not-yet-known not-yet-known not-yet-known unknown Nowadays, improvement in productivity and safety of biopharmaceuticals with lower costs are prior considerations of this industry. Utilizing reporter genes and FACS-based screening is a straightforward and fast approach to accelerate the identification of high producer cell lines. So far, random integration has been widely applied to generate recombinant cell lines, so applying the genetic regulatory elements such as ubiquitous chromatin-opening element (UCOE) could reduce the negative random insertional effect of the expression cassette and boost the gene of interest transcription level. Here, we used the combined strategy of FACS-based screening by green fluorescence intensity and UCOE to accelerate the clonal selection and enhance recombinant Darbepoetin alfa (DPO) productivity in Chinese Hamster Ovary (CHO) cells. In this way, two expression cassettes, pOptiVEC TM and UCOE-containing plasmid, CET1019HD, which entailed codon optimized Darbepoetin alfa-LoxP-IRES-EGFP-LoxP-IRES-DHFR fragment, were designed. To achieve stable cell line, the cassettes were linearized and transfected to the CHO DG44 cells. After stably transfected clones was obtained by changing the medium to a HT-deficient one, EGFP was used as a selection marker in FCAS to enrich the cells with the brightest green fluorescent intensity. In the following, the DPO and EGFP expressions were assessed in transcription and protein levels through qRT-PCR, FCM, western blotting, and ELISA. Expression analysis revealed that all UCOE-containing cell pools indicated higher DPO yield compared to non-UCOE populations. Indeed, FACS sorting and enrichment of the UCOE-entailing cells leads to obtaining a clone with more than 8-fold productivity. Besides, isolating high-producing cells through FACS with a simple gate resulted in a 1.5-fold improvement of target protein concentration compared to the unsorted cells. According to the results, we suggest the EGFP-FACS-based screening for sorting high-producer recombinant cell lines in a reduced time and UCOE integrated strategy to enhance protein production dramatically.

Background: Natural killer cells are critical immune cells for AML targeting. However, little is known about the relationship between using checkpoint inhibitors and Hsp70 as NK cell activators and later immune responses to control AML. We aimed to investigate the antitumor effects of NK cells pre-treated with ex-vivo Hsp70, human PD-1 blocker, and IL-15. Procedure: The NK cells were isolated from patients-derived MNCs using MACS and activated using the different combinations of Hsp70, PD-1 blocker, and IL-15. Then their killing potential and the expression pattern of PRF-1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A & B were estimated. Results: Our data revealed that the PD-1 expression was significantly reduced after NK cell activation with the different formulas of NK cell activators. Also, the expression of NKG2A was reduced, particularly in the IL-15 and IL-15 + PD-1 blocker treated groups, and adding the Hsp70 increased its expression. Moreover, the cytotoxic effect of NK cells increased in all groups, especially in IL-15 + PD-1 blocker group, in parallel with increasing in IFN-γ releasing, Granzymes, and perforin expression. All changes in IL-15 + PD-1 blocker groups were associated with the up-regulation of PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing and down-regulation of PIK3CB, AKT-1, Granzymes, and perforin. Conclusions: We suggested that combining IL-15 and PD-1 blockers could enhance the killing potential of AML-NK cells. Moreover, Hsp70, in combination with IL-15 and PD-1 blocker, interferes activation of AML-NK cells through unknown mechanisms.