Cre Mouse Model Research Articles

Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin

BackgroundSex differences exist in the prevalence and progression of major glomerular diseases. Podocytes are the essential cell-type in the kidney which maintain the physiological blood-urine barrier, and pathological changes in podocyte homeostasis are critical accelerators of impairment of kidney function. However, sex-specific molecular signatures of podocytes under physiological and stress conditions remain unknown. This work aimed at identifying sexual dimorphic molecular signatures of podocytes under physiological condition and pharmacologically challenged homeostasis with mechanistic target of rapamycin (mTOR) inhibition. mTOR is a crucial regulator involved in a variety of physiological and pathological stress responses in the kidney and inhibition of this pathway may therefore serve as a general stress challenger to get fundamental insights into sex differences in podocytes.MethodsThe genomic ROSAmT/mG-NPHS2 Cre mouse model was used which allows obtaining highly pure podocyte fractions for cell-specific molecular analyses, and vehicle or pharmacologic treatment with the mTOR inhibitor rapamycin was performed for 3 weeks. Subsequently, deep RNA sequencing and proteomics were performed of the isolated podocytes to identify intrinsic sex differences. Studies were supplemented with metabolomics from kidney cortex tissues.ResultsAlthough kidney function and morphology remained normal in all experimental groups, RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways. Interestingly, rapamycin abolished prominent sex-specific clustering of podocyte gene expression and induced major changes only in male transcriptome. Several sex-biased transcription factors could be identified as possible upstream regulators of these sexually dimorphic responses. Concordant to transcriptomics, metabolomic changes were more prominent in males. Remarkably, high number of previously reported kidney disease genes showed intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition.ConclusionsOur results highlight remarkable intrinsic sex-differences and sex-specific response patterns towards pharmacological challenged podocyte homeostasis which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. This work provides rationale and an in-depth database for novel targets to be tested in specific kidney disease models to advance with sex-specific treatment strategies.

Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines.

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

The duct of von Ebner's glands is a source of Sox10 + taste bud progenitors and susceptible to pathogen infections.

We have recently demonstrated that Sox10-expressing (Sox10 +) cells give rise to mainly type-III neuronal taste bud cells that are responsible for sour and salt taste. The two tissue compartments containing Sox10 + cells in the surrounding of taste buds include the connective tissue core of taste papillae and von Ebner's glands (vEGs) that are connected to the trench of circumvallate and foliate papillae. In this study, we performed single cell RNA-sequencing of the epithelium of Sox10-Cre/tdT mouse circumvallate/vEG complex and used inducible Cre mouse models to map the cell lineages of vEGs and/or connective tissue (including stromal and Schwann cells). Transcriptomic analysis indicated that Sox10 expression was enriched in the cell clusters of vEG ducts that contained abundant proliferating cells, while Sox10-Cre/tdT expression was enriched in type-III taste bud cells and vEG ductal cells. In vivo lineage mapping showed that the traced cells were distributed in circumvallate taste buds concurrently with those in the vEGs, but not in the connective tissue. Moreover, multiple genes encoding pathogen receptors were enriched in the vEG ducts hosting Sox10 + cells. Our data supports that it is the vEGs, not connective tissue core, that serve as the niche of Sox10 + taste bud progenitors. If this is also true in humans, our data indicates that vEG duct is a source of Sox10 + taste bud progenitors and susceptible to pathogen infections.

Vascular smooth muscle-specific YAP/TAZ deletion triggers aneurysm development in mouse aorta.

Inadequate adaption to mechanical forces, including blood pressure, contributes to development of arterial aneurysms. Recent studies have pointed to a mechanoprotective role of YAP and TAZ in vascular smooth muscle cells (SMCs). Here, we identified reduced expression of YAP1 in human aortic aneurysms. Vascular SMC-specific knockouts (KOs) of YAP/TAZ were thus generated using the integrin α8-Cre (Itga8-Cre) mouse model (i8-YT-KO). i8-YT-KO mice spontaneously developed aneurysms in the abdominal aorta within 2 weeks of KO induction and in smaller arteries at later times. The vascular specificity of Itga8-Cre circumvented gastrointestinal effects. Aortic aneurysms were characterized by elastin disarray, SMC apoptosis, and accumulation of proteoglycans and immune cell populations. RNA sequencing, proteomics, and myography demonstrated decreased contractile differentiation of SMCs and impaired vascular contractility. This associated with partial loss of myocardin expression, reduced blood pressure, and edema. Mediators in the inflammatory cGAS/STING pathway were increased. A sizeable increase in SOX9, along with several direct target genes, including aggrecan (Acan), contributed to proteoglycan accumulation. This was the earliest detectable change, occurring 3 days after KO induction and before the proinflammatory transition. In conclusion, Itga8-Cre deletion of YAP and TAZ represents a rapid and spontaneous aneurysm model that recapitulates features of human abdominal aortic aneurysms.

Interferon-alpha plays an important role in glial-mediated symptoms of neuropsychiatric systemic lupus erythematosus.

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease that leads to clinical pathology across organs, including the central nervous system (CNS). In up to 80% of patients with SLE, CNS manifestations can range from anxiety and fatigue to overt psychosis. While studies have demonstrated a hallmark increase in circulating interferon alpha (IFNα) in the peripheral blood and cerebrospinal fluid, the cellular mechanisms leading to neuropsychiatric lupus (NPSLE) remain unclear (Crow et. al., 2014). Using a mouse model of lupus which expresses an elevated IFNα signature and exhibits behaviors of NPSLE, we found a spatial pattern of interferon-stimulated genes (ISGs) in the brain parenchyma utilizing the multiplexed spatial transcriptomics platform MERFISH (Moffitt et. al., 2016). Guided by unbiased single nucleus sequencing highlighting astrocytes and oligodendrocytes as major glial cells enriched in ISG expression, we hypothesized that this spatial distribution implicates IFNα-driven glial activity against neuron function, thus disrupting regions of the CNS that manifests as NPSLE. We also investigated the role of IFNα on microglia activity and behavior changes using a cell-specific Cremouse model. Further functional assays reveal increased synapse engulfment by microglia upon IFNα activation. Despite waning ISGs levels in the periphery, levels in the brain persist along with behavior severity, pointing to a CNS source of IFNα. This body of work demonstrates how IFNα released locally by glial cells can mediate NPSLE development via disruption of neuron function. Deeper understanding of targetable pathways in IFNα signaling will expand the therapeutic potential of type 1 interferon-driven neuropsychiatric diseases.

Constitutive bone marrow adipocytes suppress local bone formation.

BM adipocytes (BMAd) are a unique cell population derived from BM mesenchymal progenitors and marrow adipogenic lineage precursors. Although they have long been considered to be a space filler within bone cavities, recent studies have revealed important physiological roles in hematopoiesis and bone metabolism. To date, the approaches used to study BMAd function have been confounded by contributions by nonmarrow adipocytes or by BM stromal cells. To address this gap in the field, we have developed a BMAd-specific Cre mouse model to deplete BMAds by expression of diphtheria toxin A (DTA) or by deletion of peroxisome proliferator-activated receptor gamma (Pparg). We found that DTA-induced loss of BMAds results in decreased hematopoietic stem and progenitor cell numbers and increased bone mass in BMAd-enriched locations, including the distal tibiae and caudal vertebrae. Elevated bone mass appears to be secondary to enhanced endosteal bone formation, suggesting a local effect caused by depletion of BMAd. Augmented bone formation with BMAd depletion protects mice from bone loss induced by caloric restriction or ovariectomy, and it facilitates the bone-healing process after fracture. Finally, ablation of Pparg also reduces BMAd numbers and largely recapitulates high-bone mass phenotypes observed with DTA-induced BMAd depletion.

Comparative Evaluation of Inducible Cre Mouse Models for Fibroblast Targeting in the Healthy and Infarcted Myocardium.

Several Cre recombinase transgenic mouse models have been generated for cardiac fibroblast (CF) tracking and heart regulation. However, there is still no consensus on the ideal mouse model to optimally identify and/or regulate these cells. Here, a comparative evaluation of the efficiency and specificity of the indirect reporter Cre-loxP system was carried out in three of the most commonly used fibroblast reporter transgenic mice (Pdgfra-CreERT2, Col1a1-CreERT2 and PostnMCM) under healthy and ischemic conditions, to determine their suitability in in vivo studies of cardiac fibrosis. We demonstrate optimal Cre recombinase activity in CF (but also, although moderate, in endothelial cells (ECs)) derived from healthy and infarcted hearts in the PDGFRa-creERT2 mouse strain. In contrast, no positive reporter signal was found in CF derived from the Col1a1-CreERT2 mice. Finally, in the PostnMCM line, fluorescent reporter expression was specifically detected in activated CF but not in EC, which leads us to conclude that it may be the most reliable model for future studies on cardiovascular disease. Importantly, no lethality or cardiac fibrosis were induced after tamoxifen administration at the established doses, either in healthy or infarcted mice of the three fibroblast reporter lineages. This study lays the groundwork for future efficient in vivo CF tracking and functional analyses.

Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits.

To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.

Tetracycline response element driven Cre causes ectopic recombinase activity independent of transactivator element

ObjectiveTamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity. MethodsAdipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity. ResultsTamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass. ConclusionsThese results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

Obesity has become a global pandemic. Identification of key factors in adipogenesis helps to tackle obesity and related metabolic diseases. Here, we show that DDB1 binds the histone reader BRWD3 to promote adipogenesis and diet-induced obesity. Although typically recognized as a component of the CUL4-RING E3 ubiquitin ligase complex, DDB1 stimulates adipogenesis independently of CUL4. A DDB1 mutant that does not bind CUL4A or CUL4B fully restores adipogenesis in DDB1-deficient cells. Ddb1+/- mice show delayed postnatal development of white adipose tissues and are protected from diet-induced obesity. Mechanistically, by interacting with BRWD3, DDB1 is recruited to acetylated histones in the proximal promoters of ELK1 downstream immediate early response genes and facilitates the release of paused RNA polymerase II, thereby activating the transcriptional cascade in adipogenesis. Our findings have uncovered a CUL4-independent function of DDB1 in promoting the transcriptional cascade of adipogenesis, development of adipose tissues, and onset of obesity.

Oncogenic mutation in RAS-RAF axis leads to increased expression of GREB1, resulting in tumor proliferation in colorectal cancer.

BRAFV600E mutation accounts for up to 90% of all BRAF mutations in human colorectal cancer (CRC), and constitutively activates the MEK‐MAPK pathway. It is recognized that neutralizing mAbs for epidermal growth factor receptor alone are not effective for CRC with BRAFV600E mutation. Therefore, there is increasing interest in identification of the possible therapeutic targets in downstream of BRAF mutation in CRCs. To address this, we studied genome engineered mouse models for colonic neoplasia that has BrafV600E mutation on the basis of Apc inactivation, induced in 2 distinct Cre mouse models, CDX2P‐G22Cre and CDX2P‐CreERT2 mice. We carried out oligonucleotide microarray analysis for colonic neoplasia generated in these mouse models, and compared gene expression profiles among Kras/Braf WT, Kras‐mutated, and Braf‐mutated mouse colon tumors to seek new molecular targets corresponding to the KRAS‐BRAF‐MAPK axis. We found that the expression of the growth regulation by estrogen in breast cancer protein 1 (Greb1) was the most upregulated gene in Braf‐mutated mouse tumors compared to Kras/Braf WT counterparts. The silencing of GREB1 significantly reduced the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GREB1 promoted cell proliferation. Although GREB1 was first identified as a hormone‐responsive gene mediating estrogen‐stimulated cell proliferation in endometriosis, breast, and ovarian cancers, these results suggest that RAS‐RAF‐MAPK signaling upregulates GREB1 expression in CRC, resulting in cellular proliferation. Thus, GREB1 is a possible therapeutic target for CRCs with BrafV600E mutation.

Abstract A12: Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA)

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption, and pancreatitis, all of which share the ability to generate oxidative stress—a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre (KC) model. For our studies, KC mice received either tap water or tap water containing 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&E stain), Alcian Blue (AB) staining, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Amylase, lipase, PFOA, and inflammatory cytokines were quantified in serum. Our results show that PFOA accumulates in the serum and pancreas of treated KC mice. In 6-month-old KC mice, exposure to PFOA increased the total AB+ lesion area from 4.87% ± 1.65% to 7.71% ± 3.01%. While the average lesion size was similar between control and PFOA-treated groups, the number of lesions per mm2 increased from 8.73 ± 3.19 to 16.73 ± 6.31, demonstrating that PFOA exposure leads to increased lesion development. qPCR analysis of pancreata verified increased expression of lesion markers CK19 and Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Collectively, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model, which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biologic persistence, suggest that it may participate in promoting pancreatic cancer progression. Citation Format: Barbara A. Hocevar, Lisa M. Kamendulis. Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA) [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A12.

Endothelial-Specific Cre Mouse Models.

The field of vascular biology has gained enormous insight from the use of Cre and inducible Cre mouse models to temporally and spatially manipulate gene expression within the endothelium. Models are available to constitutively or inducibly modulate gene expression in all or a specified subset of endothelial cells. However, caution should be applied to both the selection of allele and the analysis of resultant phenotype: many similarly named Cre models have divergent activity patterns while ectopic or inconsistent Cre or inducible Cre expression can dramatically affect results. In an effort to disambiguate previous data and to provide a resource to aid appropriate experimental design, here we summarize what is known about Cre recombinase activity in the most widely used endothelial-specific Cre and Cre/ERT2 mouse models.

Abstract 3095: Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA)

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption and pancreatitis; all of which share the ability to generate oxidative stress - a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~ 4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre model. For our studies, LSL-KRasG12D;Pdx-1Cre mice received either tap water or tap water supplemented with 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&E stain), immunohistochemistry for CK19, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Serum was utilized for quantitation of amylase, lipase, PFOA and inflammatory cytokine levels. Our preliminary results show that PFOA accumulates in the serum and pancreas of treated LSL-KRasG12D;Pdx-1 Cre mice. In addition, exposure to PFOA increased the CK19+ lesion area in LSL-KRasG12D;Pdx-1 Cre mice indicating an acceleration of pancreatic cancer progression. qPCR analysis of pancreata verified increased expression of both CK19 and the early lesion marker Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Together, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biological persistence, suggest that it could be a prominent agent promoting pancreatic cancer progession. Citation Format: Barbara A. Hocevar, Lisa M. Kamendulis. Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3095.

Oestrogen fuels the growth of endometrial hyperplastic lesions initiated by overactive Wnt/β-catenin signalling.

Unopposed oestrogen is responsible for approximately 80% of all the endometrial cancers. The relationship between unopposed oestrogen and endometrial cancer was indicated by the increase in the number of endometrial cancer cases due to the widespread use of oestrogen replacement therapy. Approximately 30% of the endometrial cancer patients have mutations in the Wnt signalling pathway. How the unbalanced ratios of ovarian hormones and the mutations in Wnt signalling pathway interact to cause endometrial cancer is currently unclear. To study this, we have developed a uterine epithelial cell-specific inducible cre mouse model and used 3D in vitro culture of human endometrial cancer cell lines. We showed that activating mutations in the Wnt signalling pathway for a prolonged period leads to endometrial hyperplasia but not endometrial cancer. Interestingly, unopposed oestrogen and activating mutations in Wnt signalling together drive the progression of endometrial hyperplasia to endometrial cancer. We have provided evidence that progesterone can be used as a targeted therapy against endometrial cancer cases presented with the activating mutations in Wnt signalling pathway.

Generation of Elf5-Cre knockin mouse strain for trophoblast-specific gene manipulation.

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5-Cre mice, we mated Elf5-Cre mice with Rosa26mT/mG reporter mice, and found that Elf5-Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5-Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5-Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.

Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes

Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting andprotein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs ofglomerular disease or off-target effects under basalconditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.

Global or Granulosa Cell-Specific Pten Mutations in Combination with Elevated FSH Levels Fail to Cause Ovarian Tumours in Mice.

Phosphatase and tensin homologue (PTEN) is a known tumour suppressor. To explore the role of Pten in ovarian tumorigenesis, we used transgenic (Tg) SOX2. Cre and AMH. Cre mouse models to direct global Pten haploinsufficiency (Pten +/-) or ovary-specific granulosa cell (GC) Pten disruption (Pten GC ). Pten mutant models were combined with progressively rising Tg-follicle-stimulating hormone (TgFSH) levels to study the tumorigenic potential of combined genetic/endocrine modification in vivo. Global Pten +/- mice exhibited grossly detectable tumours in multiple organs including uterine and mammary tissue and displayed reduced survival. Despite extra-ovarian tumorigenesis, Pten +/- females had no detectable ovarian tumours, although elevated corpus luteum numbers increased ovary size and estrous cycling was altered. Combined TgFSH/Pten +/- mice also had no ovarian tumours, but early survival was reduced in the presence of TgFSH. Ovary-specific Pten GC ±TgFSH females exhibited no detectable ovarian or uterine tumours, and corpus luteum numbers and estrous cycling remained unchanged. The non-tumorigenic ovarian phenotypes in Pten +/- and Pten GC ±TgFSH mice support the proposal that multi-hit genetic mutations (including ovarian and extra-ovarian tissue) initiate ovarian tumours. Our findings suggest that elevated FSH may reduce early cancer survival; however, the ovary remains remarkably resistant to Pten-induced tumorigenic changes even in the presence of uterine and reproductive cancers.

TH-AB-204-02: Preliminary Study of Up-Converting Nanocrystal (UCNC) Imaging in Optical-ECT

Purpose: Optical-emission computed tomography (optical- ECT) enables high-resolution 3D imaging of emission in optically cleared tissues on the order of 1 cm3. This work demonstrates capabilities of optical-ECT to quantify the red fluorescent protein (RFP) tdTomato in rat lung, and presents preliminary data on the feasibility of imaging near-IR absorptive up-converting nanocrystals (UCNCs) with optical-ECT. Methods: The nanocrystal NaYF4:Yb/Er studied in this work up-converts 980nm light to visible light peaking sharply at ∼530nm. Collimation of light using telecentric lenses coupled with a CCD array allows for inverse radon 3D reconstruction of fluorescent molecules in optically cleared tissue or whole-organ samples in emission mode. As demonstration of the typical capability of the system, tdTomato RFP emission in the lung of a Cre mouse model is reconstructed in 3D. Compatibility of UCNC with optical clearing is assessed with agarose phantom, to which 1mg/mL of 100-nm diameter NaYF4:Yb/Er coated with polyethyleneimine was injected at two different locations before the agarose was fixed with ethanol and cleared with methyl salicylate. Results: Emission mode imaging of the tdTomato-expressing mouse lung reveals in high-resolution (25.8µm3/voxel) information unseen in transmission imaging. UCNCs are shown to emit in ethanol and methyl salicylate, detected by fluorimetry. Projection images of injected UCNC crystals in agarose confirm that the crystal is unaffected by optical clearing. One-second exposure images of the excited nanocrystals yield maximum CCD counts of ∼3800 with a 530nm filter, a significant increase compared to three-second exposure image of tdTomato yielding ∼1200 counts. Conclusion: Optical-ECT imaging enables 3D emission mapping of un-sectioned tissue or whole organ on the order of 1cm3. UCNCs are not only confirmed to be unaffected by optical clearing but also yield high intensity fluorescence. Since UCNC is biocompatible, 3D imaging of the behavior of cancer cells in small animal models with UCNCs is currently under exploration.