A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells

Abstract

MicroRNAs (miRNAs) are a novel class of short non-coding RNAs, which negatively regulate target gene expression at post-transcriptional level. They mediate an important layer of control in the global regulation of gene networks, controlling a broad range of physiological as well as patho-physiological pathways including development, cancer, metabolism, proliferation, and stress resistance. So far, more than 365 miRNA genes have been identified in CHO cells. The functional analysis of the physiological effect of such large numbers of miRNAs, however, requires an efficient functional screening method. In the current study, we therefore established and evaluated a protocol to perform miRNA overexpression and to screen their effect on bio-industrially relevant phenotypes, such as growth, viability and productivity, using a recombinant, Epo-Fc producing CHO cell line. For protocol optimization, four CHO miRNAs (cgr-miR-17, cgr-miR-221, cgr-miR-21, and cgr-miR-210) were cloned into small hairpin vectors including a GFP cassette and transfected. After transfection cells were analyzed for growth and productivity over a 4-day period. Even from this small set of four miRNAs, the overexpression of miR-17, one of the members of the oncogenic miR-17-92 cluster, gave proof of principle that this method enables the identification of miRNA engineering candidates as its overexpression increased the speed of cell proliferation without negatively impacting specific productivity. The here presented method is applicable for medium-throughput screening for microRNA, miR-sponge, siRNA, or mRNA overexpression along with detailed functional characterization using the same experimental set up. As the same procedure can be applied to different production cell lines, the protocol can also be used to test for individual, cell line specific responses to microRNAs. Thus our system represents a general platform to functionally screen candidates for rational cell factory design.

Generation of monoclonal antibody-producing mammalian cell lines

“…Overexpression of cgr-miR-7 produced effects similar to growth arrest obtained by temperature-shifting and increased specific productivity [72]. Overexpression of miRNAs in the cgr-miR-17-92 cluster enhanced cell growth [73].…”

Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation

“…To improve the reliability of the assay, the expression of each miRNA was normalized using two internal references (cgr-miR-185-5p and Actr5 ). The miRNA cgr-miR-185-5p was used as an internal control previously (Jadhav et al 2012), and the mRNA Actr5 showed very stable expression in the microarray experiment across all CHO cell lines used in this study. Additionally, Actr5 was described as a suitable internal control gene before (Bahr et al 2009).…”

“…In mammalian cells, miRNAs are predicted to regulate or fine-tune gene expression of ~50 % of all protein-coding genes (Krol et al 2010). In CHO cells, it has already been shown that miRNAs can be utilized to improve growth (Jadhav et al 2012), apoptosis resistance (Druz et al 2013), and specific productivity (Barron et al 2011; Jadhav et al 2014; Strotbek et al 2013). …”

5.2 Functional -Omics for Cell Lines and Processes: The -Omics Technologies on the Example of CHO Cells

“…To express miRNAs from plasmids, artificial constructs comprising the mature miR-5p and miR-3p sequence and universal loop sequence from a highly expressed miRNA can be synthesized and cloned into specific miRNA expression constructs that contain miRNA flanking regions, RNA-Polymerase II (Pol II) promoter, and a 3'polyadenylation site. This system was also used to transiently assess the influence of 4 different miRNAs on growth and productivity in CHO cells [115]. Alternatively, miRNAs can be PCR-amplified from genomic locations including 100 to 200 nucleotides up-and downstream of the miRNA stem-loop and cloned into similar expression constructs.…”