Conditional Mutagenesis Research Articles

Mouse mutants and phenotypes: Accessing information for the study of mammalian gene function

Recent advances in high-throughput gene targeting and conditional mutagenesis are creating new and powerful resources to study the in vivo function of mammalian genes using the mouse as an experimental model. Mutant ES cells and mice are being generated at a rapid rate to study the molecular and phenotypic consequences of genetic mutations, and to correlate these study results with human disease conditions. Likewise, classical genetics approaches to identify mutations in the mouse genome that cause specific phenotypes have become more effective. Here, we describe methods to quickly obtain information on what mutant ES cells and mice are available, including recombinase driver lines for the generation of conditional mutants. Further, we describe means to access genetic and phenotypic data that identify mouse models for specific human diseases.

O31. Genetic analysis of Hippo signaling in mammalian organ size regulation and tumorigenesis

The Hippo signaling pathway was identified through genetic screens in Drosophila that were designed to identify novel genes involved in organ size control. In flies, mutations in core components of the Hippo signaling pathway, including the serine-threonine kinases Hippo and Warts lead to tissue overgrowth, in part by enhancing proliferation and inhibiting apoptosis. Direct orthologs of these and other core Hippo signaling components are found in mammals and they have been shown to function in an analogous kinase cascade to inhibit the activity of Yap and Taz, transcriptional co-activators orthologous to Drosophila yorkie. Despite conservation at the biochemical level, little is known about the requirements for core Hippo signaling pathway components in mammalian growth control. Here we describe our recent studies that employ conditional mutagenesis to delineate essential requirements for core Hippo signaling components in mammalian organ size regulation and in tumor suppression.

A mouse tool for conditional mutagenesis in ovarian granulosa cells

Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER(T)² fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1-iCreER(T)² transgenic mice express the iCreER(T)² fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with "floxed" Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1-iCreER(T)² mice using a Cre reporter line demonstrated that Cre-mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1-iCreER(T)² mice should be a useful tool to analyze the gene functions in ovarian granulosa cells.

Meningioma mouse models

Meningiomas, although mostly benign, may sometimes present aggressive features and raise issues concerning alternative treatment options besides surgery. In order to gain new insights in meningioma biology and develop alternative treatments, several meningioma mouse models have been engineered during the past two decades. As rodents very rarely develop spontaneous meningiomas, animal models have been first developed by implanting human meningioma cells derived from a primary tumor and meningioma cell lines subcutaneously into athymic mice. Induction of de novo meningiomas in rodents with mutagens, such as nitrosourea, has also been reported. Advances in our understanding of molecular genetics of meningioma have pinpointed the central role of NF2 tumor suppressor gene in the pathogenesis of those tumors. These discoveries have led to the creation of a genetically engineered model utilizing conditional mutagenesis to specifically inactivate the mouse Nf2 gene in arachnoidal cells, resulting in the formation of intracranial meningothelial hyperplasia and meningiomas and thus reproducing the main mechanism of human meningeal tumorigenesis. This powerful new technology significantly improves on prior models and may open avenues of investigation never before possible in meningioma research. We present here a review of current meningioma mouse models used in translational therapeutics with associated imaging and pre-clinical studies.

MLL2 Is Required in Oocytes for Bulk Histone 3 Lysine 4 Trimethylation and Transcriptional Silencing

Author SummaryIt is well established that gametes and early mammalian embryos undergo extensive epigenetic changes, which are changes in phenotype or gene expression that do not entail changes in DNA sequence. However, the machinery responsible for epigenetic modification in these situations is poorly understood. In mice, we conditionally deleted the histone 3 lysine 4 (H3K4) methyltransferase Mll2, an enzyme that alters DNA structure and packaging, either in gametes or in somatic cells of the ovary and also produced a mouse hypomorph expressing low levels of MLL2. We show that MLL2 is required in oocytes during gametogenesis and is also needed as a maternally derived factor during early development. Oocytes deficient in Mll2 display decreased methylation of H3K4 (H3K4me3) and show abnormal maturation and gene expression, in particular of pro-apoptotic factors. In addition, we demonstrate that embryonic genome activation is compromised in the absence of Mll2. Together our results identify MLL2 as one of the key players in the epigenetic reprogramming required for female fertility in the mouse.

ROSA26Flpo deleter mice promote efficient inversion of conditional gene traps in vivo

Gene trap mutagenesis in embryonic stem cells is an important tool to help elucidate gene function in current mouse mutagenesis efforts. Vector systems based on inversion of the gene trap module have recently been devised to allow for conditional mutagenesis. However, additional efforts are needed to improve this technology including improving the efficiency of site-specific recombinases required to manipulate these conditional vectors in vivo. Here we describe a mouse line carrying the codon-optimized FLP recombinase Flpo at the ROSA26 locus that functions at higher efficiency than a similar Flpe line in mediating the DNA inversion of a conditional gene trap cassette in vivo.

Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage

Mutations in the transcription factor SOX10 cause neurocristopathies, including Waardenburg-Hirschsprung syndrome and peripheral neuropathies in humans. This is partly attributed to a requirement for Sox10 in early neural crest for survival, maintenance of pluripotency, and specification to several cell lineages, including peripheral glia. As a consequence, peripheral glia are absent in Sox10-deficient mice. Intriguingly, Sox10 continues to be expressed in these cells after specification. To analyze glial functions after specification, we specifically deleted Sox10 in immature Schwann cells by conditional mutagenesis. Mutant mice died from peripheral neuropathy before the seventh postnatal week. Nerve alterations included a thinned perineurial sheath, increased lipid and collagen deposition, and a dramatically altered cellular composition. Nerve conduction was also grossly aberrant, and neither myelinating nor nonmyelinating Schwann cells formed. Instead, axons of different sizes remained unsorted in large bundles. Schwann cells failed to develop beyond the immature stage and were unable to maintain identity. Thus, our study identifies a novel cause for peripheral neuropathies in patients with SOX10 mutations.

Abstract 4182: A screen for genes that define the strong gender bias nature of hepatocellular carcinoma using a transposon mutagenesis murine model

Abstract Introduction: Hepatocellular carcinoma (HCC) is a deadly disease where many of the underlying associated genetic mechanisms remain undiscovered. Although HCC has been relatively rare in the US, the growing number of patients with cirrhosis (due to hepatitis infection and/or obesity-related fatty liver) has accounted for a surge in the incidence of HCC. Using the conditional Sleeping Beauty (SB) transposon somatic insertional mutagenesis system in transgenic mice, a novel unbiased HCC genetic screen was performed to elucidate the important genes responsible for this disease1. Methods: Triple transgenic (containing the conditional SB transposase, transposon mutagenic vector and liver-specific Cre transgenes) and quadruple transgenic (as above plus a conditional dominant negative p53 allele) mice were generated and aged for tumorigenesis1. Results: Using this system combined with a conditional p53-deficient background, we were able to identify many HCC cancer candidate genes. In addition, liver tumor nodules were also isolated from experimental mice without a predisposed genetic background. Liver tumors were prevalent in male experimental mice, mimicking the strong gender bias in the incidence of HCC seen in humans. Liver tumors were isolated from female experimental mice with and without a p53-deficient background, only after a prolonged latency (>432-days). From wild-type female liver tumors, we identified 17 candidate genes including the Pten tumor suppressor gene. From p53-deficient female liver tumors, we identified 6 candidate genes including some genes that are implicated in the miRNA18a pathway. This miRNA pathway has been associated with increased incidence of HCC in female patients. Conclusion: SB system allows for the identification of putative genes involved in HCC development in female mice. Finally, our ultimate goal is that candidate genes will be useful in generating possible therapeutic agents for the treatment of HCC and increase our knowledge in defining the underlying genetic mechanisms associated with the strong gender bias nature of this deadly disease. 1. Keng, V.W., et al. (2009). A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma. Nat Biotechnol. 27, 264-74. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4182.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of β-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1lacZ (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1−/− mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures.

Novel aspects of the roles of Rac1 GTPase in the cardiovascular system

Rac1 GTPase is an established master regulator of cell motility through cortical actin re-organization and of reactive oxygen species generation through regulation of NADPH oxidase activity. Numerous molecular and cellular studies have implicated Rac1 in various cardiovascular pathologies: vascular smooth muscle proliferation, cardiomyocyte hypertrophy, and endothelial cell shape change. The physiological relevance of these in vitro findings, however, is just beginning to be reassessed with the newly developed, conditional mouse mutagenesis technology. Conditional gene targeting has also revealed unexpected, cell type-specific roles of Rac1. The aim of this review is to summarize the recent advance made in Rac1 research in the cardiovascular system, with special focus on its novel roles in the regulation of endothelial function, angiogenesis, and endothelium-mediated neuroprotection.

Control of Cognition and Adaptive Behavior by the GLP/G9a Epigenetic Suppressor Complex

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

Role of STAT5 in controlling cell survival and immunoglobulin gene recombination during pro-B cell development

STAT5 and interleukin 7 (IL-7) signaling are thought to control B lymphopoiesis by regulating the expression of key transcription factors and by activating variable (V(H)) gene segments at the immunoglobulin heavy-chain (Igh) locus. Using conditional mutagenesis to delete the gene encoding the transcription factor STAT5, we demonstrate that the development of pro-B cells was restored by transgenic expression of the prosurvival protein Bcl-2, which compensated for loss of the antiapoptotic protein Mcl-1. Expression of the genes encoding the B cell-specification factor EBF1 and the B cell-commitment protein Pax5 as well as V(H) gene recombination were normal in STAT5- or IL-7 receptor alpha-chain (IL-7Ralpha)-deficient pro-B cells rescued by Bcl-2. STAT5-expressing pro-B cells contained little or no active chromatin at most V(H) genes. In contrast, rearrangements of the immunoglobulin-kappa light-chain locus (Igk) were more abundant in STAT5- or IL-7Ralpha-deficient pro-B cells. Hence, STAT5 and IL-7 signaling control cell survival and the developmental ordering of immunoglobulin gene rearrangements by suppressing premature Igk recombination in pro-B cells.

Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development

The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4 EGFPCre knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26 LacZ reporter with the Wnt4 EGFPCre allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4 EGFPCre -activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26 YFP -positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4 EGFPCre ; Rosa26 LacZ -marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4 EGFPCre allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells.

The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mutant myoblasts correlates with a deficit in the recruitment of actin fibers and vinculin to myoblast contact sites. Comparison of the changes observed in mutant myogenic cells indicates that Rac1 and Cdc42 function in a nonredundant and not completely overlapping manner during the fusion process. Our genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts.

Generation of a mouse line expressing Sox17‐driven Cre recombinase with specific activity in arteries

The HMG-box transcription factor Sox17 has been shown to play important roles in both endoderm formation and cardiovascular development. To conditionally inactivate genes in these domains, we have targeted a codon improved Cre Recombinase (iCre) into exon 1 of the Sox17 gene. Surprisingly, Cre-mediated recombination in the Rosa26 reporter mouse line revealed largely specific activity within the vasculature rather than in endoderm-derived tissues. Here we report a new Cre knock-in mouse line, Sox17(iCre) with activity in the vascular endothelial cells of arteries in the cardiovascular system but not in veins. Cre-mediated recombination was also strongly detected in the liver and spleen, the two organs associated with hematopoiesis. Thus, these results indicate that the Sox17(iCre) would be an appropriate tool for conditional mutagenesis of genes in the vasculature and could be used in studies of blood vessel development and angiogenesis. Additionally, we provide evidence that two different promoters drive Sox17 expression in the endodermal and vascular system.

Manual annotation of the mouse genome: HAVANA and EUCOMM

AbstractThe European Conditional Mouse Mutagenesis (EUCOMM) project aims to establish a mutant resource containing up to 13,000 conditional mouse mutations in C57BL/6N embryonic stem cells. As part of this resource, up to 8,000 targeted conditional mutations will be generated for genes that can not be readily trapped by random gene trapping methods. Non-symmetrical critical exons are identified as part of the targeting approach, which when deleted produce a knockout (KO) transcript that is susceptible to nonsensemediated decay (NMD). The success of this approach is dependent on detailed and accurate genome annotation, which is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute. Based on a combination of EST, cDNA and protein evidence, HAVANA produces high quality manual annotation of gene structures, that includes alternative splice variants, polyadenylation features, pseudogenes and gene family clusters. Using this annotation we are able to identify suitable exons for targeting, whilst minimising potential disruption to adjacent genomic features. Annotation of both gene and KO transcripts can be viewed through the Vertebrate Genome Annotation (VEGA) browser ("http://vega.sanger.ac.uk":http://vega.sanger.ac.uk).More information on the EUCOMM resource and the targeted genes is available at "http://www.eucomm.org":http://www.eucomm.org. Targeting vector design information can also be viewed through the Ensembl genome browser ("http://www.ensembl.org":http://www.ensembl.org) as DAS resources ("http://das.sanger.ac.uk/das/KO_designs":http://das.sanger.ac.uk/das/KO_designs ; "http://das.sanger.ac.uk/das/KO_vectors":http://das.sanger.ac.uk/das/KO_vectors). In addition to the EUCOMM project, HAVANA also contributes towards the mouse Consensus Coding DNA Sequence (CCDS) project and is involved in collaborations with RefSeq and MGI.

Manual annotation of the mouse genome: HAVANA and EUCOMM

The European Conditional Mouse Mutagenesis (EUCOMM) project aims to establish a mutant resource containing up to 13,000 conditional mouse mutations in C57BL/6N embryonic stem cells. As part of this resource, up to 8,000 targeted conditional mutations will be generated for genes that can not be readily trapped by random gene trapping methods. Non-symmetrical critical exons are identified as part of the targeting approach, which when deleted produce a knockout (KO) transcript that is susceptible to nonsensemediated decay (NMD). The success of this approach is dependent on detailed and accurate genome annotation, which is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute. Based on a combination of EST, cDNA and protein evidence, HAVANA produces high quality manual annotation of gene structures, that includes alternative splice variants, polyadenylation features, pseudogenes and gene family clusters. Using this annotation we are able to identify suitable exons for targeting, whilst minimising potential disruption to adjacent genomic features. Annotation of both gene and KO transcripts can be viewed through the Vertebrate Genome Annotation (VEGA) browser ("http://vega.sanger.ac.uk":http://vega.sanger.ac.uk).More information on the EUCOMM resource and the targeted genes is available at "http://www.eucomm.org":http://www.eucomm.org. Targeting vector design information can also be viewed through the Ensembl genome browser ("http://www.ensembl.org":http://www.ensembl.org) as DAS resources ("http://das.sanger.ac.uk/das/KO_designs":http://das.sanger.ac.uk/das/KO_designs ; "http://das.sanger.ac.uk/das/KO_vectors":http://das.sanger.ac.uk/das/KO_vectors). In addition to the EUCOMM project, HAVANA also contributes towards the mouse Consensus Coding DNA Sequence (CCDS) project and is involved in collaborations with RefSeq and MGI.

Clonal Conditional Mutagenesis in Malaria Parasites

We describe here an efficient method for conditional gene inactivation in malaria parasites that uses the Flp/FRT site-specific recombination system of yeast. The method, developed in Plasmodium berghei, consists of inserting FRT sites in the chromosomal locus of interest in a parasite clone expressing the Flp recombinase via a developmental stage-specific promoter. Using promoters active in mosquito midgut sporozoites or salivary gland sporozoites to drive expression of Flp or its thermolabile variant, FlpL, we show that excision of the DNA flanked by FRT sites occurs efficiently at the stage of interest and at undetectable levels in prior stages. We applied this technique to conditionally silence MSP1, a gene essential for merozoite invasion of erythrocytes. Silencing MSP1 in sporozoites impaired subsequent merozoite formation in the liver. Therefore, MSP1 plays a dual role in the parasite life cycle, acting both in liver and erythrocytic parasite stages.

A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma

We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)-associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC. These encode genes, such as EGFR and MET, previously associated with HCC and others, such as UBE2H, that are potential new targets for treating this neoplasm. Our system, which could be modified to drive transposon-based insertional mutagenesis wherever tissue-specific Cre expression is possible, promises to enhance understanding of cancer genomes and identify new targets for therapeutic development.

Developmental Signaling in Myocardial Progenitor Cells: A Comprehensive View of Bmp- and Wnt/β-Catenin Signaling

The tight regulation of different signaling systems and the transcriptional and translational networks during embryonic development have been the focus of embryologists in recent decades. Defective developmental signaling due to genetic mutation or temporal and region-specific alteration of gene expression causes embryonic lethality or accounts for birth defects (e.g., congenital heart disease). The formation of the heart requires the coordinated integration of multiple cardiac progenitor cell populations derived from the first and second heart fields and from cardiac neural crest cells. This article summarizes what has been learned from conditional mutagenesis of Bmp pathway components and the Wnt effector, beta-catenin, in the developing heart of mice. Although Bmp signaling is required for cardiac progenitor cell specification, proliferation, and differentiation, recent studies have demonstrated distinct functions of Wnt/beta-catenin signaling at various stages of heart development.