Cre-mediated Gene Deletion Research Articles

Tamoxifen exerts direct and microglia-mediated effects preventing neuroinflammatory changes in the adult mouse hippocampal neurogenic niche.

Tamoxifen-inducible systems are widely used in research to control Cre-mediated gene deletion in genetically modified animals. Beyond Cre activation, tamoxifen also exerts off-target effects, whose consequences are still poorly addressed. Here, we investigated the impact of tamoxifen on lipopolysaccharide (LPS)-induced neuroinflammatory responses, focusing on the neurogenic activity in the adult mouse dentate gyrus. We demonstrated that a four-day LPS treatment led to an increase in microglia, astrocytes and radial glial cells with concomitant reduction of newborn neurons. These effects were counteracted by a two-day tamoxifen pre-treatment. Through selective microglia depletion, we elucidated that both LPS and tamoxifen influenced astrogliogenesis via microglia mediated mechanisms, while the effects on neurogenesis persisted even in a microglia-depleted environment. Notably, changes in radial glial cells resulted from a combination of microglia-dependent and -independent mechanisms. Overall, our data reveal that tamoxifen treatment per se does not alter the balance between adult neurogenesis and astrogliogenesis but does modulate cellular responses to inflammatory stimuli exerting a protective role within the adult hippocampal neurogenic niche.

Detecting Photoactivatable Cre-mediated Gene Deletion Efficiency in Escherichia coli.

Gene deletion is one of the standard approaches in genetics to investigate the roles and functions of target genes. However, the influence of gene deletion on cellular phenotypes is usually analyzed sometime after the gene deletion was introduced. Such lags from gene deletion to phenotype evaluation could select only the fittest fraction of gene-deleted cells and hinder the detection of potentially diverse phenotypic consequences. Therefore, dynamic aspects of gene deletion, such as real-time propagation and compensation of deletion effects on cellular phenotypes, still need to be explored. To resolve this issue, we have recently introduced a new method that combines a photoactivatable Cre recombination system and microfluidic single-cell observation. This method enables us to induce gene deletion at desired timings in single bacterial cells and to monitor their dynamics for prolonged periods. Here, we detail the protocol for estimating the fractions of gene-deleted cells based on a batch-culture assay. The duration of blue light exposure significantly affects the fractions of gene-deleted cells. Therefore, gene-deleted and non-deleted cells can coexist in a cellular population by adjusting the duration of blue light exposure. Single-cell observations under such illumination conditions allow the comparison of temporal dynamics between gene-deleted and non-deleted cells and unravel phenotypic dynamics provoked by gene deletion.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

Isolating cells from adult murine brain for validation of cell-type specific cre-mediated deletion

BackgroundTheCre/loxP system allows for the temporal and spatial investigation of the expression of a single gene in the nervous system. Current methods of validating conditional knock-out mouse models rely on heterogeneous brain tissue or primary culture. These methods may assess the extent of genetic knockdown in the brain but do not provide age-appropriate, cell-type specific information. New methodWe isolated specific cell types from adult murine brain using FACS to assess cell type-specific gene expression in conditional mouse models. ResultsWe identified robust but incomplete genetic knockdown in microglia isolated from two separate microglia-specific knockout models. Comparisonwith existing methods(s): Genetic knockdown in isolated adult microglia differed significantly from cultured primary microglia. ConclusionsDifferences observed in primary cultured microglia compared to isolated adult microglia suggest that current methods used to validate microglia-specific gene deletion over-estimate deletion efficiency. Assessment of gene expression in isolated adult microglia provides a more accurate assessment of Cre-mediated gene deletion.

Genetically engineered mouse models to evaluate the role of Wnt secretion in bone development and homeostasis.

Alterations in components of the Wnt signaling pathway are associated with altered bone development and homeostasis in several human diseases. We created genetically engineered mouse models (GEMMs) that mimic the cellular defect associated with the Porcupine mutations in patients with Goltz Syndrome/Focal Dermal Hypoplasia. These GEMMs were established by utilizing mice containing a conditionally inactivatable allele of Wntless/GPR177 (a gene encoding a protein required for the transport of Porcupine-modified ligand to the plasma membrane for secretion). We crossed this strain to another which drives cre-mediated gene deletion in mature osteoblasts (Osteocalcin-cre) resulted in mice lacking the ability to secrete Wnt ligands in this cell type. These mice displayed severely reduced bone mass and provide a model to understand the effects of disrupting the ability to secrete Wnt ligands on the skeletal system.

A versatile modular vector system for rapid combinatorial mammalian genetics.

Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases.

The PPARγ Agonist Efatutazone Increases the Spectrum of Well-Differentiated Mammary Cancer Subtypes Initiated by Loss of Full-Length BRCA1 in Association with TP53 Haploinsufficiency

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have anticancer activity and influence cell differentiation. We examined the impact of the selective PPARγ agonist efatutazone on mammary cancer pathogenesis in a mouse model of BRCA1 mutation. Mice with conditional loss of full-length BRCA1 targeted to mammary epithelial cells in association with germline TP53 insufficiency were treated with efatutazone through the diet starting at age 4 months and were euthanized at age 12months or when palpable tumor reached 1 cm(3). Although treatment did not reduce percentage of mice developing invasive cancer, it significantly reduced prevalence of noninvasive cancer and total number of cancers per mouse and increased prevalence of well-differentiated cancer subtypes not usually seen in this mouse model. Invasive cancers from controls were uniformly estrogen receptor α negative and undifferentiated, whereas well-differentiated estrogen receptor α-positive papillary invasive cancers appeared in efatutazone-treated mice. Expression levels of phosphorylated AKT and CDK6 were significantly reduced in the cancers developing in efatutazone-treated mice. Efatutazone treatment reduced rates of mammary epithelial cell proliferation and development of hyperplastic alveolar nodules and increased expression levels of the PPARγ target genes Adfp, Fabp4, and Pdhk4 in preneoplastic mammary tissue. Intervention efatutazone treatment in mice with BRCA1 deficiency altered mammary cancer development by promoting development of differentiated invasive cancer and reducing prevalence of noninvasive cancer and preneoplastic disease.

Cre transgene results in global attenuation of the cAMP/PKA pathway

Use of the cre transgene in in vivo mouse models to delete a specific ‘floxed' allele is a well-accepted method for studying the effects of spatially or temporarily regulated genes. During the course of our investigation into the effect of cyclic adenosine 3′,5′-monophosphate-dependent protein kinase A (PKA) expression on cell death, we found that cre expression either in cultured cell lines or in transgenic mice results in global changes in PKA target phosphorylation. This consequently alters gene expression profile and changes in cytokine secretion such as IL-6. These effects are dependent on its recombinase activity and can be attributed to the upregulation of specific inhibitors of PKA (PKI). These results may explain the cytotoxicity often associated with cre expression in many transgenic animals and may also explain many of the phenotypes observed in the context of Cre-mediated gene deletion. Our results may therefore influence the interpretation of data generated using the conventional cre transgenic system.

Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.

Apollo Contributes to G Overhang Maintenance and Protects Leading-End Telomeres

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response.

Fibulin-4 conducts proper elastogenesis via interaction with cross-linking enzyme lysyl oxidase

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.

Critical Role for Transcription Coactivator Peroxisome Proliferator-activated Receptor (PPAR)-binding Protein/TRAP220 in Liver Regeneration and PPARα Ligand-induced Liver Tumor Development

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (Pbp(DeltaLiv)) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G(2)/M phase in Pbp(DeltaLiv) hepatocytes after partial hepatectomy. Pbp(DeltaLiv) hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl(4)-induced hepatocellular proliferation and hepatotoxicity occurred in Pbp(DeltaLiv) mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl(4) activation. Pbp(DeltaLiv) mice, chronically exposed to Wy-14,643, a PPARalpha ligand, revealed a striking proliferative response and clonal expansion of a few Pbp(fl/fl) hepatocytes that escaped Cre-mediated gene deletion in Pbp(DeltaLiv) livers, but no proliferative expansion of PBP null hepatocytes was observed. In these Pbp(DeltaLiv) mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBP(DeltaLiv) hepatocytes; all liver tumors developing in Pbp(DeltaLiv) mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.

Targeted viral delivery of Cre recombinase induces conditional gene deletion in cardiovascular circuits of the mouse brain.

The Cre/loxP system has shown promise for investigating genes involved in nervous system function and pathology, although its application for studying central neural regulation of cardiovascular function and disease has not been explored. Here, we report for the first time that recombination of loxP-flanked genes can be achieved in discrete cardiovascular regulatory nuclei of adult mouse brain using targeted delivery of adenovirus (Ad) or feline immunodeficiency virus (FIV) bearing Cre recombinase (Ad-Cre, FIV-Cre). Single stereotaxic microinjections of Ad-Cre or FIV-Cre into specific nuclei along the subfornical organ-hypothalamic-hypophysial and brain stem-parabrachial axes resulted in robust and highly localized gene deletion as early as 7 days and for as long as 3 wk in a reporter mouse model in which Cre recombinase activates beta-galactosidase expression. An even greater selectivity in Cre-mediated gene deletion could be achieved in unique subpopulations of cells, such as vasopressin-synthesizing magnocellular neurons, by delivering Ad-Cre via retrograde transport. Moreover, Ad-Cre and FIV-Cre induced gene recombination in differential cell populations within these cardiovascular nuclei. FIV-Cre infection resulted in LacZ activation selectively in neurons, whereas both neuronal and glial cell types underwent gene recombination upon infection with Ad-Cre. These results establish the feasibility of using a combination of viral and Cre/loxP technologies to target specific cardiovascular nuclei in the brain for conditional gene modification and suggest the potential of this approach for determining the functional role of genes within these sites.

Cre-Mediated Cerebellum- and Hippocampus-Restricted Gene Mutation in Mouse Brain

Using the phage P1-drived Cre/loxP recombination system, we have created a line of cre-transgenic mice in which the Cre-mediated gene deletion is restricted to granule cells of cerebellum and dentate gyrus of hippocampus. Low levels of deletion were also present in pyramidal cells of hippocampal CA1 and CA3 fields. The Cre/loxP recombination occurred prenatally. The recombination efficiencies in the granular layer of the cerebellum, the granular layer of the dentate gyrus, and the CA1 and CA3 pyramidal cells of the hippocampus were 34.0%, 23.1%, 3.0%, and 9.8%, respectively. This line of cre-transgenic mice should be conducive to studies of the effect of a gene mutation upon brain development and plasticity.

Cre-mediated gene deletion in the mammary gland.

To delete genes specifically from mammary tissue using the Cre-lox system, we have established transgenic mice expressing Cre recombinase under control of the WAP gene promoter and the MMTV LTR. Cre activity in these mice was evaluated by three criteria. First, the tissue distribution of Cre mRNA was analyzed. Second, an adenovirus carrying a reporter gene was used to determine expression at the level of single cells. Third, tissue specificity of Cre activity was determined in a mouse strain carrying a reporter gene. In adult MMTV-Cre mice expression of the transgene was confined to striated ductal cells of the salivary gland and mammary epithelial cells in virgin and lactating mice. Expression of WAP-Cre was only detected in alveolar epithelial cells of mammary tissue during lactation. Analysis of transgenic mice carrying both the MMTV-Cre and the reporter transgenes revealed recombination in every tissue. In contrast, recombination mediated by Cre under control of the WAP gene promoter was largely restricted to the mammary gland but occasionally observed in the brain. These results show that transgenic mice with WAP-Cre but not MMTV-Cre can be used as a powerful tool to study gene function in development and tumorigenesis in the mammary gland.

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction.

The biosynthesis of complex asparagine (N)-linked oligosaccharides in vertebrates proceeds with the linkage of N-acetylglucosamine (GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:beta-D-mannoside beta 1-4 N-acetylglucosaminyltransferase III (GlcNAc-TIII, EC2.4.1.144) catalyzes the addition of GlcNAc to the mannose that is itself beta 1-4 linked to underlying N-acetylglucosamine. GlcNAc-TIII thereby produces what is known as a 'bisecting' GlcNAc linkage which is found on various hybrid and complex N-glycans. GlcNAc-TIII can also play a regulatory role in N-glycan biosynthesis as addition of the bisecting GlcNAc eliminates the potential for alpha-mannosidase-II, GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and core alpha 1-6-fucosyltransferase to act subsequently. To investigate the physiologic relevance of GlcNAc-TIII function and bisected N-glycans, the mouse gene encoding GlcNAc-TIII (Mgat3) was cloned, characterized, and inactivated using Cre/loxP site-directed recombination. The Mgat3 gene is highly conserved in comparison to the rat and human homologs and is normally expressed at high levels in mammalian brain and kidney tissues. Using fluorescence in situ hybridization (FISH), the Mgat3 gene was regionally mapped to chromosome 15E11, near the Scn8a sodium channel gene at 15F1. Following homologous recombination in embryonic stem cells and Cre mediated gene deletion, Mgat3-deficient mice were produced that lacked GlcNAc-TIII activity and were deficient in E4-PHA visualized GlcNAc-bisected N-linked oligosaccharides. Nevertheless, GlcNAc-TIII deficient mice were found to be viable and reproduced normally. Moreover, such mice exhibited normal cellularity and morphology among organs including brain and kidney. No alterations were apparent in circulating leukocytes, erythrocytes or in serum metabolite levels that reflect kidney function. We thus find that GlcNAc-TIII and the bisecting GlcNAc in N-glycans appear dispensable for normal development, homeostasis and reproduction in the mouse.