Type

Journal Article

Authors

M Clynes
F O'Sullivan
P Meleady
C Clarke
P Doolan
N Sanchez
N Kumar
N Barron

Subjects

Biochemistry

Topics
cho cells micrornas recombinant proteins chemistry cricetulus mirn7 microrna human sequence homology nucleic acid gene expression profiling animals metabolism base sequence cell culture techniques oligonucleotide array sequence analysis biotechnology cricetinae molecular sequence data humans

Engineering CHO cell growth and recombinant protein productivity by overexpression of miR-7. (2010)

Abstract The efficient production of recombinant proteins by Chinese Hamster Ovary (CHO) cells in modern bioprocesses is often augmented by the use of proliferation control strategies. The most common method is to shift the culture temperature from 37 °C to 28-33 °C though genetic approaches to achieving the same effect are also of interest. In this work we used qRT-PCR-based expression profiling using TLDA™ cards to identify miRNAs displaying differential expression 24h after temperature-shift (TS) from 37 °C to 31 °C. Six miRNAs were found to be significantly up-regulated (mir-219, mir-518d, mir-126, mir-30e, mir-489 and mir-345) and four down-regulated (mir-7, mir-320, mir-101 and mir-199). Furthermore, qRT-PCR analysis of miR-7 expression over a 6 day batch culture, with and without TS, demonstrated decreased expression over time in both cultures but to a significantly greater extent in cells shifted to a lower culture temperature. Unexpectedly, when miR-7 levels were increased transiently by transfection with miR-7 mimic in CHO-K1 cells, cell proliferation at 37 °C was effectively blocked over a 96 h culture period. On the other hand, transient inhibition of endogenous miR-7 levels using antagonists had no impact on cell growth. The exogenous overexpression of miR-7 also resulted in increased normalised (per cell) production at 37 °C, though the yield was lower than cells grown at reduced temperature. This is the first report demonstrating a functional impact of specific miRNA disregulation on CHO cell behavior in batch culture and provides some evidence of the potential which these molecules may have in terms of engineering targets in CHO production clones. Finally, we report the cloning and sequencing of the hamster-specific cgr-miR-7.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

M Clynes, F O'Sullivan, P Meleady, C Clarke, P Doolan, N Sanchez, N Kumar, N Barron

Experts in our system

1
Martin Clynes
Dublin City University
Total Publications: 209
 
2
Finbarr O'Sullivan
Dublin City University
Total Publications: 21
 
3
Paula Meleady
Dublin City University
Total Publications: 95
 
4
Colin Clarke
Dublin City University
Total Publications: 26
 
5
Padraig Doolan
Dublin City University
Total Publications: 37
 
6
Niall Barron
Dublin City University
Total Publications: 44