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The last decade has witnessed an enormous rise in the interest for retinoid signalling and its cognate receptors, because of their central role in the coordination of development and homeostasis, through their ability to orchestrate the expression of numerous target genes. These receptors include six nuclear receptor (NR) family members, the retinoic acid receptor (RAR) α, β and γ, and the retinoid X receptor (RXR) α, β and γ, which are expressed in many cell types in mammals. Analysis of the development of mouse embryos bearing retinoid receptor null mutations demonstrated that these receptors transduce the effects of retinoic acid (RA, the active derivative of vitamin A) in vivo, and revealed impressive complexity. However, frequent redundancy in receptor functions and lethality of compound RAR-null mutants, as well as of RXRα-null mutants, precluded the characterisation of the functions of these receptors during late development and postnatally. We illustrate here how recent developments of conditional targeted somatic mutagenesis have opened new avenues in analysing the physiological functions of retinoid X receptor signalling in a variety of tissues and cell types, as well as in exploring the pathophysiological consequences of their alteration that led to novel mouse models of human diseases. [ABSTRACT FROM AUTHOR]

The G-protein-coupled receptor signaling system, consisting of a huge variety of receptors as well as of many G-proteins and effectors, operates in every cell and is involved in many physiological and pathological processes. The versatility of this system and the involvement of specific components makes G-protein-coupled receptors and their signaling pathways ideal targets for pharmacological interventions. Classical mouse knockout models have often provided important preliminary insights into the biological roles of individual receptors and signaling pathways and they are routinely used in the process of target validation. The recent development of efficient conditional mutagenesis techniques now allows a much more detailed analysis of G-protein-mediated signaling transduction processes. This review summarizes some of the areas in which progress has recently been made by applying conditional mutagenesis of genes coding for G-proteins and G-protein-coupled receptors. [ABSTRACT FROM AUTHOR]

Ca2+ influx through various ion channels is an important determinant of the cytosolic Ca2+ concentration, which plays a pivotal role in countless cellular processes. The cardiac L-type Ca2+ channel, Cav1.2, represents a major pathway for Ca2+ entry and is in many cells expressed together with other high- and low-voltage-activated Ca2+ channels. This article will focus on the use of conditional transgenic mouse models to clarify the roles of Ca2+ channels in several biological systems. The phenotypes of conditional Ca2+ channel transgenic mice have provided novel, and often unexpected, insights into the in vivo function of L-type and T-type Ca2+ channels as mediators of signaling between cell membrane and intracellular processes in blood pressure regulation, smooth muscle contractility, insulin secretion, cardiac function, sleep, learning, and memory. [ABSTRACT FROM AUTHOR]

This chapter will review conditional mouse model systems that have been developed to study gene function in skeletal, cardiac, and vascular smooth muscle cells in vivo with an emphasis on the utility of these models for investigating developmental and pathophysiological gene function in muscle. In general, these systems have utilized muscle-specific/selective promoter-enhancers in conjunction with site-specific DNA recombinases, e.g., Cre-loxP, and fusion proteins with these recombinases that confer temporal control, such as tamoxifen-inducible CreER systems. A major focus of this chapter will be to discuss unique challenges of studying Cre-mediated mutagenesis/gene targeting in these muscle types during development and in the adult animal, some of which are inherent of the muscle cell type being studied. For example, unlike cardiac and skeletal muscle cells, the vascular SMC is extremely plastic and able to undergo rapid phenotypic modulation to various environmental cues in vivo. Thus, employing SMC marker gene promoter enhancers for conditional gene targeting in SMCs must take into account the possibility and/or certainty that the particular SMC promoter enhancers used may or may not be transcriptionally active in SMCs of a vessel wall under normal and some pathophysiological conditions. Moreover, individual floxed loci within the same muscle cell type and tissue have different degrees of sensitivity to Cre, most likely dependent on the chromatin state of that particular gene, i.e., closed/condensed state or open/active state. Thus, Cre recombination may be ineffective for specific floxed gene DNA. Lastly, rigorous efforts must be made to confirm the degree of recombination in a tissue, taking into full account the multicellularity of the tissue, to understand the extent of the physiological effect in that organ. [ABSTRACT FROM AUTHOR]

The advent of technologies that allow conditional mutagenesis has revolutionized our ability to explore gene functions and to establish animal models of human diseases. Both aspects have proven to be of particular importance in the study of lipid-related disorders. Classical approaches to gene inactivation by conventional gene targeting strategies have been successfully applied to generate animal models like the LDL receptor- and the apolipoprotein E-knockout mice, which are still widely used to study diverse aspects of atherosclerosis, lipid transport, and neurodegenerative disease. In many cases, however, simply inactivating the gene of interest has resulted in early lethal or complex phenotypes which are difficult to interpret. In recent years, additional tools have therefore been developed that allow the spatiotemporally controlled manipulation of the genome, as described in detail in Part I of this volume. Our aim is to provide an exemplary survey of the application of different conditional mutagenesis techniques in lipid research in order to illustrate their potential to unravel physiological functions of a broad range of genes involved in lipid homeostasis. [ABSTRACT FROM AUTHOR]

Conditional systems have proven to be efficient and powerful to delineate several aspects of cardiac pathophysiology and diseases. The possibility of addressing a particular time point in animal life is certainly an important breakthrough allowed by conditional strategies with temporal control of either transgene expression or gene modifications. The purpose of this review is to present various mouse models for cardiovascular diseases based on conditional approaches. [ABSTRACT FROM AUTHOR]

Friedreich ataxia (FRDA), the most common recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts and cardiomyopathy. It is caused by severely reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biosynthesis. Mouse models have been important tools in dissecting the steps of pathogenesis in FRDA. Furthermore, animal models that reproduce some of the key events in a pathology are essential for the development of effective therapies, both pharmacological and gene therapy approaches. This chapter presents an overview of the current mouse models that have been developed for FRDA. [ABSTRACT FROM AUTHOR]

Here we demonstrate the feasibility of a doubly regulatable transgenic mouse design that allows for gene manipulation by both Cre-recombinase and the tetracycline inducible system. Using a knock-in strategy to insert both elements of the tetracycline inducible system and a neomycin (neo) cassette flanked by loxP sequences (floxed) into the wild-type locus, we generated mice that express the 5-HT1B receptor in a conditional manner. In the presence of a floxed neo-cassette, receptor expression was silenced. Removal of this cassette by Cre-mediated recombination led to 5-HT1B receptor expression, which was highly regulatable when doxycycline, a derivative of tetracycline, was administered to the mice. This system allowed for a determination of an in vivo time course of receptor half-life and recovery. Physiological studies also demonstrated that rescued 5-HT1B receptors were functional, and that this functionality was reversible upon treatment with doxycycline. Crossing mice where the 5-HT1B, or the 5-HT1A, receptors were silenced by the neo-cassette, with mice expressing either Cre-recombinase or the tetracycline transactivator (tTA) under the control of tissue-specific promoters, led to tissue-specific re-expression of these receptors. Our studies thus demonstrate the potential of this strategy for achieving both a classic knockout, as well as subsequent tissue-specific and/or inducible knockouts. [ABSTRACT FROM AUTHOR]

In the postgenomic era, a primary focus of mouse genetics is to elucidate the role of individual genes in vivo. However, in the nervous system, studying the contribution of specific genes to brain functions is difficult because the brain is a highly complex organ with multiple neuroanatomical structures, orchestrating virtually every function in the body. Further, higher-order brain functions such as learning and memory simultaneously recruit several signaling cascades in different subcellular compartments and have highly fine-tuned spatial and temporal components. Conditional transgenic and gene targeting methodologies, however, now offer valuable tools with improved spatial and temporal resolution for appropriate studies of these functions. This chapter provides an overview of these tools and describes how they have helped gain better understanding of the role of candidate genes such as the NMDA receptor, the protein kinase CaMKIIα, the protein phosphatases calcineurin and PP1, or the transcription factor CREB, in the processes of learning and memory. This review illustrates the broad and innovative applicability of these methodologies to the study of brain plasticity and cognitive functions. [ABSTRACT FROM AUTHOR]

Evolutionarily conserved, widely expressed genes provide the functional backbone of most, if not all, cell types. Although mouse mutants created by germ line gene inactivation are instrumental in establishing the importance of such genes in vivo, distortion of embryonic development or multiple body systems often preclude detailed functional studies. To overcome this limitation, DNA recombination systems such as Cre/loxP of bacteriophage P1, have been adapted for use in mammalian cells. The mutagenic event is restricted to the tissue or cell type in question leaving other body systems undisturbed. Conditional inactivation of Csk or Socs3, for example, established their key role in the prevention of inappropriate inflammation, while unexpected immunoregulatory activities emerged from studies of the NF-κB and AP-1 pathways. Also, cell types responsible for protective or pathogenic TNFα production have been identified. Inactivation of immunoregulatory receptors in leukocyte subsets can provide robust experimental systems revealing the conceptual simplicity underlying the modulation of complex signaling pathways during homeostatic responses. As illustrated for TGF-β receptor, such system-guided approaches can provide a comprehensive picture of the regulatory events driving in vivo phenotype and specific responses of primary cells. This in turn facilitates the identification of novel regulatory mechanisms, targets for therapeutic intervention and prediction of side effects. With the increasing evidence for a role of somatic mutations in a wider range of human diseases, conditional mouse models are set to play a continuing part in the identification of pathogenic mechanisms for restoration of normal cellular processes in diseases including cancer, inflammation and autoimmunity. [ABSTRACT FROM AUTHOR]

The development of inducible and conditional technologies allowed us to generate transgenic mouse models that faithfully recapitulate human tumorigenesis. It is possible to control, in time and space, the development of tumors in almost every mouse tissue. The result is that now we have available mouse models for all major human cancers. Novel noninvasive approaches to tumor imaging will enable us to follow tumor development and metastasis in vivo, as well as the effects of candidate therapeutic drugs. Such new generation tumor models, which accurately emulate the disease state in situ, should provide a useful platform with which to experimentally test drugs targeted to specific gene products, or combinations of genes that control rate-limiting steps of tumor development. In this review, we focus on the different mouse models for colon cancer. [ABSTRACT FROM AUTHOR]

Explorations into the molecular embryology of the mouse have played a vital role in our understanding of the basic mechanisms of gene regulation that govern development and disease. In the last 15 years, these mechanisms have been analyzed with vastly greater precision and clarity with the advent of systems that allow the conditional control of gene expression. Typically, this control is achieved by silencing or activating the gene of interest with site-specific DNA recombination or transcriptional transactivation. In this review, I discuss the application of these technologies to mouse development, focusing on recent innovations and experimental designs that specifically aid the study of the mouse embryo. [ABSTRACT FROM AUTHOR]

The combination of two powerful technologies, the Cre/loxP recombination system and the protein transduction technique, holds great promise for the advancement of biomedical and genome research by enabling precise and rapid control over mutation events. Protein transduction is a recently developed technology to deliver biologically active proteins directly into mammalian cells. It involves the generation of fusion proteins consisting of the cargo molecule to be delivered and a so-called protein transduction domain. Recently, the derivation of cell permeable variants of the DNA recombinase Cre has been reported. Cre is a site-specific recombinase that recognizes 34 base pair loxP sites and has been widely used to genetically engineer mammalian cells in vitro and in vivo. Recombinant cell-permeable Cre recombinase was found to efficiently induce recombination of loxP-modified alleles in various mammalian cell lines. Here we review recent advances in conditional expression and mutagenesis employing cell-permeable Cre proteins. Moreover, this review summarizes recent findings of studies aimed at deciphering the molecular mechanism of the cellular uptake of cell-permeable fusion proteins. [ABSTRACT FROM AUTHOR]

Viruses are intracellular parasites with simple DNA or RNA genomes. Virus life revolves around three steps: infection of a host cell, replication of its genome within the host cell environment, and formation of new virions; this process is often but not always associated with pathogenic effects against the host organism. Since the mid-1980s, the main goal of viral vectorology has been to develop recombinant viral vectors for long-term gene delivery to mammalian cells, with minimal associated toxicity. Today, several viral vector systems are close to achieving this aim, providing stable transgenic expression in many different cell types and tissues. Here we review application characteristics of four vector systems, derived from adeno-associated viruses, adenoviruses, retroviruses and herpes simplex virus-1, for in vivo gene delivery. We discuss the transfer capacity of the expression vectors, the stability of their transgenic expression, the tropism of the recombinant viruses, the likelihood of induction of immunotoxicity, and the ease (or difficulty) of the virus production. In the end, we discuss applications of these vectors for delivery of three molecular systems for conditional mutagenesis, two for inducible transcriptional control of transgenic expression (the tet and the dimerizer systems), and the third one for inducible control of endogenous gene expression based on RNA interference. [ABSTRACT FROM AUTHOR]

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for functional genomics in mammalian cells. Furthermore, RNAi holds promise as a simple, fast and cost-effective approach to studying mammalian gene function in vivo and as a novel therapeutic approach. This review provides an overview of the progress of RNAi in vivo, with emphasis on systemic/local siRNA delivery, viral shRNA vectors, shRNA vector transgenic mice and conditional systems to control shRNA vectors. Taken together, the data from 80 in vivo studies show that RNAi is a useful tool that offers new opportunities for functional genomics in mice. [ABSTRACT FROM AUTHOR]

Our ability to genetically manipulate the mouse has had a great impact on medical research over the last few decades. Mouse genetics has developed into a powerful tool for dissecting the genetic causes of human disease and identifying potential targets for pharmaceutical intervention. With the recent sequencing of the human and mouse genomes, a large number of novel genes have been identified whose function in normal and disease physiology remains largely unknown. Government-sponsored multinational efforts are underway to analyze the function of all mouse genes through mutagenesis and phenotyping, making the mouse the interpreter of the human genome. A number of technologies are available for the generation of mutant mice, including gene targeting, gene trapping and transposon, chemical or radiation-induced mutagenesis. In this chapter, we review the current status of gene trapping technology, including its applicability to conditional mutagenesis. [ABSTRACT FROM AUTHOR]

Murine embryonic stem (ES) cells have become an indispensable tool for investigating genetic function both in vitro and, importantly, in vivo. Recent advances, including tetraploid aggregation, new site-specific recombinases and RNAi, have enabled more sophisticated manipulation of the ES cell genome. For instance, it is now possible to control gene expression in both a temporally and spatially restricted manner. Such new technologies are answering complex questions surrounding the function and interaction of an increasing number of genes. This chapter will review both the history and recent technological progress that has been made in mouse ES cell derivation, genetic manipulation and the generation of ES cell-derived chimaeric animals. [ABSTRACT FROM AUTHOR]

Controlling gene activity in space and time represents a cornerstone technology in gene and cell therapeutic applications, bioengineering, drug discovery as well as fundamental and applied research. This chapter provides a comprehensive overview of the different approaches for regulating gene activity and product protein formation at different biosynthetic levels, from genomic rearrangements over transcription and translation control to strategies for engineering inducible secretion and protein activity with a focus on the development during the past 2 years. Recent advances in designing second-generation gene switches, based on novel inducer administration routes (gas phase) as well as on the combination of heterologous switches with endogenous signals, will be complemented by an overview of the emerging field of mammalian synthetic biology, which enables the design of complex synthetic and semisynthetic gene networks. This article will conclude with an overview of how the different gene switches have been applied in gene therapy studies, bioengineering and drug discovery. [ABSTRACT FROM AUTHOR]

Unlike recombinase-mediated gene manipulations, tetracycline (Tet)-controlled genetic switches permit reversible control of gene expression in the mouse. Trancriptional activation can be induced by activators termed tTA (Tet-Off) or rtTA (Tet-On) in the absence and presence of Tet, respectively. The Tet-Off and Tet-On systems are complementary, and the decision to choose one over the other depends on the particular experimental strategy. Both systems were optimized over the years and can now be used to develop mouse models. [ABSTRACT FROM AUTHOR]

Together with numerous other genome modifications, chromosome engineering offers a very powerful tool to accelerate the functional analysis of the mammalian genome. The technology, based on the Cre/loxP system, is used more and more in the scientific community in order to generate new chromosomes carrying deletions, duplications, inversions and translocations in targeted regions of interest. In this review, we will present the basic principle of the technique either in vivo or in vitro and we will briefly describe some applications to provide highly valuable genetic tools, to decipher the mammalian genome organisation and to analyze human diseases in the mouse. [ABSTRACT FROM AUTHOR]

In the last decade, site-specific recombinases (SSRs), such as Cre and Flp, have emerged as indispensable tools for the precise in vivo manipulation of the mouse genome. It is now feasible to control, in space and time, the onset of gene knockouts in almost any tissue of the mouse, thus greatly facilitating the creation of sophisticated animal models for human disease and drug development. This review describes the basic principles and current status of the SSR technology, with a focus on strategies for conditional somatic mutagenesis using the Cre/lox system and ligand-activated Cre recombinases. Practical hints for generating and analysing conditional mouse mutants will be given and exciting novel applications of the SSR technology will be discussed, such as cell fate mapping and the combined use of Cre, Flp and other biotechnological tools. It will be shown how genetic manipulation of the mouse by site-specific recombination can provide new solutions to old problems in the analysis of human physiology and pathophysiology and how it can be employed for drug discovery and development. [ABSTRACT FROM AUTHOR]