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. 2017 Apr 18;114(7):1495–1510. doi: 10.1002/bit.26280

Figure 2.

Figure 2

Generation of stable miRNA expressing CHO host cells. CHO host cells were stably transfected with miRNA expression plasmids followed by antibiotic selection in order to generate stable miRNA overexpressing cells. (A) Overview on the pcDNA6.2‐GW/emGFP‐miR expression vector system used for this study. For strong expression of mature miR‐557 a tandem expression construct comprising two pre‐miR‐557 expression cassettes was used. Respective miRNA sequences, flanking regions as well as restriction enzyme cleavage sites are indicated. (B) FACS analysis of emGFP expression of CHO host cells stably expressing pcDNA6.2‐GW/emGFP‐miR expression plasmids. Non‐transfected wildtype CHO host cells served as negative control. (C) Analysis of mature miRNA expression of stable engineered CHO host cells using qRT‐PCR. QRT‐PCR amplifications curves are shown for miR‐557 (black triangles) as well as miR‐NT expressing control cells (grey squares). Total RNA was isolated from exponentially growing host cells followed by reverse transcription. Expression of mature miR‐557 (upper graph) was normalized to the expression levels of U6 snoRNA (lower graph). Analysis was performed in technical triplicates which are indicated by separate amplification curves.