Type

Journal Article

Authors

Niall Barron
Martin Clynes
Catherine M Greene
Nga T Lao
Sebastian Vencken
Paul S Kelly
Alan Griffith

Subjects

Biochemistry

Topics
cell growth mammalian cells cho cells chinese hamster ovary cells reporter assay dna antisense x linked inhibitor genetic engineering

miR-CATCH Identifies Biologically Active miRNA Regulators of the Pro-Survival Gene XIAP, in Chinese Hamster Ovary Cells. (2017)

Abstract Genetic engineering of mammalian cells is of interest as a means to boost bio-therapeutic protein yield. X-linked inhibitor of apoptosis (XIAP) overexpression has previously been shown to enhance CHO cell growth and prolong culture longevity while additionally boosting productivity. The authors confirmed this across a range of recombinant products (SEAP, EPO, and IgG). However, stable overexpression of an engineering transgene competes for the cells translational machinery potentially compromising product titre. MicroRNAs are attractive genetic engineering candidates given their non-coding nature and ability to regulate multiple genes simultaneously, thereby relieving the translational burden associated with stable overexpression of a protein-encoding gene. The large number of potential targets of a single miRNA, falsely predicted in silico, presents difficulties in identifying those that could be useful engineering tools. The authors explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH protocol. A biotin-tagged antisense DNA oligonucleotide for XIAP mRNA is designed and used to pull down and capture bound miRNAs. Two miRNAs are chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both results in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increases XIAP protein levels. A 3'UTR reporter assay confirms miR-124-3p to be a bona fide regulator of XIAP in CHO-K1 cells. This method demonstrates a useful approach to finding miRNA candidates for CHO cell engineering without competing for the cellular translational machinery.
Collections Ireland -> Dublin City University -> PubMed

Full list of authors on original publication

Niall Barron, Martin Clynes, Catherine M Greene, Nga T Lao, Sebastian Vencken, Paul S Kelly, Alan Griffith

Experts in our system

1
Niall Barron
Dublin City University
Total Publications: 44
 
2
Martin Clynes
Dublin City University
Total Publications: 209
 
3
Catherine M Greene
Royal College of Surgeons in Ireland
Total Publications: 150
 
4
Nga T Lao
Dublin City University
Total Publications: 7
 
5
Sebastian Vencken
Royal College of Surgeons in Ireland
Total Publications: 13
 
6
Paul S. Kelly
Dublin City University
Total Publications: 11