Recent advances in inducible expression in transgenic mice
Introduction
Before the pioneering work of Palmiter, Brinster, and coworkers1., 2. which resulted in the capacity to genetically modify mice (and ultimately other mammals), scientists had to rely on naturally occurring or artificially induced, heritable genetic traits in order to study the genes involved in physiologically relevant processes. The ability to modify the mouse genome and to either introduce, modify or ablate a specific gene in vivo has become a powerful tool in understanding the role(s) of specific gene products in normal growth and development of cells and tissues and has allowed for the establishment of mouse models of human diseases, including neurological syndromes, metabolic disorders, and cancer. The utility of transgenic models to examine the function of a target gene is limited, however, by several practical considerations. The establishment of mice which harbor constitutively over expressed gene products or mutated or ablated alleles during development, while informative, share the risk of exhibiting embryonic lethality, loss of precursor cell populations, infertility and/or systemic compensation for the genetic abnormality. Furthermore, many ES cell-derived knockout mice fail to develop normally.
Genetically altered mice frequently exhibit a spectrum of phenotypes, ranging from the expected to strain-dependent alterations in gene function3 and the differentiated function of many tissues is often compromised. As an example, mammary gland development is impaired4., 5. in mice deleted of such divergent genes as the progesterone receptor,6 estrogen receptor α,7 Stat5a and Stat5b,8., 9. Wnt-4,10 LEF1,11 prolactin12., 13. or prolactin receptor,14 Id2,15 the EGF receptor,16 A-Myb,17 C/EBPβ,18., 19. the osteoprotegerin-ligand20 and cyclin D1.21., 22. The developmental defects (including retarded growth, retinopathy and failure of mammary gland development) following ablation of the cyclin D1 gene, for example, are profound.21., 22. The abnormal mammary gland phenotype in cyclin D1 deficient mice was discernible by early pregnancy and included a redistribution of progesterone receptor A and B ratios, a severe reduction in Stat5 phosphorylation and attenuated alveolar development, leading to a near complete failure in lactation. These observations culminated in the conclusion that cyclin D1 is absolutely crucial for mammary gland growth and differentiation.23 In recent experiments,24 cyclin D1−/− mice failed to develop mammary adenocarcinomas when mated to transgenic mice predisposed to mammary gland adenocarcinoma (through over-expression of the Neu or Ras oncogenes). These studies are consistent with the experiments in which cyclin D1 antisense reduced ErbB-2 induced mammary tumor growth.25 The failure of the cyclin D1−/− mice to undergo critical aspects of mammary gland development, as a direct or indirect result of the loss of the cyclin D1 gene, raises concerns about the interpretation of these data. Developing a mouse model in which cyclin D1 function could be reduced or ablated before, during or after the onset of cancer in otherwise normal adult mammary tissue may provide more definitive information.
To overcome these limitations, systems have been engineered which inducibly regulate the transgene of interest or excise the targeted gene in the tissue of choice. Such events can be temporally and spatially controlled during growth and development as well as at any point during the lifespan of the animal. The characteristics of an ideal inducible transgenic system must include low basal level expression and robust induction of the transgene, the lack of secondary or deleterious effects of the inducing agent, tissue specific targeting and the ability to sustain transgene induction. These characteristics are particularly important in the delivery of embryonically lethal, transforming or otherwise toxic genes. For example, in loxP containing mice, the inopportune expression of Cre during either embryogenesis and/or development could result in a complex mosaic genetic architecture, complicating any analyses. With potent oncogenes, such as Myc, which exhibits a wide range of biologic effects,26 the ability to control both the temporal expression profile and the activity of the gene is critical. Binary systems are used to address these issues, where one mouse line contains an activator of expression under the control of a tissue or developmentally regulated promoter and a second mouse line contains the silenced gene of interest. Importantly, postnatal genetic manipulation in a defined tissue at a specific time is more likely to recapitulate either the developmental or oncogenic mechanism(s) being investigated, therefore, providing a more informative model for the study of a specific genes normal or disregulated function.
The early transgenic lines relied on the administration of heavy metals or naturally occurring steroid hormones, such as glucocorticoids, to induce transgene activity.27 However, heavy metals are toxic and the glucocorticoids regulate endogenous genes, complicating interpretation. Inducible systems have been developed in which the ligand does not, theoretically, affect endogenous genes. The inducing agents include tetracycline, which is used in the tet operon system,28 IPTG, which is used in the Lac operon repressor system,29 the chemical inducer of dimerization, FK1012, which is used in the FKBP inducible system30 and ecdysone receptor (EcR) agonists, used in the EcR inducible system.31
We review here recent developments in the primary methodologies for inducible gene expression in transgenic mice (nuclear receptor fusion proteins, tetracycline regulation, ponasterone A induction and conditional Cre expression), with a major focus on their use in adult mice.
Section snippets
Conditional nuclear receptor fusion protein-inducible expression
Nuclear receptor fusion proteins have been used to regulate transgene activity. The chimeric transgene is comprised of the target transgenic protein fused to the ligand-binding domain (LBD) of steroid receptors, most commonly those for estrogen or progesterone. Administration of ligand results in the reversal of functional inhibition of the unliganded receptor-chimera and has been successfully applied to both the Cre- and Myc-oncogene systems (Table 1).32., 33., 34., 35., 36., 37., 38., 39., 40.
Conditional tetracycline-inducible expression
Transgenic approaches engineered to reversibly control target genes have used transactivators, such as the VP16 and Gal4/UAS activation system, driven by tissue-specific promoters.51., 52. The tetracycline-regulated system, developed by Dr Bujard in Germany,53., 54. provides excellent temporal and spatial control of transgene expression. In the absence of ligand, the potent tetracycline receptor (tetR) transactivator, tTA, is bound to a tetracycline operator sequence (tetO), which is placed
Conditional ponasterone A-inducible expression
The ecdysone-inducible expression system developed by Dr Evans at the Salk Institute31 has several characteristics that are well suited to inducible transgenics. Based on the EcR, for which there is no known mammalian homologue, the system is essentially heterologous and studies in mammalian cells have shown that the system has low basal activity in the absence of ligand and robust induction by ecdysteroids.31., 87., 88., 89., 90. Ecdysone is an insect molting hormone that is responsible for
Conditional expression of Cre
By placing the Cre-recombinase cassette, through ‘knock in’ technology, under the control of an endogenous, conditionally regulated gene promoter, accurate temporal and spatial delivery of Cre can be achieved. Cross breeding of conditional-Cre mice with floxed allele mice yields doubly transgenic mice capable of undergoing targeted in vivo genetic manipulation in those cells with sufficient Cre activity. Furthermore, Cre activity can be monitored by sequential breeding with a Cre-reporter mouse,
Discussion and future directions
Sophisticated molecular modeling strategies can now be developed, through the combination of multiple inducible transgenic systems, to more accurately recapitulate human diseases.
By combining different inducible transgenic systems, specific types of biological questions can be addressed and the technical limitations that exist in the current systems may be overcome (Figure 3). In principle, single inducible systems can be used to address issues of initiation versus progression of tumorigenesis (
Acknowledgements
We would like to thank Dr Eric Bouhassira for a critical reading of the manuscript. This work was supported by grants from NIH R03AG20337 (to C.A.) (RO1CA70896, RO1CA75503, RO1CA86072), the Pfeiffer Foundation, The Susan Komen Breast Cancer Foundation, (to R.G.P.) and CA536340 (J.M.H.). Work conducted at the Albert Einstein College of Medicine was supported by Cancer Center Core National Institute of Health grant 5-P30-CA13330-26.
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