Purelink hipure plasmid filter purification kit

FBN1 allelic minigene fragments (2798 bp), including either the reference (Ref) or mutated (Mut) sequence, have been generated by PCR using the patient genomic DNA as template, Phusion Hot Start II high-fidelity DNA polymerase (Thermo Scientific), and primers 516-519 (Table 1). The agarose-gel purified PCR fragments were cloned into a linearized dual tagged (N-terminal 3xFLAG and C-terminal c-Myc) expression vector (p3xFLAG-Myc-CMV-26, Sigma) as detailed in Supp. Figure 1A. Plasmids grown in XL1-Blue Supercompetent E. coli cells (Stratagene) were purified by using a PureLink HiPure plasmid filter purification kit (Invitrogen) according to the manufacturer's protocol. All the FBN1 constructs were verified by full-length insert sequencing (for primers, see Table 1). Plasmid 1283 was selected as the FBN1-Ref minigene, and plasmid 1288 was selected as the FBN1-Mut minigene (Supp. Figure 1B). Plasmids were formulated at a final DNA concentration of 1 mg/ml in sterile isotonic saline.

To generate promoter deletion constructs, primary monocyte genomic DNA was extracted from the interphase and phenol layer using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. To obtain 5′ end promoter deletion products, primers (Supplementary Table S1) flanking the desired promoter regions were used for PCR amplification, using the purified genomic DNA as template. PCR was performed using iProof High Fidelity DNA Polymerase (Bio-rad) under the following parameters: initial denaturation at 98°C; 40 cycles of amplification, denaturation at 98°C for 10 s, annealing at Tm of primer pair +3°C for 30 s, elongation at 72°C for 2.5 min; followed by a final extension at 72°C.
The isolated promoter lengths were separately cloned into pGL4.20 vector (Promega) using standard molecular cloning techniques. The restriction enzymes used were XhoI and HindIII (Thermo Fisher Scientific). T4 DNA ligase was from Roche. For small-scale purification of plasmids, AxyPrep Plasmid Miniprep kit (Axygen Biosciences) was used. Large-scale purification of plasmids intended for transfection was carried out using PureLink HiPure Plasmid Filter Purification kit (Invitrogen). The full-length sequence of each promoter construct was confirmed by sequencing using Big Dye Terminator cycle sequencing kit and ABI Prism 3100 Genetic Analyzer (Life Technologies).

Mice were deeply anaesthetized by an intraperitoneal injection of 85 mg/kg ketamine and 10 mg/kg xylazine. A minor incision was done on hindlimb to expose the tibialis anterior (TA) muscle, which was injected along its length with 30 μL of 0.9% saline containing purified plasmid DNA (PureLink HiPure Plasmid Filter Purification Kit, Invitrogen, Carlsbad, CA, USA). To evaluate the hypertrophic capacity of the cachectic muscles, 15 μg of a plasmid coding for a constitutively active form of Akt protein (myr-Akt) or p70 ribosomal S6 kinase 1 (S6K1) were injected into muscle. In order to monitor the gene delivery in the electrotransfer experiments, a plasmid encoding green fluorescent protein (Snap-GFP) was co-transfected into the muscle. Thereafter, two stainless steel spatula electrodes were placed at each side of the isolated TA muscle belly, and electric pulses were applied using an electroporator (AVS Projetos). Five square-wave pulses with a pulse length of 20 and 200 ms intervals between each pulse were delivered at 21 volts [29 (link)]. Muscle damage was minimal, and myofibers with abnormal morphology were excluded from the analysis. The myr-Akt plasmid was purchased from AddGene (Addgene plasmid #9008; http://n2t.net/addgene:9008, accessed on 11 January 2018). S6K1 and GFP plasmids were a gift from Dr. Bert Blaauw (University of Padua, Padua, Italy).

Pig Pitx2a (sequence deduced from the swine genomic sequence NW_003610943) and Pitx2c (NM_001206435) were amplified from oligo-dT-primed cDNA from left atrium of newborn piglets; pig Pitx2b (sequence deduced from the swine genomic sequence NW_003610943) was amplified from skeletal muscle cDNA of 20-day-old neonatal animals. Each of the full-length Pitx2 constructs was directionally cloned into p3xFLAG-CMV-14 expression vector (Sigma, Madrid, Spain) at the EcoRI (5′) and BamHI (3′) restriction sites and verified by sequencing [28] (link). Mouse full-length Pitx2c (NM_001042502.1) was amplified from Sol8 myoblast cDNA by PCR with specific primers containing HindIII and XbaI restriction sequences [27] (link). Subsequently, the PCR product was inserted into the pcDNA3.1/Zeo(-) plasmid (Invitrogen, Barcelona, Spain), which was modified to generate the V5-tagged Pitx2c as reported [29] (link). The plasmids were purified by using a PureLink HiPure plasmid filter purification kit (Invitrogen, Barcelona, Spain) according to the manufacturer's protocol. Purified plasmids were formulated in nuclease-free water (Ambion, Madrid, Spain).