Cre Expression Research Articles

Synthetic biology approach to developing all-in-one baculovirus vector using mammalian introns and miRNA binding sites

BackgroundInsect baculovirus is a promising vector for gene delivery into mammalian cells. We developed a Cre/loxP-based hybrid baculovirus comprising two viruses: one expressing Cre recombinase and the other harboring the transgene flanked by two loxP sites. Co-transduction of mammalian cells with two viruses confers Cre expression, which excises the loxP-flanking cassette off baculovirus genome and catalyzes DNA minicircle formation, thereby prolonging transgene expression. Two separate baculoviruses avoid undesirable recombination and loss of transgene during virus production process, but reduces gene delivery efficiency and complicates applications. MethodsTo tackle this problem, we exploited synthetic biology strategy to control Cre expression and develop an all-in-one baculovirus harboring both cre and loxP-flanking transgene cassette. We controlled cre transcription with a mammalian EF-1α promoter, and regulated cre translation with an intron inserted within the coding region and bantam miRNA binding site at the 3′UTR. Significant findingsThe all-in-one baculovirus vector selectively conferred Cre/loxP-mediated recombination in mammalian cells, but not in E. coli and Sf-9 cells, hence circumventing transgene excision from the virus genome during gene cloning in E. coli and virus amplification in insect cells. The all-in-on vector enabled formation of DNA minicircle in mammalian cells at efficiencies exceeding 80% and implicated its potential for gene delivery. Our design may be expanded to other gene delivery systems that require two separate vectors and is beneficial to gene therapy applications.

AAV-mediated gene editing lights up the lung

AAV-mediated gene editing lights up the lung

Parecoxib ameliorates renal toxicity and injury in sepsis‐induced mouse model and LPS ‐induced HK ‐2 cells

Parecoxib is a selective COX-2-specific inhibitor, which has been demonstrated to inhibit sepsis-induced systemic inflammation, but its role in sepsis-induced acute kidney injury has not been studied. This study was designed to investigate the effects of Parecoxib on sepsis-induced acute kidney injury. In this study, the mice sepsis model was established using an internationally recognized cecal ligation and puncture (CLP). Hematoxylin-eosin staining was performed to examine kidney injury. Biochemical kit was used to detect the expression of BUN and Cre in serum, and ELISA was used to detect the expression of inflammatory factors in renal tissue. Tunel staining was used to detect tissue apoptosis. Furthermore, CCK-8 assay was used to detect the cell viability of HK-2 cells and RT-qPCR was used to detect the expression of LPS-induced inflammatory factors in HK-2 cells.TUNEL staining was used to detect the level of cell apoptosis. Finally, the expressions of COX-2, p-NF-kB P65, p-IKKβ, NF-kB P65, IKKβ, Kim1, NGAL, iNOS, VEGF, VEGFR2, CD31 and apoptosis-related proteins in renal tissues and HK-2 cells were detected by Western blot. We discovered that parecoxib could alleviate renal pathological changes, reduce renal function injury, and inhibit renal pathology to inhibit the release of inflammatory factors in renal tissue. Parecoxib inhibited sepsis induced microvascular damage and apoptosis in renal tissue. Parecoxib reduced the inflammation and apoptosis of renal tubular epithelial cells induced by LPS. Our data suggest that Parecoxib ameliorates sepsis-induced kidney injury, and may have potential as a novel therapeutic method for treating sepsis-induced kidney injury.

Abstract 12114: Endothelial Extracellular Signal-Regulated Kinase 2 Contributed to the Development of Pulmonary Arterial Hypertension in Mice Through the Impairment of Endothelial Nitric Oxide Synthase Function

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease, characterized by abnormal muscularization and endothelial dysfunction in pulmonary artery (PA). Previous study showed that endothelial extracellular signal-regulated kinase 2 (eERK2) released vasoconstrictive factors and decreased NO bioactivity resulting in obese-induced hypertension and endothelial dysfunction. However, the role of eERK2 in the pathogenesis of PAH remains to be elucidated. Hypothesis: We hypothesized eERK2 impaired eNOS function and progressed the pathogenesis of PAH. Methods and Results: We generated eERK2 knock out mice (EE2KO) crossing Tie2-Cre mice and ERK2 flox mice and induced PAH by exposing mice to chronic hypoxia (10%) for 4 weeks. The littermate ERK2 flox mice without Cre expression were used as Control (Ctr). Immuno-histochemical staining showed the deficiency of eERK2 expression in the PA of EE2KO. We assessed the medial thickness of pulmonary arteries (MTPA) and measured right ventricular systolic pressure (RVSP). Although Ctr exhibited the progression of PA muscularization with an increase in MTPA, EE2KO revealed the significantly attenuated MTPA under hypoxic condition. Moreover, we found the lower RVSPs in EE2KO than those in Ctr under hypoxia in spite of similar RVSPs in normoxia. In RT-PCR, lung endothelin1 (ET1) gene expression was significantly lower in EE2KO than Ctr under hypoxia. Interestingly, hypoxia increased eNOS protein expression both in Ctr and EE2KO with Western blot analysis. The phosphorylation of AMPKα and eNOS were up-regulated in the lungs of EE2KO relative to Ctr under hypoxia. In addition, eNOS dimer/monomer ratios with non redusing SDS-PAGE was significantly increased in EE2KO compared to the Ctr under hypoxia (1.29±0.10 vs. 0.87±0.02, P=0.02, dimer / monomer ratios), suggesting improved eNOS uncoupling. Conclusions: These findings suggest that hypoxia increased eNOS expression and function, and that eERK2 impaired eNOS function by decreasing the phosphorylation of AMPKα and eNOS, and inducing eNOS uncoupling. In addition, ET1 synthesis through eERK2 might contribute to these pathogenesis of PAH with hypoxia.

Conditional Deletion of Activating Rearranged During Transfection Receptor Tyrosine Kinase Leads to Impairment of Photoreceptor Ribbon Synapses and Disrupted Visual Function in Mice.

Purpose: The rearranged during transfection (RET) receptor tyrosine kinase plays a key role in transducing signals related to cell growth and differentiation. Ret mutant mice show abnormal retinal activity and abnormal levels and morphology of bipolar cells, yet die on the 21st day after birth as a result of renal underdevelopment. To extend the observation period, we generated the Ret conditional knockout Chx10-Cre;C-Retlx/lx mouse model and analyzed the retinal function and morphological changes in mature and aging Chx10-Cre;C-Retlx/lx mice.Methods: Retina-specific depletion of Ret was achieved using mice with floxed alleles of the Ret gene with CHX10-driven Cre recombinase; floxed mice without Cre expression were used as controls. Retinal function was examined using electroretinography (ERG), and 2-, 4-, 12-, and 24-month-old mice were analyzed by hematoxylin staining and immunohistochemistry to evaluate retinal morphological alterations. The ultrastructure of photoreceptor synapses was evaluated using electron microscopy.Results: The results of the ERG testing showed that b-wave amplitudes were reduced in Chx10-Cre;C-Retlx/lx mice, whereas a-waves were not affected. A histopathological analysis revealed a thinner and disorganized outer plexiform layer at the ages of 12 and 24 months in Chx10-Cre;C-Retlx/lx mice. Moreover, the data provided by immunohistochemistry showed defects in the synapses of photoreceptor cells. This result was confirmed at the ultrastructural level, thus supporting the participation of Ret in the morphological changes of the synaptic ribbon.Conclusion: Our results provide evidence of the role of Ret in maintaining the function of the retina, which was essential for preserving the structure of the synaptic ribbon and supporting the integrity of the outer plexiform layer.

Germline DDX41 mutations cause ineffective hematopoiesis and myelodysplasia.

DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Neuron-specific ablation of eIF5A or deoxyhypusine synthase leads to impairments in growth, viability, neurodevelopment, and cognitive functions in mice

Eukaryotic initiation factor 5A (eIF5A)†, ‡ is an essential protein that requires a unique amino acid, hypusine, for its activity. Hypusine is formed exclusively in eIF5A post-translationally via two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase. Each of the genes encoding these proteins, Eif5a, Dhps, and Dohh, is required for mouse embryonic development. Variants in EIF5A or DHPS were recently identified as the genetic basis underlying certain rare neurodevelopmental disorders in humans. To investigate the roles of eIF5A and DHPS in brain development, we generated four conditional KO mouse strains using the Emx1-Cre or Camk2a-Cre strains and examined the effects of temporal- and region-specific deletion of Eif5a or Dhps. The conditional deletion of Dhps or Eif5a by Emx1 promotor–driven Cre expression (E9.5, in the cortex and hippocampus) led to gross defects in forebrain development, reduced growth, and premature death. On the other hand, the conditional deletion of Dhps or Eif5a by Camk2a promoter–driven Cre expression (postnatal, mainly in the CA1 region of the hippocampus) did not lead to global developmental defects; rather, these KO animals exhibited severe impairment in spatial learning, contextual learning, and memory when subjected to the Morris water maze and a contextual learning test. In both models, the Dhps-KO mice displayed more severe impairment than their Eif5a-KO counterparts. The observed defects in the brain, global development, or cognitive functions most likely result from translation errors due to a deficiency in active, hypusinated eIF5A. Our study underscores the important roles of eIF5A and DHPS in neurodevelopment.

Cre driver mouse lines for thalamocortical circuit mapping.

Subpopulations of neurons and associated neural circuits can be targeted in mice with genetic tools in a highly selective manner for visualization and manipulation. However, there are not well-defined Cre "driver" lines that target the expression of Cre recombinase to thalamocortical (TC) neurons. Here, we characterize three Cre driver lines for the nuclei of the dorsal thalamus: Oligodendrocyte transcription factor 3 (Olig3)-Cre, histidine decarboxylase (HDC)-Cre, and corticotropin-releasing hormone (CRH)-Cre. We examined the postnatal distribution of Cre expression for each of these lines with the Cre-dependent reporter CAG-tdTomato (Ai9). Cre-dependent expression of tdTomato reveals that Olig3-Cre expresses broadly within the thalamus, including TC neurons and interneurons, while HDC-Cre and CRH-Cre each have unique patterns of expression restricted to TC neurons within and across the sensory relay nuclei of the dorsal thalamus. Cre expression is present by the time of natural birth in all three lines, underscoring their utility for developmental studies. To demonstrate the utility of these Cre drivers for studying sensory TC circuitry, we targeted the expression of channelrhodopsin-2 to thalamus from the CAG-COP4*H134R/EYFP (Ai32) allele with either HDC-Cre or CRH-Cre. Optogenetic activation of TC afferents in primary visual cortex was sufficient to measure frequency-dependent depression. Thus, these Cre drivers provide selective Cre-dependent gene expression in thalamus suitable for both anatomical and functional studies.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

The missing 'link'? β-Catenin's role in skeletal muscle glucose uptake during exercise and contraction.

Androgen-induced insulin resistance is ameliorated by deletion of hepatic androgen receptor in females.

Androgen excess is one of the most common endocrine disorders of reproductive-aged women, affecting up to 20% of this population. Women with elevated androgens often exhibit hyperinsulinemia and insulin resistance. The mechanisms of how elevated androgens affect metabolic function are not clear. Hyperandrogenemia in a dihydrotestosterone (DHT)-treated female mouse model induces whole body insulin resistance possibly through activation of the hepatic androgen receptor (AR). We investigated the role of hepatocyte AR in hyperandrogenemia-induced metabolic dysfunction by using several approaches to delete hepatic AR via animal-, cell-, and clinical-based methodologies. We conditionally disrupted hepatocyte AR in female mice developmentally (LivARKO) or acutely by tail vein injection of an adeno-associated virus with a liver-specific promoter for Cre expression in ARfl/fl mice (adLivARKO). We observed normal metabolic function in littermate female Control (ARfl/fl ) and LivARKO (ARfl/fl ; Cre+/- ) mice. Following chronic DHT treatment, female Control mice treated with DHT (Con-DHT) developed impaired glucose tolerance, pyruvate tolerance, and insulin tolerance, not observed in LivARKO mice treated with DHT (LivARKO-DHT). Furthermore, during an euglycemic hyperinsulinemic clamp, the glucose infusion rate was improved in LivARKO-DHT mice compared to Con-DHT mice. Liver from LivARKO, and primary hepatocytes derived from LivARKO, and adLivARKO mice were protected from DHT-induced insulin resistance and increased gluconeogenesis. These data support a paradigm in which elevated androgens in females disrupt metabolic function via hepatic AR and insulin sensitivity was restored by deletion of hepatic AR.

Abstract MP55: Bardet-biedl Syndrome 3 Gene In Pomc Neurons Regulates Glucose Homeostasis And Baroreflex Sensitivity

Bardet-Biedl syndrome (BBS) proteins have emerged as critical regulators of various physiological functions including energy and glucose homeostasis, autonomic function and blood pressure. BBS3 protein is a cilia related protein that mediates the ciliary localization of the BBSome, a protein complex composed of eight BBS protein. Consistent with this, we found that BBS3 gene knockout in human RPE1 cells disrupt the ciliary localization of BBS2 and BBS9 proteins, indicating a loss of BBSome-mediated cargo trafficking to cilia. We previously demonstrated that disruption of the BBSome through Bbs1 gene deletion in the anorexigenic pro-opiomelanocortin (POMC) neurons altered energy homeostasis and sympathetic nerve traffic. However, the significance of BBS3-mediated ciliary localization of the BBSome in POMC neurons is not clear. To address this, we investigated the consequence of Bbs3 gene deletion in POMC neurons . We generated mice lacking the Bbs3 gene in POMC neurons by crossing Bbs3 fl/fl mice with mice expressing inducible Cre in POMC neurons (POMC ERCre ). To visualize Cre recombinase we further crossed POMC ERCre /Bbs3 fl/fl mice with tdTomato reporter mice. To induce Cre expression tamoxifen (75 mg/kg for 5 days) was injected at 6 weeks of age. Interestingly, both male and female POMC ERCre /Bbs3 fl/fl mice did not develop obesity as indicated by the lack of difference in body weight (male 28.1 + 0.5 vs 28.3 + 1.2g, female 21.9 + 0.7 vs 21.4 + 0.5g), fat mass (male 2.9 + .0.4 vs 3.4 + 0.5g, female 2.7 + .3 vs 2.3 + 0.2g) and weight of white and brown fat pads. Insulin and glucose tolerance tests revealed that POMC ERCre /Bbs3 fl/fl mice have glucose intolerance, whereas insulin sensitivity was normal. Blood pressure and renal SNA were comparable between POMC ERCre /Bbs3 fl/fl and control mice measured in the conscious state. In contrast, measurement of renal SNA response to changes in arterial pressure evoked by infusion of sodium nitroprusside and phenylephrine showed reduced baroreflex sensitivity in POMC ERCre /Bbs3 fl/fl mice. These findings demonstrate that POMC neuron BBS3, that mediate cargo transport to cilia, is not necessary for the regulation of energy homeostasis, but is involved in the regulation of glucose homeostasis and baroreflex sensitivity.

Distal Lck Promoter-Driven Cre Shows Cell Type-Specific Function in Innate-like T Cells.

Innate-like T cells, including invariant NKT cells, mucosal-associated invariant T (MAIT) cells, and γ δ T (γδT) cells, are groups of unconventional T lymphocytes. They play important roles in the immune system. Because of the lack of Cre recombinase lines that are specific for innate-like T cells, pan–T cell Cre lines are often used to study innate-like T cells. In this study, we found that distal Lck promoter–driven Cre (dLckCre) in which the distal Lck gene promoter drives Cre expression in the late stage of thymocyte development has limited function in the innate-like T cells using ROSA26floxed-Stop-tdTomato reporter. Innate-like T cells differentiate into mature functional subsets comparable to the CD4+ Th subsets under homeostatic conditions. We further showed that dLckCre-expressing γδT cells are strongly biased toward γδT1 phenotype. Interestingly, the γδT cells residing in the epidermis and comprising the vast majority of dendritic epidermal T cells nearly all express dLckCre, indicating dLckCre is a useful tool for studying dendritic epidermal T cells. Taken together, these data suggest that Lck distal promoter has different activity in the conventional and unconventional T cells. The use of dLCKcre transgenic mice in the innate-like T cells needs to be guided by a reporter for the dLckCre function.

The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair

Increased permeability is implicated in the pathogenesis of intestinal disease. Invitro and invivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested invivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial-specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1-deficient colonic epithelia failed to up-regulate proliferation in response to damage invivo or Wnt signaling invitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. ZO-1 makes critical, tight junction-independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

Epididymal initial segment-specific Cre recombinase activity in Lcn8-Cre knock-in mice.

Sperm acquire the ability to fertilize ova through a complex process of epididymal maturation. To identify the functions of genes expressed in the proximal epididymis, mouse models specific to this region are needed. A Lcn8-Cre knock-in mouse line was generated using CRISPR/Cas9 technology. A 37bp coding sequence of Lcn8 from the ATG start codon was replaced by an NLS-Cre-polyA cassette, resulting in Cre expression and the absence of Lcn8. Epididymal initial segment-specific Cre expression was identified using RT-PCR and western blotting, and the spatial-temporal Cre activity was further confirmed by using the Rosa26tdTomato reporter mice. Immunofluorescence staining showed that active Cre recombinase was present in the principal cells. Histological analyses of sperm and epididymides, and the four-month mating tests, were used to confirm that Cre expression did not affect normal development and male fecundity. The novel Lcn8-Cre mice can be used to establish epididymal initial segment-specific conditional knock-out mouse models.

A novel mouse line with epididymal initial segment-specific expression of Cre recombinase driven by the endogenous Lcn9 promoter.

Spermatozoa released from testes undergo a maturation process and acquire the capacity to fertilize ova through epididymal transit. The epididymis is divided into four regions, each with unique morphology, gene profile, luminal microenvironment and distinct function. To study the functions of relevant genes in the epididymal initial segment (IS), a novel IS-specific mouse model, Lcn9-Cre knock-in (KI) mouse line was generated via CRISPR/Cas9 technology. The TAG stop codon was replaced by a 2A-NLS-Cre cassette, resulting in the co-expression of Lcn9 and Cre recombinase. IS-specific Cre expression was first observed from postnatal day 17. Using the Rosa26tdTomato reporter mice, the Cre-mediated DNA recombination was detected exclusively in principal cells. The epididymal IS-specific Cre activity in vivo was further confirmed using Lcn9-Cre mice crossed with a mouse strain carrying Tsc1 floxed alleles (Tsc1flox/+). Cre expression did not affect either normal development or male fecundity. Different from any epididymis-specific Cre mice reported previously, the novel Lcn9-Cre mouse line can be used to introduce entire IS-specific conditional gene editing for gene functional study.

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.

Abstract LB247: A forward genetic screen to identify drivers of neuroendocrine prostate cancer

Abstract Neuroendocrine prostate cancer (NEPC) is a deadly variant of prostate cancer for which no cure is yet available. Up to 20 percent of the patients with castration resistant prostate cancer (CRPC), will progress to NEPC after treatment with standard-of-care therapies, such as abiraterone and enzalutamide, that target the androgen receptor (AR) pathway. However, the molecular mechanisms that drive the emergence of NEPC are largely unknown, contributing to a lack of treatment options for patients with NEPC. We implemented a forward genetic approach to identify novel drivers of NEPC, by using the Sleeping Beauty (SB) transposon-based forward genetic mutagenesis screening system in our Pten-, Trp53-deficient mouse model that is primed to develop NEPC at a low frequency. We generated a mouse strain (NPp53SBT2Onc) in which the Nkx3.1 promoter regulates the expression of tamoxifen-inducible Cre specifically in the mouse prostate epithelium, resulting in transposition of the T2Onc2 transposable element and conditional deletion of Pten and Trp53 in mouse prostate epithelium. We observed an increased NEPC incidence in our NPp53SBT2Onc mouse model compared to control mice without the transposon T2Onc2, suggesting that the SB-mediated insertional mutagenesis events are driving the development of NEPC. To elucidate the genetic events that are driving the observed NEPC phenotype, we identified the common insertion sites (CISs) of the transposon insertions, which revealed a total of 330 CIS-associated genes. We further performed an integrative analysis using RNA-seq data from our SB cohort, a second mouse dataset and a metastatic CRPC patient cohort, which allowed us to identify the Master Regulator (MR) proteins that are associated with NEPC, as well as the upstream genomic events that modulate these MRs and the emergence of NEPC. Altogether, our approach resulted in the identification of CIS-associated genes that are upstream of these MRs associated to NEPC and that are likely to drive this lethal subtype of prostate cancer. We will further validate and elucidate the role of these candidate genes in prostate cancer progression to NEPC, in both mouse and human using CRISPR technology. In summary, we have developed a Sleeping Beauty forward genetic screen in our mouse model of CRPC, which not only accelerates disease progression, but also increases the incidence of NEPC. Our integrative analysis identified a panel of CIS-associated genes that are likely to be functionally involved in progression to NEPC. Validation of candidate drivers resulting from our analysis will shed light on mechanisms that this aggressive disease uses to evade treatments and may inform the development of novel therapies. Citation Format: Francisca Nunes de Almeida, Alessandro Vasciaveo, Min Zou, Matteo Di Bernardo, Andrea Califano, Cory Abate-Shen. A forward genetic screen to identify drivers of neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB247.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Who am I? (re-)Defining fibroblast identity and immunological function in the age of bioinformatics.

Expression of Functional Human Proteinase Activated Receptor (PAR)-1 on Mouse Platelets

Thrombin activates platelets by specific cleavage of PARs leading to platelet aggregation. Human (h) platelets express PAR-1 and PAR-4, each with a different sensitivity to thrombin and specific kinetics of activation/deactivation influencing thrombogenesis and thrombus stability. PAR-1 and PAR-4 are candidates for the generation of anti-thrombotic drugs; indeed, a PAR-1 inhibitor (Vorapaxar) is already in clinical use. However, side effects such as bleeding warrant a continuous effort towards the generation of novel alternative inhibitors. In this regard, human PAR-4 may be a more selective antithrombotic target with a lesser impact on hemostasis. Mice are commonly used to test anti-thrombotic drugs and their mechanisms of action, but cannot be used in this instance because mouse (m) platelets express different PAR subtypes than human, i.e. PAR-3 and PAR-4. Moreover, mPAR-3 cannot be cleaved by thrombin and only acts as a co-receptor enhancing mPAR-4 response. In human platelets, PAR-1 and PAR-4 can act independently of one another. Yet, a different platelet membrane receptor, unrelated to PARs, which binds thrombin with high affinity - glycoprotein (GP) Ibα in the GPIb-IX-V complex - enhances PAR-1 response to low-dose thrombin through a mechanism only partially elucidated. Humanizing mouse platelets with respect to PAR expression would greatly facilitate explaining the functional interplay of GPIb with PAR-1 and PAR-4 and, notably, the role of these different PARs in hemostasis and thrombosis. Attempts to introduce hPAR-1 in mouse platelets have been so far unsuccessful (French et al. 2016, Arachiche et al. 2014) and claims have been made that obstacles to achieve this goal may be unsurpassable. Using a different approach, we introduced a floxed hPAR-1 transgene in the ROSA26 locus of C57BL6/J mice under the strong CAG promoter. The presence of an IRES sequence and a second transgene for eGFP allowed us to screen efficiently for platelets expressing hPAR1. Mice positive for the floxed-transgene were then bred with either a germ-line Cre recombinase-expressing strain (EIIa-Cre) or one in which Cre expression was linked to a platelet specific receptor (PF4-Cre). The percentage of platelets isolated from EIIa-Cre/PAR1+/- mice expressing eGFP was between 35 to 40% and breeding to homozygosity did not increase transgene expression. In contrast, eGFP expression was positive in nearly all PF4-Cre/PAR1+/-mice, suggesting that suppression of hPAR1 expression at the germ-line level could be advantageous for megakaryocyte development. As a functional test for transgene-expressed hPAR-1 we measured aggregation and intracellular calcium increase in washed platelets from PF4-Cre/PAR1+/+ mice stimulated by 5 μmol/liter of a specific PAR-1 activation peptide (TFLLRN, P1-AP). In both assays, the response of human or hPAR-1 expressing mouse platelets was indistinguishable; littermate control platelets lacking transgene expression (floxed hPAR1) did not respond to a 10 times higher concentration of peptide. Interestingly, when stimulated with a low dose of thrombin (0.25 nM) aggregation and intracellular calcium increase of PF4-Cre/PAR1+/+ platelets was similar to littermate controls. We reasoned that this could be a consequence of the presence of mPAR3, owning to its high affinity for thrombin. Thus we proceeded to cross PF4-Cre/PAR1+/+ with mPAR3-/- mice, so to obtain mice that express only mPAR4 and hPAR1. Aggregation induced with P1-AP (5 μM) was similar with platelets expressing PF4-Cre/PAR1+/+ and mPAR3-/-/PF4-Cre/PAR1+/+. As expected from the cofactor role of mPAR3, the minimal amount of thrombin necessary to aggregate fully platelets from mPAR3-/-/PF4-Cre/PAR1-/- was higher than in wild-type mice (2 nM vs 0.25 nM); however, 2 nM was also the minimum thrombin concentration necessary to activate mPAR3-/-/PF4-Cre/PAR1+/+ platelets. Therefore, the presence of a functional hPAR1 responsive to P1-AP does not influence the response of mouse platelets to low thrombin concentrations mediated by mPAR4, indicating that hPAR-1 and mPAR-4, like the human homologue, respond to thrombin independently of one another. In conclusion, to date we have shown that expression of a functional hPAR1 in mouse platelets is possible and should provide a versatile animal model to address several open questions on the role of platelet PAR-1 and GPIbα in hemostasis and thrombosis. DisclosuresNo relevant conflicts of interest to declare.