Strategies to achieve conditional gene mutation in mice

Abstract

The laboratory mouse is an ideal model organism for studying disease because it is physiologically similar to human and also because its genome is readily manipulated. Genetic engineering allows researchers to introduce specific loss-of-function or gain-of-function mutations into genes and then to study the resulting phenotypes in an in vivo context. One drawback of using traditional transgenic and knockout mice to study human diseases is that many mutations passed through the germline can profoundly affect development, thus impeding the study of disease phenotypes in adults. New technology has made it possible to generate conditional mutations that can be introduced in a spatially and/or temporally restricted manner. Mouse strains carrying conditional mutations represent valuable experimental models for the study of human diseases and they can be used to develop strategies for prevention and treatment of these diseases. In this article, we will describe the most widely used DNA recombinase systems used to achieve conditional gene mutation in mouse models and discuss how these systems can be employed in vivo.

FIGURE 1

Cre and FLP recognition sequences and mechanism of recombination. (A) The Cre recombinase recognition sequence is termed loxP (black arrow), while the Flp recombinase recognition sequence is termed FRT (gray arrow). Both loxP and FRT contain two 13-bp inverted repeats that are separated by an 8-bp spacer region. (B) Cre mediates excision or circularization of a DNA segment (white rectangle) when flanked by two loxP sites (black triangles) in a cis (same DNA strand) arrangement and oriented in the same direction. (C) Cre mediates inversion of a DNA segment (black arrow) when flanked by two loxP sites (black triangles) in a cis arrangement and oriented in the opposite direction. (D) Cre mediates translocation of a DNA segment (rectangles) when the loxP sites (black triangles) are located on different strands of DNA (trans arrangement) and are oriented in the same direction.

FIGURE 2

Breeding schemes for generating Cre-lox conditional, tissue-specific mutant mice. (A) Classical Cre-lox strategy to obtain conditional gene knockout in a tissue-specific manner. Mice harboring the conditional allele are crossed with Cre transgenic mice, in which Cre recombinase is under the control of a tissue-specific promoter (TSP). Breeding of these two mice will produce heterozygous offspring with excision of the floxed gene of interest only in the Cre-expressing cells/tissue (gray), while the gene of interest remains functional in other cell/tissue types (white). Offspring that are heterozygous for the conditional allele and also contain the Cre transgene (G1) are crossed to homozygous conditional mutant mice. Offspring that are homozygous for the conditional allele and heterozygous for the Cre transgene (G2) represent the conditional deletion mice, which can be generated at a frequency of 25%. Littermates that are homozygous for the conditional allele but do not carry the Cre transgene can be used as experimental controls. (B) Ligand-inducible strategy to obtain conditional gene knockout. Mice harboring the conditional allele are crossed with mice carrying a CreER fusion protein that is under the control of a tissue specific promoter (TSP). Breeding of these two mice will produce heterozygous offspring that contain the floxed gene of interest and the CreER fusion transgene. These animals are crossed to homozygous conditional mutant mice (G1). Offspring that are homozygous for the conditional allele and heterozygous for the CreER transgene (G2) can be generated at a frequency of 25% and represent the experimental animals. In the presence of ligand (tamaxofin [4-OH]) the floxed gene of interest will be excised only in the CreER-expressing cells/tissue (gray), while the gene of interest remains functional in other cell/tissue types (white). Control animals are treated with vehicle and the floxed gene of interest remains functional in all cell/tissue types since the CreER transgene is not activated by the ligand.

FIGURE 3

Cre expression mediated by the “tissue-specific” liver fatty acid binding protein (Fabpl) promoter. (A) High magnification of β-galactosidase staining in whole mount of the distal small intestine from Fabpl-Cre/+; R26R/+ mice. Cre-positive portions of the tissue stain blue when exposed to X-Gal. The Fabpl-Cre transgene directs expression of Cre recombinase to the distal small intestine and colon. (B) β-galactosidase expression in a paraffin section of the distal colon from Fabpl-Cre/+; R26R/+ mice. The expression of the Cre transgene is mosaic and therefore normal (white) and mutant (blue) crypts can be analyzed in the same mouse. (C) High magnification of β-galactosidase staining in whole mount of the urinary tract from Fabpl-Cre/+; R26R/+ mice. The positive staining (blue) illustrates the off-target effects that can be attained using this system.