Conditional Transgenic Mouse Models: From the Basics to Genome-Wide Sets of Knockouts and Current Studies of Tissue Regeneration

Abstract

Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene expression; Cre or Flp recombinase-mediated modifications, including excision, inversion, insertion and interchromosomal translocation; combination of the tet and Cre systems, permitting inducible knockout, reporter gene activation or activation of point mutations; the avian retroviral system based on delivery of rtTA specifically into cells expressing the avian retroviral receptor, which enables cell type-specific, inducible gene expression; the tamoxifen system, one of the most frequently applied steroid receptor-based systems, allows rapid activation of a fusion protein between the gene of interest and a mutant domain of the estrogen receptor, whereby activation does not depend on transcription; and techniques for cell type-specific ablation. The diphtheria toxin receptor system offers the advantage that it can be combined with the 'zoo' of Cre recombinase driver mice. Having described the basics we move on to the cutting edge: generation of genome-wide sets of conditional knockout mice. To this end, large ongoing projects apply two strategies: gene trapping based on random integration of trapping vectors into introns leading to truncation of the transcript, and gene targeting, representing the directed approach using homologous recombination. It can be expected that in the near future genome-wide sets of such mice will be available. Finally, the possibilities of conditional expression systems for investigating gene function in tissue regeneration will be illustrated by examples for neurodegenerative disease, liver regeneration and wound healing of the skin.

Mouse Primordial Germ Cells

“…Instead, genetic lineage tracing systems using transgenic mice with a drug-inducible, conditional reporter (Bockamp et al, 2008) may provide the only viable alternative. For example, in a system using a modified Cre recombinase (Danielian et al, 1998;Hayashi and McMahon, 2002), Cre expression is driven by the promoter from the gene of interest.…”

Bio-applications Derived from Site-Directed Genome Modification Technologies

“…By limiting the expression of Cre or Flp recombinases to a speci fi c tissue or cell type, and/or for a given time, based on the use of an inducible expression system, it has been possible to control the inactivation of a gene (Miller 2011 ;Wang 2009 ;Bockamp et al 2008 ) . The common strategy for conditional knockout of alleles is to fl ank with loxP or FRT sites at a proximal exon within the target gene, leading to frameshift and null allele mutations following Cre-mediated recombination.…”

Databases, Internet Resources, and Genetic Nomenclature

“…Although a variety of genetically tractable model systems are currently emerging, the mouse is still the only vertebrate for which highly versatile and systematic phenotype-and gene-driven mutagenesis strategies are well established (see Chapters 2À8). Genome manipulation tools include traditional, BAC/YAC, and lentiviral transgenesis, gene targeting by homologous, site-and/or time-specific recombination, gene trapping, chemical-and transposon-induced mutagenesis, nuclease-based engineering, and RNA-mediated interference (reviewed in Bockamp et al, 2008;Doyle et al, 2012;Gama Sosa et al, 2010;Miller, 2011). Such tools enable the creation of spatially or temporally restricted or induced mutations, ranging from single nucleotide changes to complex genomic rearrangements, that can provide unparalleled insight into individual gene function.…”