Endofree plasmid maxi kit

The construction of expression plasmids for gH, gL, gQ1, and gQ2 (pCAGGS-gH, pCAGGS-gL, pCAGGS-gQ1, and pCAGGS-gQ2) was described previously [11 (link)]. The plasmid was amplified in DH5a Escherichia coli and purified using QIAGEN EndoFree Plasmid Maxi Kit (QIAGEN), following the manufacturer's instructions. The plasmid DNA was complexed with PEI (87 kDa linear; Polysciences Inc.) based on a method described previously [35 (link)]. PEI stock solution was diluted to 1 mg/mL with 5% glucose solution and mixed with an equal volume of glucose solution containing 0.2 mg/mL plasmid DNA to achieve a nitrogen-to-phosphate ratio (N/P) of 7.5. The mixture was incubated for at least 15 min at room temperature for complex formation before use.

shRNA-expressing pDNAs driven by human U6 promoter were constructed from pBAsi-hU6 Neo vector (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. Target sequences of human ZIP6 (GenBank accession no. NM_012319) and ZIP10 (GenBank accession no. NM_020342) were designed by Takara siRNA Design Support System (Takara Bio Inc.). The pBAsi-hU6 Neo vector, which transcribes a non-related sequence of RNA with partial duplex formation, was used as a control pDNA throughout the present study. Each pDNA was amplified in Escherichia coli top 10 (Invitrogen) and purified using a QIAGEN Endofree Plasmid Maxi Kit (QIAGEN GmbH, Hilden, Germany).

Plasmid DNA (pDNA) encoding for the therapeutic gene SDF-1α (pSDF-1α) was obtained from InvivoGen, San Diego, USA. The plasmids were first amplified by transforming chemically competent DH5α E. coli cells (Biosciences, Dublin, Ireland) according to the manufacturer’s protocol. Transformed cells were then expanded in Lysogeny broth (LB) plates containing 100 μg/mL of blasticidin as the selective antibiotic for pSDF-1α. After 24 h at 37 °C, bacterial colonies were harvested and amplified in LB broth containing the appropriate antibiotic and cultured overnight in a shaker incubator at 37 °C. Plasmid purification was performed using a QIAGEN^® EndoFree^®Plasmid Maxi kit (Qiagen, Sussex, UK) and final nucleic acid concentration was determined using NanoDrop 1000 spectroscopy. Plasmids were further diluted in TE buffer to obtain a working concentration of 0.5 μg/μL and stored at −20 °C until use. Plasmid DNA (pDNA) encoding a non-therapeutic Gaussia luciferase (pLuc) purchased from New England Biolabs, Massachusetts, USA, was similarly amplified using ampicillin as the selective antibiotic. Based on our previous study [34 (link)], polyplex particles were formulated by initially mixing a specified amount of branched cationic 25 kDa PEI (Sigma-Aldrich, Wicklow, Ireland) and anionic pDNA (fixed at a dose of 2 μg) to give an N/P ratio of 10.

We derived the TCR construct from a pUC57-s183cys b2Aa vector that we had previously made, and subcloned it into the pVAX1 vector [42 (link)]. The plasmid was propagated and purified from Escherichia coli using the One Shot Top10 E. coli kit (ThermoFisher Scientific), purified using QIAGEN EndoFree Plasmid Maxi Kit (Qiagen, Hilden, Germany), and linearized using the XbaI restriction enzyme (New England Biolabs, Ipswich, MA). The linearized DNA was used to produce the TCR mRNA using the mMESSAGE mMACHINE T7 Ultra kit (ThermoFisher Scientific) following the manufacturer's instructions.

The following substances, materials, and reagents (and suppliers) were used in this study: the miRCURY LNA™ Universal RT microRNA PCR system including universal cDNA synthesis kit, SYBR™ Green Master Mix, and the primers for has-miR-29s and 5 s RNA (Exiqon, Vedbaek, Denmark); Endofree Qiagen plasmid maxi kit (QIAGEN Gmbh, Hilden, Germany); Dual-Glo™ luciferase kit and FuGENE™ HD transfection reagent, pGEM-T vector (Promega Branch, Beijing, China); Ex Taq and restriction enzymes (Takara, Dalian, China); Lipofectamine™ 2000 transfection reagent, Trizol, DNaseI (Invitrogen, Shanghai, China); miR-29a, b and c mimics (Ambion, Austin, Texas, United States); pMIR-REPORT™ vector (Applied Biosystems, Foster City, California, United States); 3-amino-9 ethylcarbazole (AEC) and VECTASTAIN Elite ABC kit (Vector Laboratories, Burlingame, California, United States); MMP-2 antibodies (Cell Signaling Technology, Beverly, Massachusetts, United States; Lifespan Biosciences, Atlanta, Georgia, United States); CD31 antibody (BD PharMingen, San Diego, California, United States); ARPE-19 and HEK 293 cells (ATCC, Manassas, Virginia, United States); Dulbecco’s Modified Eagle Medium (DMEM), DMEM/F-12, Fetal Bovine Serum (FBS), Opti-MEM (Gibco, Gaithersburg, Maryland, United States); and Neg-50 frozen section media (Microm BioResource, Auburn, California, United States).