Characterization of three loci for homologous gene targeting and transgene expression

• Published in: Biotechnology and bioengineering Vol. 110; no. 8; pp. 2225 - 2235
• Main Authors: Eyquem, Justin, Poirot, Laurent, Galetto, Roman, Scharenberg, Andrew M., Smith, Julianne
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.08.2013; Wiley Subscription Services, Inc
• Subjects: Biotechnology; Biotechnology - methods; Cell Line; Gene Expression; Gene Targeting; Genes, Reporter; Genetic engineering; genome engineering; Genomic Instability; Humans; Integration; integration cassette design; meganuclease; Mutagenesis, Insertional - methods; Promoter Regions, Genetic; Transgenes
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.24892

Integrative gene transfer is widely used for bioproduction, drug screening, and therapeutic applications but usual viral methods lead to random and multicopy insertions, contribute to unstable transgene expression and can disturb endogenous gene expression. Homologous targeting of an expression cassette using rare‐cutting endonucleases is a potential solution; however the number of studied loci remains limited. Furthermore, the behavior and performance of various types of gene cassettes following gene targeting is poorly defined. Here we have evaluated three loci for gene targeting, including one locus compatible with the proposed Safe Harbor criteria for human translational applications. Using optimized conditions for homologous gene targeting, reporter genes under the control of different promoters were efficiently inserted at each locus in both sense and antisense orientations. Sustainable expression was achieved at all three loci without detectable disturbance of flanking gene expression. However, the promoter, the integration locus and the cassette orientation have a strong impact on transgene expression. Finally, single targeted integrations exhibited greatly improved transgene expression stability versus multicopy or random integration. Taken together, our data suggest a potential set of loci for site‐specific transgene integration, suitable for a variety of biotechnological applications. Biotechnol. Bioeng. 2013; 110: 2225–2235. © 2013 Wiley Periodicals, Inc. To identify potential integration sites for efficient transgene expression, the authors performed homologous gene targeting at three new loci chosen to represent different genomic environments. For clones carrying single copy insertions, they observed sustainable expression of a GFP cassette without disturbing the expression of flanking genes. Differences in expression were observed as a function of the locus, the promoter and the transgene orientation within the cassette but, the expression levels observed among individual clones with the same expression cassette at the same locus were highly reproducible and predictable.