The total amount of product produced in each culture was then calculated and normalized in concentration to the starting culture volume. biotherapeutics, namely, through the co-transfection of and the product-coding gene. CHO-S cells were co-transfected with the product-coding gene and a vector containing using polyethylenimine. Cells co-transfected with showed reduced levels of apoptosis, increased specific productivity, and an overall increase in product yield of approximately 100%. Similar results were produced by employing another anti-apoptotic protein, Bcl-2 delta in CHO cells, or through the co-transfection with bcl-xL using HEK-293E cells. This work provides an alternative method for increasing yields of therapeutic proteins in TGE applications without generating a prior stable cell line and subsequent screening which are both time and resource consuming. without having to undergo the lengthy process of clonal isolation and screening. 2 Materials and Methods 2.1 Cell lines/maintenance Cell lines tested for TGE included CHO-S (Invitrogen), a CHO-S cell line stably expressing Bcl-xL created using the same Dihydrostreptomycin sulfate vector used for the transient expression of Bcl-xL as described below, and the HEK-293E (ATCC) cell line. The HEK-293E cell line is a suspension adapted HEK293 cell line stably expressing the EpsteinCBarr virus nuclear antigen (EBNA-1) allowing for episomal replication of ori-P containing plasmids, and has been shown to increase transgene expression . CHO cells were maintained in SFM4CHO (Hyclone) media supplemented with 8 mmol L-glutamine and 10 ml/L HT supplement while HEK-293E cells were maintained in a 50/50 mixture of SFM4HEK 293 (Hyclone) and FreeStyle 293 (Gibco). These media are here to after referred to as maintenance medium. All cultures were grown in a 37C incubator with 5% CO2 and shaken at 125 rpm either in 125 mL shake flasks or a six-well plate and passaged at a seeding density of 2 105 cells/mL every 3-4 days. Dihydrostreptomycin sulfate Viable cell counts were assessed using the Nova Bioprofile flex (Nova Biomedical) or the Guava EasyCyte plus system (Millipore) with the Nexin or viacount assay per the manufacturer’s instructions. 2.2 Plasmids for expression of product and anti-apoptoticproteins The product expression plasmid was constructed in the Biopharmaceutical Development Program of the SAIC-Frederick Inc. and Frederick National Laboratory for Cancer Research. The plasmid contains the product-encoding sequence to express the model product, a fusion protein, cytokine IL-2 fused with the Fc fragment of Immunoglubulin G 1 (IL-2/Fc) that originally was from Dr. Terry Strom at Beth Israel Deaconess Medical Center (BIDMC), Harvard Medical School. The fusion protein expression is driven by a CMV promoter. To determine transfection efficiency a yellow fluorescent protein (YFP) containing vector, peYFP-c1 (Clontech) was used. For TGE, and delta were cloned into pcDNA3.1 +/zeo (Invitrogen). The human wt gene was cloned between the XhoI-Xba as described previously . The delta gene lacking the Tal1 coding sequence for proteins 32-80 from the individual outrageous type gene was extracted from Craig Thompson (School of Chicago) and cloned between your Xba limitation site using PCR primers 5-GGC GGC tctaga ATG GCG CAC GCT GGG AGA -3 and 5-GGC GGC tctaga TCA CTT GTG GCC CAG ATA GGC-3 as the 5 and 3 primers respectively. The construct was sequenced to make sure proper insertion path then. The pcDNA 3.1+/zeo vector was used being a null control. All plasmids had been ready using an Endo-free Maxi-prep package (Qiagen) following manufacturer’s guidelines and kept at a focus of 0.5-1 mg/mL in endotoxin-free TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.4) 2.3 Transfection A generic procedure was employed for transfection of both cell lines with variations given below. For both CHO-S and HEK-293E cells the maintenance moderate was employed for development, however, had not been ideal for transfection. Freestyle 293 and CHO-SSFMII was employed for transfecting HEK-293E and CHO-S cells respectively. Linear PEI with molecular fat (MW) of 25,000 (Polysciences) was utilized being a transfection reagent. A share solution of just one 1 mg/mL pH 7 was ready in Milli-Q drinking water and Dihydrostreptomycin sulfate sterile filtered. Exponentially growing cells were passaged to at least one 1 106 cells/mL in maintenance medium in the entire day ahead of transfection. On the entire time of transfection, cells had been spun down (100 g, 5mins) and re-suspended in transfection moderate on the indicated cell thickness in 90% of the ultimate culture quantity at transfection (we.e. if the ultimate cell concentration is normally 1 106 cells/mL and the ultimate volume was to become 2 mL after that 2 106 cells had been resuspended in 1.8 mL of transfection medium). DNA and PEI solutions had been prepared individually in 5% Dihydrostreptomycin sulfate of the ultimate culture quantity in 150 mM NaCl so the final concentration from the IL-2/Fc vector or PEI during transfection was as indicated. For dual transfections both DNA vectors had been put into the same pipe without raising the ultimate volume. The quantity of non-product-containing DNA utilized during co-transfection is normally indicated being a fat percentage from the IL-2/Fc vector DNA. Generally, 3.5 g/mL of product-coding-containing DNA was used.
The total amount of product produced in each culture was then calculated and normalized in concentration to the starting culture volume
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