E z n a endo free plasmid maxi kit

This study utilized a third-generation packaging system consisting of two packaging lentivectors [(pMDLg/pRRE, Addgene #12251), and (pRSV-Rev, Addgene #12252)], pMD2.G (Addgene #12259) envelope plasmid, and pLTV-mC transfer plasmid under the direction of EF-1α promoter. The transgene(s) were cloned into the transfer plasmid and transformed into Stbl3 competent cells, cultured for 16 h at 37 °C, 220 rpm in LB medium augmented with 100 µg/ml ampicillin. The bacteria culture was pelleted and subjected to plasmid extraction and endotoxin treatment using the EZNA endo-free plasmid maxi kit (OMEGA-Biotek, cat# D6926-03). 293T cells pre-seeded 24 h earlier with 7 × 10⁶ in 100 mm plate were subjected to polyethyleneimine-mediated transfection comprising 2 μg each of the packaging vectors and 6 μg of the transfer vector mixed with Opti-MEM medium (Gibco, cat# 31985-070) were grown at 37 °C, 5% CO₂ for 8–10 h. The medium was substituted with DMEM containing 10% FBS and further incubated in the same conditions. Lentiviral supernatant was harvested at 48 h and 72 h periods and concentrated with 50 kDa centrifugal concentrators (SARTORIUS, cat# VS04T31). 1.5 × 10⁴ J.RT3-T3.5 cells transduced with serially-diluted virus and cultured for 72 h were cytometrically examined to titer the viruses.

The plasmids used for hydrodynamic injection, including pT3‐EF1α‐HA‐myr‐AKT and pCMV‐sleeping beauty transposase (SB), were gifts from Dr. Xin Chen of the University of California, San Francisco, CA, USA. Before mouse injection or transient transfection, the plasmids were prepared using an E.Z.N.A.® Endo‐Free Plasmid Maxi Kit (Omega Bio‐Tek). Celecoxib (CAS # 169590–42‐5) and fructose (purity ≥99%) were purchased from Aladdin Reagent Co., Ltd. Bovine insulin was purchased from Sigma‐Aldrich. A Cell Counting Kit‐8™ was obtained from Dojindo Laboratories for the colorimetric cell viability assay. The antibodies used in this study and their applications are listed in Table S1. All other chemicals were of analytical grade.

Briefly, the CD::UPRT::GFP plasmid was constructed using the vector backbone of pCpGfree-Lucia (InvivoGen). Lucia was replaced by CD::UPRT and GFP using pSELECT-zeo-FcyFur and CpG-free GFP:Sh as template. The functional plasmid of interest, CD:UPRT:GFP, was constructed using cross-lapping in vitro assembly cloning method as described [50 (link)]. The construct (Additional file 1) was transformed using heat shock method with chemically competent Escherichia coli GT115 (Invivogen) and propagated under Zeocin antibiotic selection. The plasmids were then extracted and purified with E.Z.N.A. endo-free plasmid maxi kit according to the manufacturer’s instruction (Omega Bio-tek).

For the knock‐down expression of TBX5 in SW982 cells, si‐TBX5 (F: 5′ GGG CAC GGA AAU GAU CAU ATT 3′; R: 5′ UAU GAU CAU UUC CGU GCC CTT 3′) and si‐NC (F: 5′ UUC UCC GAA CGU GUC ACG UTT 3′; R: 5′ ACG UGA CAC GUU CGG AGA ATT 3′) were purchased from Shanghai GenePharma. To overexpress the TBX5 expression in SW982 cells, the overexpressing TBX5 plasmid pCMV3‐TBX5‐GFPSpark and control vector (pCMV3‐C‐GFPSpark‐CV) were purchased from Sino Biological Inc. These plasmids were then transfected to DH5α, and constructs were prepared using EZNA™ Endo‐free Plasmid Maxi Kit (Omega BioTek, Norcross, USA).

After designing the consensus immunogens, the four consensus sequences were subjected to codon/RNA optimization to enhance gene expression in mammal cells as described previously [24 (link),25 (link)]. The four H7 COBRA HAs were synthesized (Sangon Biotech, Shanghai, China) and cloned into the eukaryotic expression plasmid pVAX1 (Invitrogen, San Diego, CA, USA) using XhoI and EcoRI with a Kozak translation initiation sequence (GCCACC) inserted before the start codon. The plasmids were propagated in Escherichia coli DH5α and purified using an E.Z.N.A.^® Endo-Free Plasmid Maxi Kit (Omega Bio-tek, Inc., Norcross, GA, USA). Sequencing was performed to ensure no mutations were in the plasmid. The HA genes of A/Shanghai/02/2013 (H7N9) and A/Phalacrocorax carbo/Hubei/HH179/2013 (H7N7) were amplified and cloned into pVAX1 to generate pH7N9 and pH7N7, following the same method for COBRA HAs.