Abstract
Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale.
Highlights
Research in the post-genomic era requires the functional characterization of proteins[1,2]
We have clearly demonstrated that FEEST is a highly efficient method for the generation of transgenic mouse models and that it guarantees transgene expression, as shown by our proof-of-principal demonstration with CAG-lhl-tTA-GFP #6 (cl-tTA6)
Using FEEST, we have established a CAG-driven conditional tTA transgenic mouse model that efficiently elicits tumor formation in an organ-specific manner and faithfully recapitulates the clinical course of tumor formation observed in patients
Summary
Research in the post-genomic era requires the functional characterization of proteins[1,2]. The traditional method of generating transgenic mice is based on the microinjection of a construct into the pronucleus of a fertilized egg[5,6]. This technique has facilitated much of our understanding of the in vivo functions of cis-regulatory elements and proteins. This technique has several disadvantages: 1) the characterization of founder lines is labor intensive; 2) the characterization of the transgene insertion is performed in the mouse, which is time-consuming in many cases and can be limited by the physiological development of the mouse; 3) only a limited number of founder lines are characterized; 4) an adequate expression level of the transgene is not guaranteed; and 5) multiple copies of the transgene can be integrated into the mouse genome in a head-to-tail fashion at a single site, potentially resulting in silencing of the transgene[7]. We generated a CAG-lsl-EGFR L858R transgenic mouse line to demonstrate that this technique can be adapted to other genes
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