Modified Estrogen Receptor Research Articles

Optical control of protein activity and gene expression by photoactivation of caged cyclofen.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.

Control of Protein Activity and Gene Expression by Cyclofen-OH Uncaging.

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light-activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.

Generation of a Mouse Model with Smooth Muscle Cell Specific Loss of the Expression of PPARγ.

An inducible tissue-specific knockout (KO) technique has been used to study the role of genes in the adult heart. This KO technique circumvents the developmental effect that could otherwise be observed in a tissue-specific KO. The peroxisome proliferator-activated receptor (PPAR) γ is a transcription factor that when activated has been shown to improve vascular remodeling and endothelial function in hypertensive rodents. Here we describe an inducible tissue specific KO protocol used to study the role of PPARγ in smooth muscle cells (SMC) in angiotensin (Ang) II-induced hypertension in adult mice. Inducible VSMC Pparγ KO mice are generated by crossing mice expressing a fusion protein of Cre recombinase with the modified estrogen receptor ligand binding domain (CreERT2) under the control of the smooth muscle myosin heavy chain (smmhc, myh11) with mice having loxP sites flanking exon 2 of the Pparγ gene (Pparγ Flox/Flox ). The SMC Pparγ KO is induced by treating smMHC-CreERT2/Pparγ Flox/Flox mice with the estrogen receptor antagonist tamoxifen causing recombination of the two loxP site by CreERT2. SMC KO is confirmed by determining mRNA Pparγ levels in aortic media. Presence of the loxP sites is determined by sequencing genomic DNA. Tissue specific expression is assayed using smMHC-CreERT2/reporter crossed mice.

Abstract B01: An allelic series of deregulated c-Myc expression interrogates the oncogenicity of distinct Myc levels in lung adenocarcinoma

Abstract A plethora of transgenic mouse models have found that c-Myc induces tumors inefficiently and requires cooperating mutations. Acute activation of high levels of Myc induces cellular proliferation, tempered by intrinsic tumor suppression via apoptosis. We hypothesized that low, near physiological, levels of deregulated Myc that fail to invoke tumor suppression pathways, would more effectively initiate sporadic tumors. We previously described the Rosa26lslMycER (R26MER) allele in which deregulated c-Myc is expressed at an endogenous-like level following expression of Cre-recombinase. This c-Myc is fused to a modified estrogen receptor, such that Myc function is dependent upon administration of tamoxifen. We have now generated a Rosa26CAGlslMycER (R26(C)MER) allele, in which the addition of the CAG enhancer has increased the amount of MycER driven by this locus 10-fold. Genetic combination of these alleles generates for the first time mice with tightly controlled and defined levels of switchable Myc protein. Lung cancer is the leading cause of cancer death worldwide and is commonly associated with elevated Myc levels. To determine the role of deregulated Myc expression in lung adenocarcinoma, intranasally administered Adenoviral Cre-recombinase was used to induce sporadic c-MycER expression at different levels in lung epithelial cells, and tamoxifen diet was used to activate c-MycER function. Whereas acute low levels of Myc induced proliferation, high levels of Myc induced both proliferation and apoptosis. Contrary to our hypothesis that Myc-induced apoptosis would delay tumorigenesis, mice with high Myc expression exhibited shorter tumor latency and decreased survival compared to mice with lower levels of expression. The observation that acute Myc activation stimulates p53 activity suggests that the Arf-p53 pathway would be selected against during tumorigenesis. We observed that the hyperplastic clusters of cells observed following Myc activation in R26(C)MER mice were enhanced in mice lacking functional Arf protein. However at later time points their number gradually decreased and the clusters remained small, suggesting that further mutations are necessary for tumorigenesis. Of the tumors that develop following long-term Myc activation, 40% carried activating mutations in Kras. We therefore crossed our R26(C)MER mice to the KrasLSL-G12D allele to investigate the mechanism of this oncogene cooperation. Endogenous levels of oncogenic Kras cooperated dramatically with high levels of Myc, inducing rapid growth of lesions in spite of apparent activation of the p53 tumor suppressor pathway and frequent apoptosis. We then hypothesized that Kras provided increased survival via the Akt pathway to counter Myc-induced apoptosis, as has been previously suggested. Indeed, we found that inhibition of PI3K through treatment with PF-04691502 increased the amount of apoptosis in R26(C)MER/(C)MER;KrasLSL-G12D/+ mice. Importantly, a more dramatic effect was observed when the same lesions were treated with a MEK inhibitor, PD 0325901. Despite having no effect on apoptosis, treatment with PD 0325901 significantly reduced proliferation in these tumors, clearing early lesions within a week of treatment, presumably by allowing Myc-induced apoptosis to overcome the residual proliferation. Consistent with this notion, MEK inhibition of human lung cancer cell lines is more effective at inducing apoptosis in the presence of higher levels of Myc protein. These data suggest that MEK inhibition may have a potent therapeutic role in lung adenocarcinomas that express high levels of Myc. Citation Format: Deborah L. Burkhart, Catherine H. Wilson, Megan Bywater, Roderik Kortlever, Jinyang Li, Mark J. Arends, Trevor D. Littlewood, Gerard I. Evan. An allelic series of deregulated c-Myc expression interrogates the oncogenicity of distinct Myc levels in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B01.

Rationally modified estrogen receptor protein as a bio-recognition element for the detection of EDC pollutants: strategies and opportunities.

The estrogen receptor protein (ER) can bind a vast number of organic pollutants widely spread in the environment and collectively known as Endocrine Disrupting Chemicals, EDCs. Its broad selectivity makes it an ideal bio-recognition element for the detection of EDCs. Here we describe the strategy and rationale for the design of ER based biosensors and assays that generate a signal in the presence of EDCs. The opportunity to use either natural or rationally modified ER molecules is discussed. The latter approach was successfully applied in the EU-FP7 project RADAR, with the aim to develop a novel biosensor for the detection of organic pollutants both in the environment and in commercial water products.

Mouse Insulin Cells Expressing an Inducible RIPCre Transgene Are Functionally Impaired

We used cre-lox technology to test whether the inducible expression of Cre minimize the deleterious effect of the enzyme on beta cell function. We studied mice in which Cre is linked to a modified estrogen receptor (ER), and its expression is controlled by the rat insulin promoter (RIP). Following the injection of tamoxifen (TM), CreER- migrates to the nucleus and promotes the appearance of a reporter protein, enhanced yellow fluorescent protein (EYFP), in cells. Immunocytochemical analysis indicated that 46.6 ± 2.1% insulin cells of adult RIPCreER- EYFP expressed EYFP. RIPCreER-EYFP (+TM) mice were normoglycemic throughout the study, and their glucose tolerance test results were similar to control CD-1 mice. However, an extended exposure to reagents that stimulate insulin synthesis was detrimental to the survival of IN+EYFP+cells. The administration of an inhibitor of the enzyme dipeptidyl-peptidase (DPP4i), which prevents the cleavage of glucagon-like peptide (GLP-1), to adult RIPCreER-EYFP mice lead to a decrease in the percentage of IN+EYFP+ to 17.5 ± 1.73 and a significant increase in apoptotic cells in islets. Similarly, a 2-week administration of the GLP-1 analog exendin 4 (ex-4) induced an almost complete ablation of IN+ expressing a different reporter protein and a significant decrease in the beta cell mass and rate of beta cell proliferation. Since normal beta cells do not die when induced to increase insulin synthesis, our observations indicate that insulin cells expressing an inducible RIPCre transgene are functionally deficient. Studies employing these mice should carefully consider the pitfalls of the Cre-Lox technique.

Molecular and functional analysis of a conditional knockout of activin A

Purpose: Osteoarthritis (OA) is the most common degenerative disease of the joint. Knockout mice have been a very powerful tool to elucidate the pathogenesis of OA. However, selectively deleting genes in adult animals in a tissue or time specific manner, still remains challenging. Here we have generated a conditional knockout mouse to study the role of activin A in a murine model of OA. Activin A is strongly regulated in human OA and is essential for normal musculoskeletal development (the constitutive knockout is neonatal lethal). Methods: Activin A floxed mice (Actf/f) were crossed with a line expressing Cre recombinase driven by the human ubiquitin promoter and controlled by a Tamoxifen-sensitive modified estrogen receptor (UbcCreERT2). The resulting mice (UbcCreERT2/Actf/f) were treated with Tamoxifen (TM) on 3 consecutive days at either 4 weeks (for cartilage injury studies), 8 weeks (for OA studies) or 10 weeks (for other studies) to create pan tissue activin A deletion. Activin A messenger RNA production was determined by real time PCR. Activin A protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). Femurs and tibias were collected at 18 weeks for micro CT imaging. Results: In activin A conditional knockout (Act-cKO) mice, less than 10% of activin A mRNA was detected in cartilage, knee joints, spleen, liver and kidney compared with control levels (Act f/f or UbcCreERT2/Actf/f; no TM). ELISA showed that activin A secretion upon cartilage injury in Act-cKO hips was similarly reduced. For functional validation, micro CT analysis showed that 18 week old female Act-cKO had more trabecular bone volume, thickness and trabecular number than controls (p < 0.05). We also observed that Act-cKO showed a much delayed hair re-growth on the dorsal skin after hair removal compared with controls. Conclusions: We have successfully generated an activin A conditional knockout mouse and have validated the deletion at the mRNA and protein level in a number of tissues including cartilage. We have demonstrated functional loss of activin in bone by showing that these mice have increased bone mass, a phenotype that is predicted by other in vivo/vitro studies. The retarded hair re-growth phenotype in Act-cKO mice was in agreement with the fact that constitutive activin A knockout had no whiskers. We are currently awaiting the outcome of the OA experiments to see whether disease is modulated by lack of activin A.

Characterization of mice expressing Ins1 gene promoter driven CreERT recombinase for conditional gene deletion in pancreatic β-cells

Gene manipulation using Cre-loxP recombination has proven to be an important approach for studying the impact of gene expression on pancreatic β-cell biology. We report the generation of a transgenic mouse line that enables a highly specific system for conditional gene manipulation within β-cells and achieve tissue specific and temporally regulated deletion of the Ctnnb1 (β-catenin) gene in pancreatic β-cells. cDNA encoding Cre recombinase fused to modified estrogen receptor (CreERT) under control of mouse insulin 1 gene promoter (Ins1) was used to construct the mouse line Tg(Ins1-Cre/ERT)1Lphi, also termed MIP1-CreERT. In a cross of MIP1-CreERT with a ROSA26/LacZ reporter strain, tamoxifen [Tmx] - dependent β-galactosidase expression occurred within pancreatic β-cells but not in other organ systems. Intraperitoneal glucose tolerance tests and glucose-stimulated changes in β-cell cytoplasmic calcium concentration were not adversely affected in adult MIP1-CreERT. A mouse line with floxed Ctnnb1 gene (Ctnnb1f/f) was crossed with the MIP1-CreERT line to generate a mouse model for inducible β-cell specific deletion of β-catenin gene (Ctnnb1f/f:MIP1-CreERT). Ctnnb1f/f:MIP1-CreERT mice and Ctnnb1f/f littermate controls, were injected with Tmx as adults to knock down β-catenin production in the majority of pancreatic β-cells. These mice showed normal glucose tolerance, islet cyto-architecture and insulin secretion. A novel protein fraction of 50Kd, immunoreactive with anti-β-catenin was observed in islet extracts from Ctnnb1f/f:MIP1-CreERT[Tmx] mice but not MIP1-CreERT-negative Ctnnb1f/f[Tmx] controls, indicating possible presence of a cryptic protein product of recombined Ctnnb1 gene. The MIP1-CreERT mouse line is a powerful tool for conditional manipulation of gene expression in β-cells.

Abstract 375: A New Mouse Model to Characterize the Mechanisms of Endothelin-1-Induced Vascular Injury

Objective: To develop a model permitting the study of the mechanisms of endothelin (ET-1)-induced vascular injury, we generated an inducible and endothelial cell (EC)-restricted human ET-1 ( EDN1 ) cDNA overexpressing transgenic mouse, which would accordingly be devoid of developmental effects. Methods and Results: A transgene was engineered that expresses chloramphenicol acetyltransferase ( cat ) cDNA prior to Cre-mediated excision, and EDN1 cDNA after Cre-mediated excision, under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter (CAG). Co-transfection of Cre expression vector and pCAG- cat - EDN1 caused a 30-fold increase in ET-1 production evaluated by ELISA. A DNA fragment containing the CAG- cat - EDN1 transgene was used to generate transgenic mice. PCR genotyping demonstrated that 14/132 mice were transgenic. Two of 7 transgenic lines were selected based on cardiac cat expression level, which were twice higher in line C-134 than C-170. Inducible EC-restricted EDN1 mice were generated crossing CAG- cat - EDN1 mice with mice expressing Cre recombinase fused to a tamoxifen (TAM)-inducible modified estrogen receptor ligand binding domain ( Cre ER T2 ) under control of EC-specific receptor tyrosine kinase ( Tie2 ) promoter. To investigate the extent of CreER T2 activation by TAM and tissue specificity, Tie2 -CreER T2 mice were crossed with ROSA mT-mG/mT-mG reporter mice expressing a membrane-targeted tandem dimer tomato (mT) before Cre-mediated excision, and membrane-targeted enhanced green fluorescent protein (mG) after excision. Tie2 -CreER T2 / cat - EDN1 and Tie2 -CreER T2 / ROSA mT-mG/+ mice were treated with 1 mg TAM/d SC for 5 d and sacrificed 14-16 d later. mG expression was detected in 22±3% of mesenteric artery EC of Tie2 -CreER T2 / ROSA mT-mG/+ mice. Plasma ET-1 levels were similar in vehicle-treated Tie2 -CreER T2 / cat - EDN1 (1.2±0.1 pg/mL, n=4) and wild-type mice (1.1±0.1 pg/mL, n=2), demonstrating no leaky expression. TAM induced 8-fold increase in plasma ET-1 levels in Tie2 -CreER T2 / cat - EDN1 mice (9.1 ± 0.3 pg/mL, n = 4). Conclusion: Tie2 -CreER T2 / cat - EDN1 -inducible EC-restricted EDN1 overexpressing mice allow the study of ET-1 vascular effects in adult independently of developmental effects.

Abstract 2732: The role of Cdx transcription factors in intestinal tumorigenesis.

Abstract Mutations in the Adenomatous polyposis coli (APC) gene are an early causative event in human colorectal cancer (CRC). The Min mouse, which harbors mutations of APC, is a widely used model for CRC, although it presents with benign polyps restricted largely to the small intestine. The transcription factors Cdx1 and Cdx2 are expressed throughout the intestinal epithelium from early gut development, and persist throughout the life of the animal. Cdx1 null mice exhibit no intestinal phenotype while Cdx2 null mice die around embryonic day 3.5. Cdx2 heterozygotes, however, have an intestinal phenotype characterized by lesions in the pericaecal region and proximal colon suggestive of a role for Cdx2 in posteriorizing the intestinal tract. Consistent with this, recent studies using conditional knockout approaches show loss of Cdx2 from the developing or adult intestinal epithelium results in an anteriorized intestinal phenotype. Cdx2 loss has been detected in 30% of human CRC, and Cdx2 heterozygous-APC mutants exhibit an increase in polyp formation in the colon; similar results are seen with Cdx2 heterozygotes treated with the carcinogen azoxymethane. These studies suggest that Cdx2 has tumor suppressor properties, however the mechanism by which Cdx2 elicits this effect is unknown. We are using a Cre-lox system to conditionally delete Cdx2 from the adult intestine alone or in a heterozygous Min background. The tamoxifen-regulated Cre, under the spatial control of the villin promoter, is fused to a modified estrogen receptor ligand binding domain that responds only to tamoxifen. Sub-optimal tamoxifen treatment is used to effect partial loss of Cdx2 to circumvent the lethality inherent to Cdx2 loss in the adult intestine. Animals will be sacrificed commencing at 14 weeks post-treatment for analysis of intestinal lesions and compound mutants will be compared to mice deficient for Cdx2 or Min alone. Partial tamoxifen treatment was successful at yielding mice with chimeric loss of Cdx2. In contrast to germ-line Cdx2 heterozygotes, chimeric Cdx2 deletion did not produce hamartomas. In contrast, Cdx-Min compound mutants exhibited a faster trajectory of wasting compared to Min heterozygotes, necessitating endpointing by 14 weeks post-treatment. These compound mutant mice exhibited a three-fold increase in tumor incidence compared to Min controls, with an increased tumors size. Histologically, the small intestinal tumors that developed in the Cdx-Min background have an altered histological appearance compared to the adenomatous Min tumors. Proliferative cells and nuclear beta-catenin expression, seen within the Min tumors, were expanded in the Cdx-Min tumors, consistent with activation of the Wnt pathway in both tumor types. This work illustrates that the conditional Cdx2 allele is an effective tool for studying the role of this transcription factor in the adult intestine, and reaffirms an important role for Cdx2 in the genesis of intestinal cancers. Citation Format: Alexa Hryniuk, David Lohnes, Stephanie Grainger. The role of Cdx transcription factors in intestinal tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2732. doi:10.1158/1538-7445.AM2013-2732

Mechanism of Acute Tamoxifen and 4-Hydroxytamoxifen-Mediated Contractile Dysfunction in Isolated Adult Cardiac Myocytes

Tamoxifen-inducible Cre-mediated gene excision is an important tool for spatiotemporal study of cardiac gene function, but tamoxifen administration can cause transient dilated cardiomyopathy and severely diminished contractile performance in MerCreMer transgenic mice. This phenomenon has been attributed primarily to the presence of modified estrogen receptor (MER), which binds tamoxifen but not endogenous estrogen. However, other studies point to a non-genomic acute effect of tamoxifen alone to alter functional properties of cardiac myocytes. To further determine whether tamoxifen-mediated contractile dysfunction occurs independently of MER, we measured sarcomere length and calcium transients in isolated adult rat cardiac myocytes acutely treated with tamoxifen. Previously we reported a dose-dependent decrease in the peak height of contraction and relaxation rate in tamoxifen-treated cells. This was accompanied by a decrease in peak calcium and calcium decay rate. Here, we present a comparison between tamoxifen and its active metabolite 4-hydroxytamoxifen, which is a more potent modulator of estrogen receptor signaling than tamoxifen. Results show a similar dose-dependent decrease in cardiac myocyte function with 4-hydroxytamoxifen, with decreased peak height and relaxation rate associated with decreased peak calcium and calcium decay rate. Importantly, the magnitude of effects was similar, suggesting a direct effect of tamoxifen and 4-hydroxytamoxifen to inhibit myocyte function, independent of the presence of MER. In addition to MER-dependent effects in tamoxifen-treated MerCreMer mice, the direct effect of tamoxifen should also be considered in understanding cardiotoxicity in MerCreMer transgenic mouse models.

Generation of NSE-MerCreMer Transgenic Mice with Tamoxifen Inducible Cre Activity in Neurons

To establish a genetic tool for conditional deletion or expression of gene in neurons in a temporally controlled manner, we generated a transgenic mouse (NSE-MerCreMer), which expressed a tamoxifen inducible type of Cre recombinase specifically in neurons. The tamoxifen inducible Cre recombinase (MerCreMer) is a fusion protein containing Cre recombinase with two modified estrogen receptor ligand binding domains at both ends, and is driven by the neural-specific rat neural specific enolase (NSE) promoter. A total of two transgenic lines were established, and expression of MerCreMer in neurons of the central and enteric nervous systems was confirmed. Transcript of MerCreMer was detected in several non-neural tissues such as heart, liver, and kidney in these lines. In the background of the Cre reporter mouse strain Rosa26R, Cre recombinase activity was inducible in neurons of adult NSE-MerCreMer mice treated with tamoxifen by intragastric gavage, but not in those fed with corn oil only. We conclude that NSE-MerCreMer lines will be useful for studying gene functions in neurons for the conditions that Cre-mediated recombination resulting in embryonic lethality, which precludes investigation of gene functions in neurons through later stages of development and in adult.

Tamoxifen has Acute Inhibitory Effects on Contractility in Isolated Adult Rat Cardiac Myocytes

Spatiotemporal cardiac gene targeting featuring α-MyHC-MerCreMer (MCM) transgenic mice is an important approach for studying heart-specific gene function. The modified estrogen receptor (MER) is activated by the selective estrogen receptor modulator (SERM) tamoxifen. MER activation by tamoxifen results in nuclear translocation of MCM, and Cre-mediated excision of loxP-flanked genes. A drawback to tamoxifen-dependent Cre translocation is transient cardiomyopathy, which is suggested to be independent of gene excision and dependent on the presence of MCM. However, studies using isolated cardiac myocytes reveal the presence of an acute non-genomic inhibitory effect of tamoxifen on several types of ion channels, suggesting tamoxifen also may have an effect in the absence of MCM. The goal of the present study was to determine whether the inhibition of ion channels found in previous studies translates to changes in contractility and calcium transients in isolated adult rat cardiac myocytes. One day after cell isolation, we found an acute, dose-dependent decrease in the peak height of contraction with tamoxifen concentrations ranging from 1-10μM. This was accompanied by diminished calcium transient amplitude. Additionally, the percentage of rod-shaped cells that visibly contract dose-dependently decreased over the course of one hour of pacing with tamoxifen. Raloxifene, also in the SERM class of drugs, had a pattern of effects similar to tamoxifen. In conclusion, the acute tamoxifen and raloxifene-induced inhibition of cardiac myocyte contractility may contribute to the transient cardiomyopathy seen in MCM transgenic mice. The results of this study emphasize the importance of using the minimum dose of tamoxifen required for gene excision in MCM transgenic mice, as well as incorporating appropriate controls to address tamoxifen-mediated acute cardiomyopathy.

Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One‐ and Two‐Photon Illumination

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells &lt;em&gt;In Vivo&lt;/em&gt;

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage. GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreER(T)). CreER(T) contains a modified estrogen receptor ligand binding domain which renders CreER(T) sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreER(T), which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP. Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreER(T) release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreER(T). We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders. This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreER(T);mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

Transient Activation of Wnt/β-Catenin Signaling Induces Abnormal Growth Plate Closure and Articular Cartilage Thickening in Postnatal Mice

Wnt/beta-catenin signaling is required for skeletal development and organization and for function of the growth plate and articular cartilage. To further clarify these roles and their possible pathophysiological importance, we created a new transgenic mouse model in which Wnt/beta-catenin signaling can be activated in cartilage for specific periods of time. These transgenic mice expressed a constitutive active form of beta-catenin fused to a modified estrogen receptor ligand-binding domain under the control of cartilage-specific collagen 11alpha2 promoter/enhancer. Transient Wnt/beta-catenin signaling activation in young adult mice by tamoxifen injections induced growth retardation and severe deformities in knee joints. Tibial and femoral growth plates displayed an excessive number of apoptotic cells and eventually underwent abnormal regression. Articular cartilage exhibited an initial acute loss of proteoglycan matrix that was followed by increases in thickness, cell density, and cell proliferation. In reciprocal studies, we found that conditional ablation of beta-catenin in postnatal mice using a Col2-CreER strategy led to hypocellularity in articular cartilage, growth plate disorganization, and a severe reduction in bone volume. Together, these data provide evidence that Wnt/beta-catenin signaling has important and distinct roles in growth plate and articular cartilage and that postnatal dysregulation of this signaling pathway causes diverse structural and functional changes in the two cartilaginous structures.

Inducible site‐specific recombination in neural stem/progenitor cells

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC-specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand-binding domain (ER(T2)). In the background of the Cre reporter mouse strain Rosa26(lacZ), we show that the fusion CreER(T2) recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by beta-galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs.

Regulated gene insertion by steroid-induced C31 integrase

Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and ΦC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the ΦC31 integrase is documented. In addition, fusion of the ΦC31 integrase with the ERT2 modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible ΦC31 integrase systems are important new tools in transgenesis and in safety studies of the ΦC31 integrase for gene therapy applications.

AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.

Identification of Hoxa9 Targets in Myeloid Progenitors Using Conditional Expression.

Deregulation of HOXA9 expression appears to be a common mechanism of transformation in both lymphoid and myeloid acute leukemias. Although HOXA9 overexpression is often associated with MLL rearrangements, deregulation is also seen in leukemias with other translocations or lacking known translocations, where overexpression is highly correlated with treatment failure. A wealth of evidence indicates that in isolation, HOXA9 is a relatively weak oncoprotein that at physiologic levels expands stem cells. However the combination of high level HOXA9 expression in concert with MEIS1 is potently leukemogenic. The molecular basis for this cooperativity and relevant downstream effectors are essentially unknown. To begin to resolve this important issue we immortalized murine hematopoietic precursors by retroviral transduction of bone marrow with a conditionally active form of Hoxa9. The C-terminus of Hoxa9 was fused to a modified estrogen receptor ligand binding domain (ER) so that protein activity is contigent upon 4-OHT. Proliferation and maintenance of myeloblast phenotype required continuous exposure to the 4-OHT precursor tamoxifen. Withdrawal of tamoxifen resulted in differentiation within 96 h, as evidenced by growth arrest, expression of myeloid/monocytic differentiation markers, and maturation to macrophage morphology. Similar experiments to establish conditional Meis1 regulation by ER fusion at either the N or C terminus were not successful as the fusions were non-transforming. Direct and indirect targets of Hoxa9 were identified by measuring gene expression differences after tamoxifen withdrawal. At 72 h, 346 genes showed >2-fold change (adjusted p<0.05). At later points, a cohort of genes were upregulated, corresponding with the onset of myeloid maturation. The Ets family member Ehf showed the largest drop (10-fold) with Hoxa9 withdrawal; EHF has been implicated in carcinomagenesis; its role in leukemia remains unstudied. Flt3, a known target of joint Hoxa9/Meis1 regulation with prognostic significance, shows an approximately 3-fold decrease with withdrawal of Hoxa9 in the presence or absence of Meis1. Cancer outlier profile analysis (www.oncomine.org) reveals that Flt3 and other targets identified in our analysis, including Rage, S100a10, and Bambi, are highly correlated with HOXA9 in AML/ALL microarray datasets, suggesting a downstream role in HOXA9-mediated leukemogenesis. Genes up-regulated with Hoxa9 withdrawal include JunB, a previously described target negatively regulated by Hoxa9 that antagonizes leukemic self-renewal (Steidl et al., Nat Genet (2006) 38 :1269–77). The targets Mylk and Laptm4b are notable for belonging to the 11-gene “immediate self-renewal signature” of MLL-AF9 leukemic stem cells, which also includes Hoxa9 and Meis1 (Krivtsov et al., Nature (2006) 442: 818–22), suggesting a key role for Hoxa9 in the expression hierarchy within this signature. The presence of a subset of targets, including Flt3 and Sox4, that are dependent on both Hoxa9 and Meis1 expression is consistent with a model in which their synergy involves co-regulation of similar targets, rather than parallel transcription programs. This model is currently being tested using global ChIP on chip and other approaches.