII and EcoRI sites under the control of T7 promoter. All constructed plasmids were verified by DNA sequencing. Primers were obtained from IDT and are reported in Feng et al [73 (link)]. Fluorescently labeled RNA was produced by transcribing pSP72 DNA cut with EcoRI in vitro using T7 RNA polymerase with a nucleotide mixture containing fluorescein-12-UTP (Roche Applied Science). Steady-state rotational anisotropy reactions (60 μL) were conducted in buffer containing 50 mM Tris, pH 7.5, 40 mM KCl, 10 mM MgCl₂, and 1 mM DTT and contained 10 nM fluorescein-labeled 5’UTR RNA and increasing amounts of A3 (A3H_{hap II}, 0.1–61 nM; A3C-A3H_{hap II}, 0.0045–6.040 nM; and A3G, 0.36202 nM). A QuantaMaster QM-4 spectrofluorometer (Photon Technology International) with a dual emission channel was used to collect data and calculate anisotropy. Measurements were performed at 21°C. Samples were excited with vertically polarized light at 495 nm (6-nm band pass), and vertical and horizontal emissions were measured at 520 nm (6-nm band pass). The K_d was obtained by fitting to a hyperbolic decay curve equation using SigmaPlot version 11.2 software.

Molday ION-labeled hBM-MSCs were transfected with ITGA4 mRNA as we previously described 12 (link). Briefly, the cDNA for ITGA4 gene was inserted into the pSP72 vector (P2191-Promega) and served as a template for in vitro production of mRNA capped with an anti-reverse-cap-analogue (ARCA) using the mMessage mMachine ® T7 Ultra Kit (AM1345, Ambion). Then, the mRNA-ITGA4 (0.94μg/ml) was mixed with Lipofectamine 2000 to form complexes, which were incubated with cells over 4 hours followed by triple washing with PBS and placement of cells in MSCBM medium for 4-6 hours to allow for ITGA4 protein production prior to experiments.

Flag-DAP5 was cloned from pECE-Flag vector (39 (link),40 (link)) into pCDNA3 expression vector (Invitrogen) using NotI-XbaI restriction sites. Point mutants (E862K, E862Q and N86A) were created by site-directed mutagenesis (Agilent). Plasmids were verified by sequencing.
Bcl2 monocistronic and bicistronic vectors were previously described (41 (link)) and kindly provided by R. E. Lloyd. Firefly luciferase vector was constructed by deletion polymerase chain reaction using Bcl2 monocistronic vector. Renilla luciferase vector was previously described (42 (link)). HCV (43 (link)), DAP5 (37 (link)), Apaf1 (37 (link)) and IRF7 (44 (link)) 5′UTRs were subcloned into a pSP72 vector (Promega) in which a 50-nucleotide-long 3′ poly(A) was inserted using PstI and BamHI restriction sites.
Constructs were introduced into HEK293T cells by the standard calcium-phosphate precipitation method for 24 h before lysis.

Three partially overlapping genomic fragments encompassing Nos gene locus from the fourth exon to position −6248 bp upstream of the ATG were used in transgenic assays to analyze their enhancer activity. The DNA fragments were amplified by PCR from C. robusta genomic DNA and were cloned as reported by Caccavale et al. [32 (link)]. The LacZ expression construct was made by using the pBluScript II KS containing the human beta-globin basal promoter upstream to the LacZ reporter gene and the SV40 polyA sequence [107 (link)]. Similarly, the GFP expression construct was prepared by using the pSP72 vector (Promega, Madison, Wisconsin, USA) containing the GFP gene and SV40 polyA, as described by Zeller et al. [108 (link)], to which we added the human beta-globin basal promoter.