Genetically engineered mouse models for drug development and preclinical trials

Abstract

Drug development and preclinical trials are challenging processes and more than 80% to 90% of drug candidates fail to gain approval from the United States Food and Drug Administration. Predictive and efficient tools are required to discover high quality targets and increase the probability of success in the process of new drug development. One such solution to the challenges faced in the development of new drugs and combination therapies is the use of low-cost and experimentally manageable in vivo animal models. Since the 1980's, scientists have been able to genetically modify the mouse genome by removing or replacing a specific gene, which has improved the identification and validation of target genes of interest. Now genetically engineered mouse models (GEMMs) are widely used and have proved to be a powerful tool in drug discovery processes. This review particularly covers recent fascinating technologies for drug discovery and preclinical trials, targeted transgenesis and RNAi mouse, including application and combination of inducible system. Improvements in technologies and the development of new GEMMs are expected to guide future applications of these models to drug discovery and preclinical trials.

Keywords: Drug discovery; Genetically engineered mouse models; Preclinical trials; RNAi mouse; Targeted transgenesis.

Fig. 1.

Targeted transgenesis. (A) Targeted transgenesis at the ROSA26 locus by recombinase mediated cassette exchange (RMCE) results in genomic integration of the target gene into the Rosa26.10 allele of mouse ES cells (Kleinhammer et al., 2011b). The hygromycin resistance gene was exchanged by C31 Integrase-mediated recombination of both pairs of attB and attP sites. Recombined ES cell clones were selected by the neomycin resistance gene and identified by PCR or Southern blot analysis. GOI: Gene of interest. (B) Flpe-mediated recombination in the KH2 ES cell line (Beard et al., 2006). The ES cell line contained an FRT-hygromycin-polyA homing cassette downstream of the COL1A1 locus. Upon co-electroporation of the targeting vector and an Flpe transient expression vector, recombination resulted in the loss of the neomycin cassette and the insertion of the gene of interest. This FRT-Flpe recombination also restored and conferred hygromycin resistance. (C) Southern blot analysis to identify recombined ES cell clones. For SpeI digestion, the bands representing alleles before and after recombination were 6.7 and 4.1 kb, respectively, as described in (B). Asterisks indicate recombined ES cell clones.

Fig. 2.

In vivo expression of a tetracycline-inducible gene in mice. Mice were derived from KH2 ES cells that carry both the R26-rtTA allele and the Flp-in TRE-AIMP3-HA allele (Beard et al., 2006). Doxycyline was administered to the mice in drinking water for 5 days (2 mg/ml, supplemented with sucrose 10 mg/ml). Thymocytes were harvested and analyzed by immunoblot with anti-HA or anti-AIMP3 antibodies. β-actin was used as a loading control. AIMP3: ARS-interacting multifunctional protein 3.

Fig. 3.

Construct of RNAi mice generated by recombinase mediated cassette exchange (RMCE). For stable integration of the shRNA expression vector into the ES cell genome, RMCE was used as described in Figure 1A. Constructs of commonly used RNAi mice are described as constitutive (A), conditional (B) and inducible (C).