Pamela Mellon - US grants

The anterior pituitary plays a central role in the regulation of reproductive function, growth, lactation, stress response, and endocrine homeostasis. The pituitary also serves as an excellent model in which to study the complex molecular interactions involved in development and organogenesis in mammalian systems. The anterior lobe rises from the somatic ectoderm by formation of Rathke's pouch. Within this primitive organ five distinct endocrine cell types arise. These are, in developmental order of appearance: corticotropes which produce pro- opiomelanocortin, thyrotropes which produce thyrotropin-releasing hormone (TSH), gonadotropes which produce both luteinizing hormone (LH) and follicle-stimulating hormone (FSH), somatotropes which produce growth hormone, and lactotropes which produce prolactin. Substantial information is available concerning the molecular events important for regulation of growth hormone and prolactin gene expression; however, much less is known about the determination of pituitary gene expression of the family of glycoprotein hormones, LH, FSH,and TSH. These hormones are heterodimeric proteins composed of a common a subunit and distinct beta subunits encoded by individual gene. The temporal appearance of the individual subunits is not coordinated and indicated that the alpha-subunit gene may be expressed in a common early progenitor cell for all endocrine lineages of the anterior pituitary. Thus, development of the individual cell types may involve independent activation of specific hormone genes coupled with specific restriction of gene expression. The molecular basis of regulation of the LH and FSH genes in gonadotropes could not be effectively investigated heretofore due to lack of appropriate cell lines. Tumors derived by pituitary-specific expression of the SV40 T- antigen oncogene in transgenic mice have allowed us to isolate clonal cell lines representing cells in the developmental lineage of the gonadotrope which express either the common alpha-subunit or both the alpha and LH beta-subunit genes. In this proposal, we investigate three major issues: A. The mechanisms of developmental and tissue-specific control of the gonadotropin genes in pituitary cells, including the roles of both transcriptional activation and restriction in directing unique patterns of gene expression. B. The molecular basis of hormonal regulation of gonadotropin gene expression, with emphasis on induction of gene expression by hypothalamic gonadotropin-releasing hormone and repression by gonadal steroids. C. The molecular events determining the developmental lineage of the gonadotrope in the anterior pituitary, utilizing approaches transgenic mice including targeted immortalization, cell ablation, and ectopic expression of regulatory proteins. These investigations will lead to detailed understanding of the molecular events governing the developmental and hormonal regulation of the gonadotrope and pituitary development.

DESCRIPTION (provided by applicant): Gonadotropin-releasing hormone (GnRH) drives the hypothalamic-pituitary-gonadal axis. The GnRH neurons have an extraordinary origin. In the mouse, they arise in the presumptive vomeronasal organ within the olfactory placode at e11.5. As they differentiate, they migrate past the cribriform plate, through the olfactory bulb and into the hypothalamus, finally extending their axons to the median eminence. This discrete population of ~800 neurons, scattered through the hypothalamus, controls puberty, menstrual cyclicity, fertility, and meno- pause. Disrupted migration or disregulated secretion results in failures in puberty, fertility, and reproductive function. The overall goal of this renewal application is to elucidate the molecular, epigenetic, and developmental mechanisms that regulate hypothalamic GnRH neuron migration and maturation. We will utilize two major model systems: our immortalized GnRH-secreting hypothalamic cells (GT1) and genetically modified mice. Under the support of this long-standing grant, we have identified evolutionarily conserved promoter and enhancer regions that target GnRH gene expression exclusively to cultured GT1 cells in vitro and to GnRH neurons in vivo. We have shown that these GnRH regulatory regions are controlled by transcriptional regulators: Oct1, NF1, Gata4, Otx2, Pbx, Prep, Meis, Dlx, Msx, necdin, C/EBP¿, Six6/3, and the TLE (Grg) co- repressors, and that mouse models lacking any one of a number of these fail to produce the correct population of GnRH neurons and/or disrupt migration in vivo. For this renewal application, we propose three novel aims: Aim 1 will address the differentiation and maturation of the GnRH neuron during migration determining the fate of neurons that lack expression of factors in the Dlx/Msx/Necdin pathway, and elucidate the mechanism of Msx repression of GnRH gene expression. Aim 2 will focus on specific homeodomain factors crucial for the development of the hypothalamus as a whole and determine their role in GnRH neuron migration and maturation in vivo and in vitro. Aim 3 will address the fundamental mechanisms of the neuron-specific enhancers of the GnRH gene. We have discovered that the major enhancer critical for neuron-specific GnRH expression is bound by activated Polymerase II and is transcribed into RNA. This long, noncoding RNA will be characterized in vitro and in vivo and its mechanisms of action will be determined. The homologous enhancers in human will be analyzed and screened for mutations in hypogonadal patient DNA. Our overarching hypothesis is that developmental migration and maturation of the GnRH neuron require the acquisition of a transcriptional regulatory program that relies on developmental coordination of homeodomain transcription factors and long non-coding enhancer RNA. We believe these studies will provide valuable insight into novel regulatory mechanisms and enable progression beyond the currently accepted paradigms. This multifaceted approach should yield a comprehensive understanding of the program of GnRH neuronal migration and cell fate in vivo and in vitro and provide insight into the genetics of hypogonadotropic hypogonadism.

The anterior pituitary gonadotrope is a unique endocrine cell which serves as the focal point for integration of hormonal signals in the regulation of the menstrual cycle. It expresses both FSH and LH, it responds to gonadal steroids in the classical feedback loop, to pulses of GnRH from the hypothalamus, and to the interplay of activin, inhibin, and follistatin produced not only in the gonad but in the gonadotrope itself. This remarkable confluence of interacting hormones, growth factors, and receptors within a single cell denotes an intricate set of endocrine and autocrine responses that are differentially modulated to regulate transcription and secretion of FSH and LH, in the delicately fluctuating equilibrium controlling reproductive function. The molecular basis of developmental and hormonal control in the gonadotrope could not be effectively investigated heretofore due to the lack of differentiated cell lines. Methods for targeted oncogenesis in transgenic mice have enabled us to derive immortalized endocrine cell lines from the pituitary. Our success in immortalization of cells representing progenitors for the gonadotrope and somatotrope provides the opportunity to expand this approach to the study of the FSH beta-subunit gene by targeting oncogenesis to a later stage gonadotrope. In Unit 2, we propose to develop an immortal cultured cell line representing a mature anterior pituitary gonadotrope. This cell line and the existing gonadotrope cell lines will be used to investigate the mechanisms of developmental control of the FSH beta gene in pituitary cells and to investigate the molecular basis of hormonal regulation of FSH beta gene expression by steroids, growth factors and releasing hormones. Furthermore, to understand the roles of autocrine response and regulation of sensitivity to hormonal input, we will investigate hormonal control of the activin/inhibin subunit genes, the follistatin gene, the activin receptor gene, and the GnRH receptor gene in the gonadotrope of the anterior pituitary. The role of developmental status and the interplay of hormones, growth factors, and receptors in controlling FSH beta-subunit gene expression will thus be investigated using the armamentarium of molecular, cellular, and transgenic animal technologies with a focus on understanding the role of FSH regulation in the menstrual cycle.

DNA binding protein; DNA footprinting; genetic regulation; hormone regulation /control mechanism; laboratory mouse

DESCRIPTION (provided by applicant): The UCSD Training Program in Reproductive Sciences takes a multidisciplinary approach to the training of postdoctoral scholars as physician-scientists and basic scientists in the field of neuroendocrine and endocrine control of reproductive processes. Three fellows are supported in two categories: those seeking advanced basic research experience after the Ph.D. or M.D. degree, and those seeking clinical and research training with the aim of becoming board-certified in Reproductive Endocrinology after residency in ob/gyn or in endocrinology after residency in medicine. A cohesive group of faculty members from the Division of Reproductive Endocrinology in the Department of Reproductive Medicine and the Division of Endocrinology in the Department of Medicine at UCSD with common interests and complimentary backgrounds provides basic and clinical training and fosters the careers of the trainees. Many of the faculty members are members of the UCSD NICHD Specialized Cooperative Centers Program in Reproductive Research and many of the senior faculty members are mentors in the NICHD Women's Reproductive Health Research Program for the training of junior faculty members in research careers. Thus, this Training Program is integrated with both NICHD Centers, allowing the teaching staff and fellows to interact at many levels, creating an atmosphere of cooperation, collaboration, and career support. Our research activities range from molecular to patient-oriented research utilizing models from in vitro analysis and cell culture, to whole animal and clinical research. Major foci of investigation include: pituitary and hypothalamic development, ovarian physiology, polycystic ovary disease, signal transduction, GnRH and gonadotropin gene expression and secretion, metabolic impact on reproductive function, activin and growth factors in reproduction, and estrogen replacement therapies. The Program of training for the fellows includes group meetings and presentations, journal clubs, clinical activity and training, laboratory training and independent research, seminars, national meetings, and course work in molecular biology, neuroendocrinology, biostatistics, and ethics. Our aim is to prepare physician-scientists and basic scientists to become the future leaders in academic reproductive research. The urgency and importance of producing critically needed basic, translational, and clinical scientists in the area of women's health is well recognized by the leadership of NIH, the scientific community, the national government, and the society as a whole.

Normal reproductive function requires the precise orchestration of hormonal regulation at the hypothalamic, pituitary, and gonadal levels. Within the anterior pituitary, the gonadotrope serves as the control center for integration of hormonal signals, producing luteinizing hormone (LH) and follicle-stimulating hormone to regulate reproduction. This cell responds to pulses of gonadotropin-releasing hormone (GnRH) from the hypothalamus through the GnRH receptor, to endocrine and autocrine activin and follistatin through activin receptors, and to steroid hormone feedback from the gonads. In Research Project I,. Our focus will be the cellular and molecular mechanisms of GnRH and activin regulation of LH and GnRH receptor gene expression in the gonadotrope. Our model system is the mouse, due to the facile manipulation s of the genome attainable by transgenic technology and the ability to exploit our immortal mouse pituitary gonadotrope cell lines that express both LH subunits, activin B, follistatin, and activin, GnRH, and steroid receptors. In the first two aims, these cell lines will be used to investigate the molecular basis of hormonal regulation in the gonadotrope. We have demonstrated that constant GnRH or its second messengers will down regulate, and short-term or pulsatile GnRH will induce, the LHBeta subunit gene in the LbetaT cells. In Specific Aim 1, we will determine the signaling pathways and their transcriptional targets for long-term repression, and short-term and pulsatile induction of the LHBeta gene, and the mechanism for induction of the Alpha-subunit gene by GnRH. Activin sensitizes the gonadotrope to GnRH by inducing the GnRH receptor mRNA, while follistatin counteracts this response. We have mapped the sequences responsive to activin/follistatin regulation in the mouse GnRH receptor gene and demonstrated that activin treatment induces, and follistatin represses, the activity of a nuclear DNA-binding protein. In Specific Aim 2 we will investigate the induction of the GnRH receptor gene and the repression of the alpha-subunit gene by activin. The mouse alpha-subunit gene is regulated by GnRH in alpha T3 cells through an Ets binding site. In Specific Aim 3, we will address the role of MAPKinase signaling through Ets proteins in GnRH action in transgenic mice. In addition, we will investigate the physiological role of activin signaling exclusively in the gonadotrope in transgenic mice.

The goal of the Center for the Study of Reproductive Biology and Disease will be do develop understanding of the mechanisms which govern normal and disordered function of the hypothalamic-pituitary- ovarian axis. We will continue to produce novel and important contributions os the reproductive sciences, integrating multi- disciplinary clinical and basic research to facilitate and accelerate the translation of promising new discoveries into clinical medicine. We have chosen five innovative research projects, all with experienced, internationally renowned leaders. Research project 11 will investigate the molecular mechanisms of gene regulation in the gonadotrope with specific focus on responses to GnRH and the activin/follistatin system and their interactions. Research project 12 will chart new territory in the role of the oocyte in follicular maturation studying GDF-9 in both rat and human ovaries. Research Project 13 will provide detailed molecular understanding of the signal transduction cascade of GnRH. Analyses will include the roles of G proteins both alpha and Betagamma subunits, the role of MAP kinase and ras, and differential use signaling pathways for various actions of GnRH in the gonadotrope. Research Project 14 will investigate the roles of the Bone Morphogenetic Proteins, members of the TGF-Beta superfamily, in ovarian physiology. Emphasis will be placed on testing the new hypothesis that BMPs are luteinization inhibitors and their activities are regulated by follistatin. Analyses will include cultured rat and human granulosa cells and rats in vivo. Research project 15 will focus on the influence of insulin in the endocrine control of LH release and steroid synthesis by the ovarian granulosa and theca compartments in vivo in normal women and patients with polycytic ovary syndrome. Two closed Cored are proposed: the Administrative Core and the Recombinant Protein Core. The Administrative Core will provide administrative, fiscal, planning and meeting services and administer the Enrichment Program. The Recombinant Protein Core will meet the needs of the Research Projects for production, purification and characterization of proteins and antibodies that are unavailable elsewhere. We envision that this integrated research Center will generate a repertoire of discoveries that will provide a comprehensive understanding of the molecules, cells, and systems involved in controlling reproductive function, leading to improved treatment of disease.

The goal of the Center for the Study of Reproductive Biology and Disease will be do develop understanding of the mechanisms which govern normal and disordered function of the hypothalamic-pituitary- ovarian axis. We will continue to produce novel and important contributions os the reproductive sciences, integrating multi- disciplinary clinical and basic research to facilitate and accelerate the translation of promising new discoveries into clinical medicine. We have chosen five innovative research projects, all with experienced, internationally renowned leaders. Research project 11 will investigate the molecular mechanisms of gene regulation in the gonadotrope with specific focus on responses to GnRH and the activin/follistatin system and their interactions. Research project 12 will chart new territory in the role of the oocyte in follicular maturation studying GDF-9 in both rat and human ovaries. Research Project 13 will provide detailed molecular understanding of the signal transduction cascade of GnRH. Analyses will include the roles of G proteins both alpha and Betagamma subunits, the role of MAP kinase and ras, and differential use signaling pathways for various actions of GnRH in the gonadotrope. Research Project 14 will investigate the roles of the Bone Morphogenetic Proteins, members of the TGF-Beta superfamily, in ovarian physiology. Emphasis will be placed on testing the new hypothesis that BMPs are luteinization inhibitors and their activities are regulated by follistatin. Analyses will include cultured rat and human granulosa cells and rats in vivo. Research project 15 will focus on the influence of insulin in the endocrine control of LH release and steroid synthesis by the ovarian granulosa and theca compartments in vivo in normal women and patients with polycytic ovary syndrome. Two closed Cored are proposed: the Administrative Core and the Recombinant Protein Core. The Administrative Core will provide administrative, fiscal, planning and meeting services and administer the Enrichment Program. The Recombinant Protein Core will meet the needs of the Research Projects for production, purification and characterization of proteins and antibodies that are unavailable elsewhere. We envision that this integrated research Center will generate a repertoire of discoveries that will provide a comprehensive understanding of the molecules, cells, and systems involved in controlling reproductive function, leading to improved treatment of disease.

The use of genetically engineered mice has grown exponentially. Transgenic and genetically deficient mice are now widely used and extremely valuable for the study of the mechanisms of diabetes, endocrine disorders, obesity, and microvascular and macrovascular complications of diabetes as well as elucidating the roles of individual genes in normal physiology, development, and endocrine function. In fact, the genetic manipulation of mice is used in virtually every field of biomedical investigation. It is arguably the most potent tool available to dissect gene function in physiology and pathophysiology. However, because of the prohibitive costs and the technical sophistication required to establish these procedures within individual laboratories, it is essential to support these technologies as a core facility. This requires an efficient and cost-effective common facility that enables individual investigators to have both transgenic mice expressing genes of interest and mice carrying gene disruptions or substitutions created for them. The techniques available from this Core place these potent tools for analysis in the hands of all of our investigators. When used in combination with the other modern techniques supplied by the Center's Mouse Phenotyping, Transcriptional Genomics, and Biochemistry and Molecular Assay Core Core facilities, it gives our investigators unlimited opportunities to examine these processes from several perspectives simultaneously. Our current facility has an established track record of enabling the creation of both transgenic mice and knockout mouse strains for multiple UCSD laboratories. The availability of this Core facility to the entire DERC membership will greatly facilitate the research of the entire diabetes/endocrinology community in Southern California. Without such a centralized resource, most laboratories would not be in a position to utilize these powerful models, severely limiting the research and granting opportunities for our diabetes community. The Transgenic and Knock-out Mouse Core of UCSD is widely respected in the community and regarded as an essential resource for modern research. The availability of its services at substantially discounted rates will substantially improves the quality, quantity and creativity of the research, while also enhancina cost-effectiveness and efficiency.

[unreadable] DESCRIPTION (provided by applicant): The delicate balance of hormonal influences that governs gonadotropin production by the pituitary gonadotrope cell includes activin and follistatin (derived from the gonad and from the gonadotrope itself), the pulsatile pattern of the hypothalamic releasing hormone, GnRH, and steroid hormone feedback. In this revised application, our focus will be on the molecular mechanisms of activin, GnRH, and steroid hormone regulation of FSH beta-subunit gene expression in the gonadotrope. Our overall hypothesis is that activin is critical for appropriate regulation of the FSH beta-subunit gene including full regulation by GnRH and steroid hormones. Our model system is the mouse, due to the facile manipulations of the genome attainable using genetic technology and the ability to exploit our immortalized mouse gonadotrope cell lines that express FSH, LH, activin, follistatin, and the receptors for GnRH, activin, androgens, progestins, and glucocorticoids. In Specific Aim 1, we will focus on the molecular mechanisms of induction of the FSH beta-subunit gene by activin and the physiological role of activin signaling in the gonadotrope in vivo in Smad-deficient mice. In Specific Aim 2, we will investigate the molecular mechanisms for tonic and pulsatile regulation of the FSH beta-subunit gene and study the dependence of GnRH action on the activin autocrine loop in culture. In Specific Aim 3, we will address the roles of steroid hormone signaling in regulation of the FSH beta-subunit gene focusing on androgens, progestins, and glucocorticoids. As is the case for GnRH, full steroid hormone induction of the FSH beta-subunit gene is dependent upon activin autocrine tone in the gonadotrope and synergistic with induction by activin. We will investigate the basis of this interaction at the molecular level. Finally, we will utilize targeted disruption of the steroid receptor genes in the gonadotrope in mice to assess the role of these receptors in the pituitary in vivo. Our overall goal is to address the integration of activin, GnRH, and steroid hormone signaling in the regulation of FSH in the gonadotrope. Thus, the interplay of peptide hormones, growth factors, steroids, receptors, and hypothalamic releasing factors in controlling gonadotropin gene expression will be investigated using the armamentarium of molecular, cellular, genetic, and mouse technologies with the goal of developing a detailed understanding of the molecular mechanisms mediating reproductive function at the level of the pituitary. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of the Center for Reproductive Science and Medicine at UCSD is to develop understanding of the mechanisms that govern normal and disordered function of the hypothalamic-pituitary-ovarian axis. This application represents our renewal for years 29-33. Our productivity has been outstanding with 60 papers published and 9 submitted in 3 years of a 4-year award. We will continue to produce novel, significant contributions to the reproductive sciences, integrating multidisciplinary clinical, translational, and basic research to facilitate and accelerate the translation of promising new discoveries into clinical medicine. We are proposing 3 integrated, innovative Research Projects, all with experienced, internationally renowned leaders. Project I (Pamela L. Mellon, PL) will address the hormonal control of the pituitary gonadotrope, focusing on regulation by activin, GnRH, and steroid hormones in vitro and in vivo. The emphasis will be on understanding the synergy and interdependence between these hormones in controlling transcription in model immortalized gonadotrope cells, genetically modified mice, and mouse models of precocious puberty and PCOS. Project II (Jerrold M. Olefsky, PL) will chart new territory in the role of metabolic control in fertility. A dual in vivo/in vitro approach will elucidate the mechanisms of adiponectin, SirT1, and PPARy actions in regulating reproduction, using immortalized hypothalamic GnRH-secreting neurons and gonadotrope cells, novel genetically modified mice, and mouse models of PCOS. Project III (R. Jeffrey Chang, PL) will delineate the relative roles of specific factors implicated in excess production of androgen by the ovarian theca cell in women with PCOS and undertake studies in human ovary culture systems, addressing fundamental mechanisms underlying PCOS. All Project Leaders serve as Co-l's on other components of the Center and all 3 projects include teams of very experienced investigators. The Projects are highly interactive and synergistic, creating a coherent mechanistic and translational Center. The Administrative Core supports the Center, provides the Enrichment Program, and facilitates interactions within the Center and the SCCPIR Program. The SCCPIR Human Ovary Tissue Bank provides tissue to NIH-funded investigators nation-wide.

PROJECT SUMMARY The overall mission of the Administrative Core of the UC San Diego Center for Reproductive Science and Medicine is to provide centralized and coordinated scientific, administrative, and financial management for the three Research Projects, the Pilot and Collaborative Projects, the Enrichment Program, and the Education/Outreach Core within the Center to facilitate translation of basic research information to clinical application. The Administrative Core provides many services to the Center including arranging meetings, seminars, journal clubs, and career guidance sessions. It prepares grants and progress reports. It facilitates interactions among the scientists at the Center and among the Centers nationwide. Our major goals are to ensure appropriate decision-making processes between the Center (represented by the Directors and the Executive Committee) and the Internal Advisory Committee, External Advisory Committee, the NICHD Program Officers, and the Steering Committee of the NCTRI Program. We work to integrate and facilitate the scientific interactions of the Research Projects and the Cores and their scientific staff within the UC San Diego Center, as well as to facilitate research collaborations and interactions within the NCTRI Program. The Administrative Core presents the Enrichment Program of international, national, and local seminar speakers to elevate the profile of reproductive research in the scientific and clinical communities of the San Diego region. We also serve to augment the many training programs in reproductive sciences at our Center including the WRHR NICHD K12 program for junior faculty in Reproductive Medicine, the clinical fellowship in Reproductive Endocrinology and Infertility, the NICHD T32 postdoctoral training grant ?Training in Reproductive Sciences?, the Ph.D. graduate program in Biomedical Sciences, and masters, medical, resident, and undergraduate student research programs. Thus, the Core augments the scientific training of many young individuals at a variety of levels of career advancement. We organize scientific retreats, Executive Committee meetings, Internal and External Advisory Committee meetings, the Reproductive Endocrine Journal Club, career hours, twice monthly research meetings among the faculty and the trainees of the Center, Individual Development Program (IDP) discussions for trainees, monthly research faculty lunches, and meetings of Focus Groups and Directors hosted in La Jolla. We serve as a focus to marshal institutional and community resources in support of reproductive research. In addition, the Administrative Core supervises the application for release of the set- aside funds the Pilot and Collaborative Projects that support young investigators and collaborative, translational projects between our Center and NCTRI Centers nationwide. Most importantly, it fosters outstanding science and encourages the translation of basic science to the bedside.

The delicate balance of hormonal influences that governs gonadotropin production by the pituitary gonadotrope cell includes activin and follistatin (derived from the gonad and from the gonadotrope itself), the pulsatile pattern of the hypothalamic releasing hormone, GnRH, and gonadal steroid hormone feedback. In Research Project I, our focus will be the cellular and molecular mechanisms of activin, GnRH, and steroid hormone regulation of LH and FSH beta-subunit gene expression in the gonadotrope. Our model system is the mouse, due to the facile manipulations of the genome attainable by genetic technology and the ability to exploit our immortal mouse gonadotrope cell lines that express FSH, LH, activin, follistatin, and the receptors for GnRH, activin, androgens, glucocorticoids, and progestins. In Specific Aim 1, we will focus on the induction of the FSH and LH beta-subunit genes by activin and the physiological role of activin signaling in the gonadotrope in vivo in Smad deficient mice. In Specific Aim 2, we will investigate the molecular mechanism for tonic and pulsatile regulation of the LH and FSH beta-subunit genes and study the dependence of GnRH action on the activin autocrine loop in culture. In Specific Aim 3, we will address the roles of steroid hormone signaling in regulation of the FSH beta-subunit gene in culture focusing on androgens, glucocorticoids, and progestins. As is the case for GnRH, steroid hormone induction of the FSH beta-subunit gene is dependent upon activin autocrine tone in the gonadotrope and synergistic with induction by activin. We will investigate the basis of this interaction at the molecular level. Finally, we will utilize targeted disruption of the steroid receptor genes in the gonadotrope in mice to assess the role of these receptors in the pituitary in vivo. Our overall goal is to address the integration of activin, GnRH, and steroid hormone signaling in the regulation of LH and FSH in the gonadotrope. Thus, the interplay of peptide hormones, growth factors, steroids, receptors, and hypothalamic releasing factors in controlling gonadotropin gene expression will be investigated using the armamentarium of molecular, cellular, genetic, and mouse technologies with the goal of developing a detailed understanding of the molecular mechanisms mediating reproductive function at the level of the pituitary.

DESCRIPTION (provided by applicant): It is clear that the progress of toxicological Superfund research during the coming decade will depend upon the mouse as an experimental model to investigate both basic and clinically relevant questions. The high degree of conservation in the genomes of humans and mice makes the approach of using transgene and knock-out gene technology to create models for human toxicology extremely attractive. At the same time, unique differences in metabolism and response to toxic chemicals between mouse and human make the substitution of human genes into the mouse compelling. This core provides this Superfund projects with the most advanced technologies for genetic modification of the mouse genome. Transgenic mice carrying new or novel genes and 'Knock-out' or 'Knock-in' mice lacking specific genes of interest or containing modified version of key genes are created. Conditional expression and Cre/LoxP targeted knock-out strategies are provided. The core provides a wide array of technology- and expertise-intensive services including experimental design consultation, embryonic stem cell homologous recombination, Cre transfection of embryonic stem cells for LoxP excision, blastocyst microinjection of genetically altered embryonic stem cells into blastocysts to create knock-out or knock-in mice, pronuclear injection of transgenes or bacterial artificial chromosomes to create transgenic mice, embryo injecion of Lentivirus vectors, cryopreservation of embryos, and fertility interventions such as in vitro fertilization and ovary transplant. Services are tailored for the projects with special service and high priority. This core is an outstanding example of how extraordinarily specialized techniques, highly trained dedicated personnel, and expensive equipment can be accessed by researchers who could not reasonably expect to develop them on an individual basis. The availability of this Transgenic and Knock-out Mouse Core will enable the projects to conduct versatile, cutting-edge, molecular genetic research in the mouse with a battery of multidisciplinary state-of-the-art techniques.

The use of genetically engineered mice has grown exponentially. Transgenic and genetically deficient mice are now widely used and extremely valuable for the study of the mechanisms of diabetes, endocrine disorders, obesity, and microvascular and macrovascular complications of diabetes as well as elucidating the roles of individual genes in normal physiology, development, and endocrine function. In fact, the genetic manipulation of mice is used in virtually every field of biomedical investigation. It is arguably the most potent tool available to dissect gene function in physiology and pathophysiology. However, because of the prohibitive costs and the technical sophistication required to establish these procedures within individual laboratories, it is essential to support these technologies as a core facility. This requires an efficient and cost-effective common facility that enables individual investigators to have both transgenic mice expressing genes of interest and mice carrying gene disruptions or substitutions created for them. The techniques available from this Core place these potent tools for analysis in the hands of all of our investigators. When used in combination with the other modern techniques supplied by the Center's Mouse Phenotyping, Transcriptional Genomics, and Biochemistry and Molecular Assay Core Core facilities, it gives our investigators unlimited opportunities to examine these processes from several perspectives simultaneously. Our current facility has an established track record of enabling the creation of both transgenic mice and knockout mouse strains for multiple UCSD laboratories. The availability of this Core facility to the entire DERC membership will greatly facilitate the research of the entire diabetes/endocrinology community in Southern California. Without such a centralized resource, most laboratories would not be in a position to utilize these powerful models, severely limiting the research and granting opportunities for our diabetes community. The Transgenic and Knock-out Mouse Core of UCSD is widely respected in the community and regarded as an essential resource for modern research. The availability of its services at substantially discounted rates will substantially improves the quality, quantity and creativity of the research, while also enhancina cost-effectiveness and efficiency.

[unreadable] DESCRIPTION: The anterior pituitary synthesizes hormones that control reproduction, homeostasis, growth, stress response, and lactation. Since each of these physiological responses must be regulated independently, individual pituitary endocrine cell types are specialized to produce distinct hormones. During development, each cell lineage arises in a unique spatial and temporal pattern. The gonadotrope, which regulates reproductive function through the synthesis of the gonadotropins Luteinizing Hormone (LH) and Follicle- Stimulating Hormone (FSH), arises last in this process, as defined by the appearance of the beta subunits of its hallmark proteins. However, its emergence can be traced throughout pituitary development by the sequential appearance of earlier markers of the lineage, first the alpha subunit shared by LH, FSH and Thyroid-Stimulating Hormone, followed by the orphan nuclear receptor Steroidogenic Factor 1, then Gonadotropin-Releasing Hormone Receptor, and finally the LH beta and FSH beta subunits. Using targeted tumorigenesis in transgenic mice, we have developed immortalized cell lines that represent sequential developmental stages of the gonadotrope lineage and the closely related thyrotrope. Utilizing these novel cell models coupled with genetic manipulation in vivo, we propose to elucidate the program of regulators involved in the progression of differentiation to the mature gonadotrope. In the first three aims, we will focus on three select classes of regulatory factors, GATA-binding proteins, basic helix-loop-helix proteins, and LIM-homeodomain proteins. Members of each family are known to directly regulate gonadotropin subunit genes and to be involved in specification of pituitary lineages; however, the balance, interplay, and timing of their actions and those of their co-factors remain to be delineated. These DNA- binding proteins and their associated co-regulators are themselves regulated during lineage progression. Our hypothesis is that programs of closely related factors and their associated co-regulators sequentially occupy control elements in gonadotrope target genes to determine lineage progression and maturation. In the fourth aim, we will take an unbiased approach to defining the mature gonadotrope phenotype at the molecular level, utilizing innovative bioinformatic and molecular methods to identify key components of the gonadotrope differentiation program. [unreadable] [unreadable] [unreadable]

The delicate balance of hormonal influences that governs gonadotropin production by the pituitary gonadotrope cell includes activin and follistatin (derived from the gonad and from the gonadotrope itself), the pulsatile pattern of the hypothalamic releasing hormone, GnRH, and gonadal steroid hormone feedback. In Research Project I, our focus will be the cellular and molecular mechanisms of activin, GnRH, and steroid hormone regulation of LH and FSH beta-subunit gene expression in the gonadotrope. Our model system is the mouse, due to the facile manipulations of the genome attainable by genetic technology and the ability to exploit our immortal mouse gonadotrope cell lines that express FSH, LH, activin, follistatin, and the receptors for GnRH, activin, androgens, glucocorticoids, and progestins. In Specific Aim 1, we will focus on the induction of the FSH and LH beta-subunit genes by activin and the physiological role of activin signaling in the gonadotrope in vivo in Smad deficient mice. In Specific Aim 2, we will investigate the molecular mechanism for tonic and pulsatile regulation of the LH and FSH beta-subunit genes and study the dependence of GnRH action on the activin autocrine loop in culture. In Specific Aim 3, we will address the roles of steroid hormone signaling in regulation of the FSH beta-subunit gene in culture focusing on androgens, glucocorticoids, and progestins. As is the case for GnRH, steroid hormone induction of the FSH beta-subunit gene is dependent upon activin autocrine tone in the gonadotrope and synergistic with induction by activin. We will investigate the basis of this interaction at the molecular level. Finally, we will utilize targeted disruption of the steroid receptor genes in the gonadotrope in mice to assess the role of these receptors in the pituitary in vivo. Our overall goal is to address the integration of activin, GnRH, and steroid hormone signaling in the regulation of LH and FSH in the gonadotrope. Thus, the interplay of peptide hormones, growth factors, steroids, receptors, and hypothalamic releasing factors in controlling gonadotropin gene expression will be investigated using the armamentarium of molecular, cellular, genetic, and mouse technologies with the goal of developing a detailed understanding of the molecular mechanisms mediating reproductive function at the level of the pituitary.

The precise interplay of hormonal influences that governs gonadotropin hormone production by the pituitary includes endocrine, paracrine and autocrine actions of activin and follistatin, hypothalamic gonadotropin-releasing hormone (GnRH), and gonadal steroid hormones. Our focus will be the cellular and molecular mechanisms of activin, GnRH, and steroid hormone regulation of the pituitary gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FSH), using cultured cells in vitro, and their roles in physiology and pathophysiology, in vivo, using targeted gene deletion in mice. In Aim 1, we will focus on the regulation of the gonadotropin genes by activin, its interactions with GnRH, and the physiological role of activin signaling in the gonadotrope, in vivo, in targeted Smad-deficient mice. In Aim 2, we address the roles of progestins and androgens in both negative and positive regulation of the gonadotropin genes and their interactions with activin signaling. Further, we will utilize targeted disruption of the progesterone and androgen receptor genes, in vivo, to assess their role within the pituitary and brain, in normal female reproductive function. Both clinical and animal studies underscore the prenatal origin of premature puberty and polycystic ovary syndrome with the common thread being abnormal androgen exposure in utero. We will use models of high-fat feeding and in utero exposure to androgens to invesigate the hypothesis that androgens, acting through the androgen receptor, are causative for advancement of puberty and for development of the hallmarks of polycystic ovary syndrome. Our overall goal is to understand the integration of activin, GnRH, and steroid hormone action in the regulation of LH and FSH in the gonadotrope in normal and pathopysiological states. Thus, the interplay of peptide hormones, growth factors, steroids, receptors, and hypothalamic releasing factors directly and indirectly controlling gonadotropin synthesis will be investigated using the armamentarium of molecular, cellular, genetic, and mouse technologies with the goal of developing a detailed understanding of the molecular mechanisms mediating normal and disordered female reproductive function.

Transgenic Mouse Many in the biomedical sciences strongly believe that the progress of cancer research, and the position of the UCSD Cancer Center in the biomedical community during the next decade, will depend upon the ability to use the mouse as an experimental model to investigate both basic and clinically relevant questions in cancer research. The creation of new pre-clinical models of cancer in mice by genetic manipulation is specifically cited by the NCI as one of four extraordinary opportunities they wish to exploit. Transgenic mice carrying new or novel genes are created by microinjection of DMA into the pronuclei of fertilized eggs and knock-out mice lacking specific genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric mice for breeding to homozygosity. The high degree of conservation of most sequences in genomes of humans and mice makes the idea of using mouse genetic manipulation technology to create models of human cancer pathogenesis extremely attractive. These approaches are remarkably powerful in cancer research particularly in the analysis of oncogenes, metastasis, cell-cycle control, tumor suppressor genes, and in the crafting of cancer model systems for developing new treatment regimens, and methods for drug testing and tumor imaging. The mission of this Shared Resource is to provide the highly technical aspects of manipulation of genes in embryos and embryonic stem cells as a service to our Center members, allowing them to create the genetically manipulated mouse models they need. This is an outstanding example of how specialized techniques, highly trained dedicated personnel, and expensive equipment can be accessed by researchers who could not reasonably expect to develop or obtain them on an individual basis. The availability of this Resource enables our researchers to conduct versatile, cutting-edge research with a battery of sophisticated genetic techniques.

The Training Core of the UC San Diego Superfund Program supports the interdisciplinary education of junior scientists to become the next generation of environmental researchers in the Environmental Health Sciences. It is a multidisciplinary program at the crossroads of biology, chemistry and environmental sciences which converge in our Superfund program. Our faculty members are affiliated with one or more of several graduate training programs: Chemistry & Biochemistry, Biological Sciences, Biomedical Sciences, and Neurosciences at UCSD or The Scripps Research Institute graduate program, each of which is independently organized and most are multidisciplinary and multi-departmental. Thus, while the supported graduate students may be based in dramatically different scientific disciplines, they share focused interest in environmental health sciences and participate in educational opportunities through meetings, seminar programs, the Community Engagement Core, the Research Translation Core, teaching opportunities, and required classes focused in molecular toxicology and toxicogenomics, ethics, statistics, and environmental health. Training students from a variety of graduate programs in interdisciplinary approaches to environmental sciences is novel and unique. Our program fosters predoctoral training that specifically addresses issues related to environmental health and such programs are critical for the preparation of young investigators to undertake the research so important to improving our environment and providing key information related to toxics in human health. Thus, the interdisciplinary training of Ph.D. graduate students within the extremely rich environment of the UCSD Superfund Program Center, will continue to create a cadre of dedicated, cutting-edge Environmental Health research leaders for the future.

PROJECT SUMMARY (See instructions): The progress of toxicological Superfund biomedical research during the coming decade will depend upon the mouse as an experimental model to investigate both basic and clinically relevant questions. The mouse is the central experimental model for five of the projects in this Program and all utilize genetically altered mice extensively. The Mouse Molecular Genetics Core provides this Superfund Program's biomedical projects with the most advanced technologies for genetic modification of the mouse genome. Transgenic mice carrying new or novel genes, bacterial artificial chromosomes, or siRNA expression vectors are produced. Knock-out mice lacking specific genes of interest or Knock-in mice containing a modified version of a gene or gene cluster are created. Mice with human genes substituted for their mouse homologs are developed. Transgenic mice expressing fluorescent markers in specific cells are created. Conditional expression and tissue-specific targeted knock-out strategies are provided. The core provides a wide array of technology- and expertise-intensive services including experimental design consultation, embryonic stem cell homologous recombination, blastocyst microinjection of genetically altered embryonic stem cells into blastocysts to create knock-out or knock-in mice, genetic strategies and consultation, pronuclear injection of transgenes or bacterial artificial chromosomes to create transgenic mice, cryopreservation of mouse lineages, provision of key marker and genetic manipulation strains, and fertility interventions such as in vitro fertilization and ovary transplant. Services are tailored for the projects with special services, ongoing consultation, and high priority. This Core is an outstanding example of how extraordinarily specialized techniques, highly trained, dedicated personnel, and expensive equipment, can be accessed by researchers who could not reasonably expect to develop them on an individual basis. The availability of this Mouse Molecular Genetics Core will enable our biomedical projects to continue to create key novel mouse models and conduct versatile, cutting-edge, molecular genetic research in the mouse with a battery of multidisciplinary state-of-the-art techniques.

It is clear that the progress of diabetes research during the coming decade will depend heavily upon the ability to utilize the mouse as an experimental model to investigate both basic and clinically relevant questions in diabetes research. The Transgenic and Knock-out Mouse Core (TKMC, Core A) provides investigators at UCLA, UCSD, the Salk Institute, and Cedars-Sinai with a wide array of genetic manipulations in the mouse including transgenic genes, homologous recombination in embryonic stem cells (ES cells), creation of chimeric mice from ES cells, and the most cutting-edge approaches to performing reverse genetics in the mouse. Transgenic, knock-out and knock-in mouse models are created that utilize the most advanced approaches including conditional Tet-inducible and tamoxifen-inducible transgenes, tissue-specific and conditional knock-outs using Cre-LoxP and Flp recombinases and recombination-mediated cassette exchange (RMCE), BAC transgenics, BAC-Trap, RiboTag, and other specialized technologies. This Core is an outstanding example of how extraordinarily specialized techniques, highly trained dedicated personnel, specially constructed facilities, and expensive equipment can be accessed by researchers who could not reasonably expect to develop them on an individual basis. Key objectives are: 1. To create innovative and important mouse models for studies of diabetes and its complications 2. To eliminate barriers to the most cutting-edge mouse genetic approaches for the DERC membership 3. To provide outstanding, reliable, and high quality mouse embryology and genetic services 4. To advance the technology of genetic manipulation of the mouse genome The availability of this Transgenic and Knock-out Mouse Core in coordination with the Metabolic and Molecular Physiology Core, the Genomics and Epigenetic Core, and the Novel Target identification and Assay Development Core, will enable our members to conduct versatile, cutting-edge, reverse genetic research in the mouse with a battery of multidisciplinary, state-of-the-art techniques.

? DESCRIPTION (provided by applicant): Androgen receptor signaling is crucial for normal female reproduction and hyperandrogenemia is a fundamental aspect of the reproductive disruption seen in Polycystic Ovary Syndrome (PCOS). Either lack of androgen receptor or androgen excess disrupts normal ovulation and neuroendocrine control of female reproduction, but the mechanisms for these effects are unknown. Our overall hypotheses are: 1) androgens acting in the brain and pituitary play an important role in regulating normal female fertility, and2) excess androgens, acting through androgen receptor in the hypothalamus and pituitary, are responsible for the neuroendocrine hallmarks of PCOS: high LH, low FSH, and progesterone (P4) resistance. We will test these hypotheses in a series of three independent but interrelated Aims that focus on the role of physiological androgen signaling and androgen excess in the hypothalamus and pituitary, using a combination of genetic and androgen-excess mouse models. Approaches will utilize genetic manipulation of androgen and P4 signaling in specific cell types in normal female mice and in a novel mouse model of obese PCOS. Aim 1 will focus on the brain by assessing whether androgen receptor (AR) is required specifically in neurons for normal female reproduction and for the deleterious effects of androgen excess. Using Cre-loxP technology, AR will be selectively deleted from brain neurons, and subsequently specifically from kisspeptin neurons. Females lacking AR in these neurons will be investigated for resistance to the effects of androgen excess in the new PCOS mouse model. Aim 2 will focus on the pituitary, with the hypothesis that excess androgens act directly at the gonadotrope to dysregulate LH? and FSH? and gonadotropin secretion in females. Female mice lacking AR selectively in the gonadotrope will be studied for altered reproductive function and gonadotropin secretion and in response to excess androgens in the PCOS mouse model. We will use primary pituitary cells in an innovative GnRH pulse system to directly test the actions of androgens on basal and GnRH-induced pulses of LH and FSH and gonadotropin gene expression. Aim 3 will test whether androgen excess interferes with P4 feedback at the hypothalamus and pituitary, as one attribute of PCOS women is impaired P4 feedback. We will determine if progesterone receptor (PR) is regulated by androgen excess in neuroendocrine tissues and whether P4 feedback is impaired by the actions of excess androgens. We will then remove PR selectively from brain neurons, kisspeptin neurons, or pituitary gonadotropes to investigate which cells mediate P4 negative feedback and the alterations caused by androgen excess. Together, these Aims will elucidate the role of AR and PR in the neuroendocrine system in normal female reproduction and illuminate the role of excess androgens in the dysregulation of neuroendocrine control of reproduction.

PROJECT SUMMARY Follicle-stimulating hormone (FSH) is essential for follicular development and fertility in women. Since the FSH? gene was recently reported to be associated with Polycystic Ovary Syndrome (PCOS) and age of menopause in genome-wide association studies (GWAS), we hypothesize that dysregulation of FSH? transcription contributes to female infertility including PCOS and Premature Ovarian Failure (POF). The goal of this proposal is to understand the contributions of FSH to the pathophysiology of the female hypothala- mic-pituitary-ovarian axis utilizing mechanistic analyses in immortalized gonadotrope cells, a sophisticated pri- mary pituitary perifusion system, novel genetic mouse models of PCOS and POF, and a new mouse model of PCOS. Aim 1 will focus on the impact of androgen excess on FSH? transcription and secretion in females. First, we will use primary pituitary cells in an innovative perifusion system to directly test the actions of andro- gens on basal, pulsatile GnRH- and activin-induced regulation of FSH secretion and FSH? gene expression. Then, female mice lacking AR selectively in gonadotropes will be studied for altered reproductive function including FSH? transcription and FSH secretion in response to excess androgens using a letrozole-induced PCOS mouse model. Finally, we will investigate whether decreased FSH production in our letrozole PCOS mouse model is dependent on GnRH or due to changes in the activin autocrine feedback loop. Aim 2 will char- acterize the functions of single nucleotide polymorphisms (SNPs) found within the regulatory sequences of the FSH? gene that are associated with PCOS, age at menopause, and altered LH/FSH ratios in GWAS studies. We have shown that one of these SNPs interferes with LHX3 binding to the proximal FSH? promoter and reduces FSH? gene expression. These SNPs will be analyzed for molecular mechanisms altering FSH? transcription. We will then use CRISPR/Cas9 mutagenesis to create mouse models to determine the effects of these SNP alterations on fertility and FSH secretion in vivo and in primary pituitary cells. Aim 3 will investigate the role of FOXL2 in FSH? regulation and POF. We have shown that FOXL2 interacts with SMAD proteins for activin induction of FSH? gene expression, with cJun for synergy between GnRH and activin, and with proges- terone receptor for synergy between activin and progesterone. First, we will investigate the molecular basis for these interactions and their roles in hormonal regulation of FSH?. Next, FOXL2 mutations found in POF will be studied for their effects on FSH? transcription in coordination with activin, GnRH, and steroid hormones. Lastly, these mutations will be modeled in mice using CRISPR/Cas9 mutagenesis to study effects on fertility and reproductive senescence in vivo and on FSH? gene regulation in primary pituitary cells. Together, these aims will delineate the mechanisms by which androgens and SNP mutations within the FSH? regulatory region alter FSH? gene expression and contribute to PCOS, and how dysregulation of FSH? by mutant FOXL2 proteins contributes to POF. These studies may provide insight for diagnosis and treatment of female infertility.

Core Summary/Abstract Studies of Superfund toxicants require novel mouse models, careful, high-throughput analyses of hormones, cytokines and small molecule metabolites, and informatics analyses of large datasets to pioneer new findings in the detection of environmental toxicants and health effects of exposure. The UC San Diego Superfund Research Center Genetics and Metabolomics Core facility will provide state-of-the-art molecular biology services for generation of murine models of toxicant exposure as well as cutting-edge analytical services for sensitive, high-throughput assay of hormones and small molecule metabolites and their informatic analyses. This Core will interface with all six proposed research projects. Comprehensive approaches for modifying the mouse genome are provided including DNA microinjection, embryonic stem cell homologous recombination, CRISPR/Cas9 mutagenesis, and blastocyst injection, specifically tailored to UCSD Superfund projects. Furthermore, metabolomics services will be provided, allowing for measure of hormones, growth factors, and thousands of small molecule metabolites (both targeted and untargeted) using a Luminex magnetic bead analyzer and liquid chromatography-mass spectrometry based approaches. Samples for analysis will include human and mouse biospecimens (plasma, urine, and stool) as well as community plant, water, and soil samples. Bioinformatics services will provide in-depth analyses of large datasets resulting from transcriptomics, cistromics, and metabolomics. This core provides centralized, cost-effective, efficient, technically sophisticated services that are crucial to the success of all six projects in the UC San Diego Superfund Research Center.

Nonalcoholic fatty liver disease (NAFLD) is escalating in California and the United States and is associated with the rising obesity epidemic. NAFLD is a hepatic phenotype that encompasses the more serious manifestation of steatohepatitis (NASH), considered to be a prerequisite for liver cancer (hepatocellular carcinoma/HCC). We also know from recent clinical studies that chemical toxicant exposure, in the absence of obesity, can lead to toxicant-associated steatohepatitis (TASH), which closely resembles the pathology of NASH. These findings are relevant to SRP stakeholders in Region 9, since California ranks high in National Priorities List (Superfund sites) nationally and contains thousands of additional hazardous waste sites (HWS). In addition, the UCSD NAFLD Research Center shows that American Indians and Hispanics, combined the largest non-Caucasian population in California, are at far greater risk of developing NAFLD. Over the past five years, our SRC has leveraged resources to demonstrate that TASH is greatly accelerated by consumption of high-fat diet (HFD) or after a diabetic episode in mice. These findings are relevant, since we can speculate that American Indians and Hispanics are more susceptible to TASH development, a risk amplified by unhealthy diets, poverty and health disparities. The UCSD SRC will be developing models through its Biomedical projects to characterize the mechanisms of Superfund toxicant induced TASH development and cancer, while the Environmental Science & Engineering (ES&E) projects will develop tools for NPL-toxicant detection and remediation. Biomedical and ES&E projects will be assisted in these efforts by Research Core services that will provide tools for mouse genetic production, metabolomic and sophisticated Bioinformatic analysis. A key objective of the projects is to highlight scientific findings to maximize Research Translation, which will happen with collaborations between the projects and the Research Translation Core. We believe our program is innovative and paradigm shifting, since it has the potential of linking important basic research findings regarding Superfund toxicant exposure with human disease biomarker identification through collaborations with the UCSD NAFLD Research Center, which contains one of the largest NALFD cohort studies in the world. Importantly, our Biomedical findings and tools developed in the ES&E projects will be used as resources and knowledge gained to reduce cumulative impacts and health disparities. These initiatives will be leveraged through Community Engagement Core efforts in disadvantaged Hispanic neighborhoods in San Diego and Mexico where statistics confirms that obesity is an escalating problem both in children and adults. Since the incidence of NAFLD and liver cirrhosis is on the rise in both children and adults and may be linked to toxicant chemical exposure, our findings will be communicated to SRP primary stakeholders at the U.S. Environmental Protection Agency and the Agency for Toxic Substances and Disease Registry, in addition to state and local agencies.

Summary It is clear that the progress of diabetes research during the coming decades will depend heavily upon the ability to utilize the mouse as an experimental model to investigate both basic and clinically relevant questions in diabetes research. The strong conservation between the genomes of humans and mice makes the approach of using genetic alteration mouse technology to create models for human diabetes, endocrine pathologies, and diabetes complications extremely useful. The Transgenic and Knock-out Mouse Core (TKMC) provides investigators at UCLA, UCSD, the Salk Institute, LA Biomed and Cedars-Sinai with a wide array of genetic manipulations in the mouse including transgenic genes, homologous recombination in embryonic stem cells (ES cells), targeted deletions and mutations using CRISPR/Cas9, creation of chimeric mice from ES cells, and the most cutting-edge approaches to performing reverse genetics in the mouse. Transgenic, knock-out and CRISPR mouse models are created that utilize the most advanced approaches including conditional Tet- inducible and tamoxifen-inducible transgenes, tissue-specific and conditional knock-outs using Cre-LoxP and Flp recombinases and recombination?mediated cassette exchange (RMCE), BAC transgenics, BAC-Trap, RiboTag, and other specialized technologies. This Core is an outstanding example of how extraordinarily specialized techniques, highly trained dedicated personnel, specially constructed facilities, and expensive equipment can be accessed by researchers who could not reasonably expect to develop them on an individual basis. Key objectives are: 1. To create innovative and important mouse models for studies of diabetes and its complications 2. To eliminate barriers to the most cutting-edge mouse genetic approaches for the DRC membership 3. To provide outstanding, reliable, and high quality mouse embryology and genetic services 4. To advance the technology of genetic manipulation of the mouse genome The availability of this Transgenic and Knock-out Mouse Core in coordination with the other DRC Cores, will enable our members to conduct versatile, cutting-edge, reverse genetic research in the mouse with a battery of multidisciplinary, state-of-the-art techniques.

TRANSGENIC MOUSE SHARED RESOURCE ABSTRACT The availability of the Transgenic Mouse Shared Resource (TMSR) enables our investigators at the Moores Cancer Center (MCC) to conduct versatile, cutting-edge research with a battery of sophisticated genetic techniques for manipulation of the mouse genome to create models for studies of cancer for incisive in vivo mechanistic investigations of the fundamental processes involved in the etiology of tumor development and metastasis. Transgenic mice carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs, specific deletions and mutations are created using CRISPR/Cas9 technology, and ?knock-out? mice that have deleted or modified genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric mice for breeding to homozygosity. The high degree of conservation of most sequences in genomes of humans and mice makes using mouse genetic manipulation technology to create models of human cancer pathogenesis extremely useful. These approaches are remarkably powerful in cancer research, particularly in the analysis of oncogenes, metastasis, cell-cycle control, tumor suppressor genes, and in the crafting of cancer model systems for developing new treatment regimens, and methods for drug testing and tumor imaging. The mission of this Shared Resource is to provide the highly technical aspects of manipulation of genes in embryos and mouse embryonic stem cells as a service to MCC members, to create the genetically manipulated mouse models they need. This is an example of how specialized techniques, highly trained and dedicated personnel, and expensive equipment can be accessed by researchers who could not reasonably expect to develop or obtain them on an individual basis. The TMSR provides cutting-edge, rapidly advancing, efficient, cost-effective services that adapt to the needs of our users, drive the field of mouse genetics, and continue to be very responsive to the vast creativity of the CCSG membership, fulfilling their needs for the most cutting-edge embryology and molecular genetics in the mouse.