Visit our Expo - Redox and Inflammation signaling 2012

Reliable and efficient gene Knock-down and inducible overexpression in vivo.
Ben Davies#, Kader Thiam*
# Senior Scientific consultant, *Director of Transgenic Technologies
genOway, 181 avenue Jean Jaures, 69362 Lyon cedex 07, France. info@genoway.com
Over the past decades, gene Knock-out and overexpression animal models have become
standard laboratory tools for in vivo functional gene analysis. More recently, attempts have
been made to extend the repertoire of transgenic techniques using RNA interference for gene
Knock-down and inducible promoter systems for temporally regulated gene expression,
allowing the compensation and developmental problems associated with classical approaches
to be overcome. Despite initial encouraging reports, the application of both these technologies
in vivo has been unreliable. Here we describe two new approaches which allow both
technologies to be robustly and consistently applied in a transgenic context.
A major requirement for successful and reliable shRNA mediated gene Knock-down in vivo
is a low copy number of integrating constructs. We have established and validated a
technology which allows the generation of a high number of independent founder lines with a
pre-screened copy number. Furthermore a second Knock-in based technology has been
designed which takes advantage of ready-to-use validated targeting vectors to introduce a
single copy of the shRNA cassette within a specific permissive genomic site. With both these
technologies, gene knock-down of up to 80% has been reliably demonstrated.
The tetracycline system has been used successfully in cell culture models to achieve
regulated, inducible gene expression. Unfortunately, the application of this technology in vivo
has been plagued by a lack of predictability. The resulting leakiness and inconsistency of
expression in the resulting transgenic lines necessitates the generation and analysis of many
independent founder lines before a convenient line with the appropriate induction kinetics is
found. We report the development, of a new system which uses a preferential genomic
context to achieve reliable and tight regulation of transgene expression. This reliable and
reproducible inducible systems is reversible and is not restricted to preferential tissues.
Through the use of these technologies, the transgenic tool box is now complete to address
gene function in vivo whilst minimizing complicating compensatory and developmental
effects. With these methods in place, gene activity can now be reliably and exquisitely finetuned,
allowing the most appropriate model for a specific scientific question to be designed.
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