Gene Knock Down in vivo by RNAi

Taconic has developed a powerful system to achieve RNAi-mediated gene knock down in vivo¹, which allows time- and cost efficient data production within 5-6 months. This technology yields data in short timeframes and is suited for high throughput analysis of target genes in vivo.

Taconic advanced RNAi technology now also enables researchers for the first time to achieve inducible and reversible gene knockdown in vivo², thus allowing investigators to gain insight into drug and target-related disease mechanisms. This powerful technology based upon temporal control of shRNA expression in transgenic mice allows gene knockdown to be induced in mice at any given point in time, and gene function can subsequently be turned back on again.

Multiple RNAi models have been delivered to and analyzed by both academics and pharmaceutical industry^3,4. On a broad basis this important novel methodology will enable new insights into gene function in vivo.

Generation of RNAi models consists of the following steps:

1. Identification and validation of suitable shRNAs for the target gene
2. Knock down vector construction
3. Generation of mice heterozygous for the best-performing shRNA
   
   For more details on these steps

Expected Timeline: 26 weeks

Taconic has incorporated rigorous quality control and documentation of different mouse production steps using project management software and the Laboratory Information Management System (LIMS). A designated Taconic scientific project manager will ensure optimal communication and strict adherence to the timeline and deliverables throughout the project. Taconic will provide a detailed Milestone Documentation after each of the steps mentioned above.

Upon generation of the line at Taconic, we will coordinate the delivery and further breeding of your animals to produce the research cohorts you want at the health status you need.

Learn more about our Cohort Production Packages for RNAi Models.

Learn more about in vivo mouse RNAi Models.

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For the generation of your RNAi knockdown mouse model, you will have to provide the name and accession number of your target gene.

The Taconic team will then identify a set of candidate shRNAs against your target for every mouse model to be generated by using advanced bioinformatics tools.

Candidate shRNA are introduced into a vector allowing constitutive or inducible expression of the shRNA. For inducible and reversible expression we use a single cassette, placing all necessary regulatory elements on one vector. A codon-optimized tet-repressor allows for tight and highly inducible regulation of shRNA expression by doxycylin.

The shRNA cassettes are introduced into ES cells by targeted transgenesis using RMCE. Knock down efficiency of the candidate shRNAs is determined in the targeted ES cells by qPCR analysis.

The ES cell line with highest degree of knock down will be chosen for injection into diploid or tetraploid blastocysts to create your constitutive or inducible RNAi knock down model.

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References

¹Seibler J, Küter-Luks B, Kern H, Streu S, Plum L, Mauer J, Kühn R, Brüning JC, Schwenk F.: Single copy shRNA configuration for ubiquitous gene knockdown in mice. Nucleic Acids Res. 2005 Apr 14;33(7):e67

² Seibler J, Kleinridders A, Küter-Luks B, Niehaves S, Brüning JC, Schwenk F.: Reversible gene knockdown in mice using a tight, inducible shRNA expression system. Nucleic Acids Res. 2007;35(7):e54

³Koch L, Wunderlich FT, Seibler J, Könner AC, Hampel B, Irlenbusch S, Brabant G, Kahn CR, Schwenk F, Brüning JC.: Central insulin action regulates peripheral glucose and fat metabolism in mice. J Clin Invest. 2008 Jun;118(6):2132-47.

⁴Christoph T, Bahrenberg G, De Vry J, Englberger W, Erdmann VA, Frech M, Kögel B, Röhl T, Schiene K, Schröder W, Seibler J, Kurreck J.: Investigation of TRPV1 loss-of-function phenotypes in transgenic shRNA expressing and knockout mice. Mol Cell Neurosci. 2008 Mar;37(3):579-89.