In the last several years we demonstrated that it is possible to improve mammalian cells growth and recombinant protein production by manipulating gene expression using noncoding RNAs, especially microRNAs and siRNAs. We initiated this approach working with specific microRNAs and we continued performing high throughput techniques, analyzing libraries containing hundreds of microRNAs and thousands of siRNAs. In the first phase we identified five microRNAs mimics: (hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429 and hsa-miR-2110) that improved functional expression of the tested protein by as much as 48%. In the second phase by conducing high throughput siRNA analysis of 22000 genes, 10 genes whose inhibition improved recombinant protein expression were identified. Among them the OAZ1-the gene encoding the ornithine decarboxylase antizyme1- was selected first for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of ornithine decarboxylase expression with cellular levels of putrescine and spermidine; a possible indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study demonstrated that OAZ1 is a novel target for improved expression of recombinant proteins. The genome-scale screening performed in this work can establish a foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Creating HEK cell line in which the OAZ1 gene is deleted was the next step in this project. By using crispr technology HEK cell line lacking OAZ1 was created, this cell line showed three times increased expression of luciferase without affecting its growth and metabolic activities. The work continued by deleting the CASP8AP2 gene that was included in the 10 genes list. The deletion of this gene improved luciferase expression from HEK293 by about 4-fold. Comparative RNA-seq and cell cycle analysis showed that CASP8AP2 knockout improves recombinant protein expression by altering cell cycle progression, and that the cell cycle protein, cyclin dependent kinase inhibitor 2A (CDKN2A), was identified as one of the key players. These results are being validated and investigations into other potentially relevant genes and pathways are conducted. In a different research work, we implemented a novel approach for identifying the target genes of the microRNA mir22-3p. This was done by integrating differential gene expression analysis with information obtained from our previously conducted High-Throughput siRNA screening. The identified genes were validated as being involved in improving luciferase expression by using siRNA and qRT-PCR. The target gene, HIPK1, was found to increase luciferase and GPC3 expression 3.3- and 2.2-fold, respectively and was chosen for further work. In a related projet associated with virus production, conditions were established to replace the two-stage transfection spanning multiple days with a single-step that utilizes the simultaneous implementation of reverse miRNA transfection and forward plasmid DNA transfection process of 293GP cells for production of gamma-retrovirus. This shortened the screening process by two days and reduced the workflow challenges for screening miRNAs that can potentially increase the production of gamma-retrovirus. In addition, the cell number were optimized together with the choice of transfection reagent. Of those tested, 2 of 8 commercial reagents were able to transfect 293GP cells with plasmid DNA.

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Inwood, Sarah; Betenbaugh, Michael J; Shiloach, Joseph (2018) Methods for Using Small Non-Coding RNAs to Improve Recombinant Protein Expression in Mammalian Cells. Genes (Basel) 9:
Inwood, Sarah; Buehler, Eugen; Betenbaugh, Michael et al. (2018) Identifying HIPK1 as Target of miR-22-3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen. Biotechnol J 13:
Abaandou, Laura; Shiloach, Joseph (2018) Knocking out Ornithine Decarboxylase Antizyme 1 (OAZ1) Improves Recombinant Protein Expression in the HEK293 Cell Line. Med Sci (Basel) 6:
Chu, Chia; Bottaro, Donald P; Betenbaugh, Michael J et al. (2016) Stable Ectopic Expression of ST6GALNAC5 Induces Autocrine MET Activation and Anchorage-Independence in MDCK Cells. PLoS One 11:e0148075
Xiao, Su; Chen, Yu Chi; Buehler, Eugen et al. (2016) Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells. Biotechnol Bioeng 113:2403-15
Gallagher, Emily J; LeRoith, Derek; Stasinopoulos, Marilyn et al. (2016) Polyol accumulation in muscle and liver in a mouse model of type 2 diabetes. J Diabetes Complications 30:999-1007
Kumar, Amit; Baycin-Hizal, Deniz; Shiloach, Joseph et al. (2015) Coupling enrichment methods with proteomics for understanding and treating disease. Proteomics Clin Appl 9:33-47
Kumar, Amit; Shiloach, Joseph; Betenbaugh, Michael J et al. (2015) The beta-3 adrenergic agonist (CL-316,243) restores the expression of down-regulated fatty acid oxidation genes in type 2 diabetic mice. Nutr Metab (Lond) 12:8
Xiao, Su; Shiloach, Joseph; Grisshammer, Reinhard (2015) Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1. Methods Mol Biol 1272:51-64
Xiao, Su; Chen, Yu-Chi; Betenbaugh, Michael J et al. (2015) MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK293 cells. Biotechnol Bioeng 112:1632-43

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