Projects / Programmes
The role of cholesterol biosynthesis during embryogenesis and spermatogenesis using conditional Cyp51 knockout mouse models
| Code |
Science |
Field |
Subfield |
| 4.02.01 |
Biotechnical sciences |
Animal production |
Genetics and selection |
| Code |
Science |
Field |
| B220 |
Biomedical sciences |
Genetics, cytogenetics |
| Code |
Science |
Field |
| 4.02 |
Agricultural and Veterinary Sciences |
Animal and Dairy science |
cholesterol, de novo biosinthesis, Cyp51, conditional transgenesis, embriogenesis, spermatogenesis, innate immunity, TLR, growth and development, fertility, health
Researchers (15)
Organisations (3)
Abstract
BACKGROUND. Research in animal science and biomedicine has been revolutionised about a decade ago by introduction of conditional knockout technology that allows inactivation of genes in a tissue or time specific manner. The use of these transgenic models combined with new experimental “omics” approaches provide a more comprehensive in vivo view of biological processes and expose targets for further development of applications in industry. In spite of several years of cholesterol research not all genes in the cholesterol pathway have been studied in vivo. We developed a novel conditional mouse model to characterize the in vivo role of one of the key genes in cholesterol synthesis pathway, a potential drug target for cholesterol lowering drugs. Here we propose thorough characterisation of our cholesterol gene Cyp51 knockout models to advance the knowledge in cholesterol basic and applied research.
OBJECTIVES
The project contains three main objectives with a common feature of utilisation of our recently developed transgenic models. The first objective will examine the effect of Cyp51 complete knockout and consequently lack of de novo cholesterol biosynthesis in embryogenesis. The second objective will examine the effect of conditional Cyp51 inactivation during spermatogenesis. Hypothesis tested here is that Cyp51 gene products, intermediates FF-MAS and T-MAS, play important role in germ cell development and/or meiosis. The third objective is designed to examine the effect of a complete or partial loss of Cyp51 function on immunity testing a hypothesis that individuals with reduced capacity for de novo cholesterol biosynthesis have aberrant innate immune response that can lead to increased susceptibility to infections and chronic inflammatory states.
ANALYTICAL APPROACHES. All three experiments will be based on our recently developed transgenic models. For objective 1 and 3 the models have already been generated and proved to be suitable for further characterisation, as shown by our preliminary analyses. Various phenotype screens will be performed on embryos, male gonadal tissue, immune cells or relevant organs. Molecular screens will involve DNA, RNA, protein level, sterol analyses as well as screens for responsiveness to agonists of Toll like receptors in activating innate immunity system . High-throughput mRNA analyses of embryos should reveal global changes in gene expression following Cyp51 inactivation. Various data sets and experimental data will finally be integrated with functional genomic approach.
RESEARCH GROUP
This project will integrate compatible expertise and resources of two research teams at the University of Ljubljana (UL) and a team at the National Institute of Chemistry (KI, Ljubljana). Principal investigator (S. Horvat UL, Biotechnical faculty (BF)) has experience in animal transgenesis, mouse model characterisation and genomics. Damjana Rozman (UL, Medical Faculty) holds expertise in Cyp51 and cholesterol homeostasis research as well as in the microarray technology. Roman Jerala (KI) will provide his expertise in the molecular immunology. For the three proposed objectives/experiments, we feel that we chose the most experienced research groups in Slovenia which should enable successful implementation of proposed research.
RELEVANCE.
The proposed research is within the general area of “functional genomics for health”, a priority area of our Ministry (MVZT), Slovenian Research Agency (ARRS) and recent EU framework programmes. The developed new conditional transgenic models should be of interest for the wider international community and spark new collaborations and larger grant applications within the EU funding schemes. We strongly believe that this integrative proposal should yield important insights of basic and applied research interest and could help in further promotion of Slovene science
Significance for science
17.1. For the development of science Advances in basic scientific knowledge We conclude that the results of this research project significantly contributed to the understanding of the biology of cholesterol, especially in the field of embryology, reproduction and molecular immunology. Due to the interdisciplinary nature of the project, our results will be also useful for general areas such as genetics, functional genomics, animal biotechnology and molecular biology. The results of individual work packages are described in detail in section 3, so at this point we will summarize them only briefly. The first experiment was to clarify the importance of Cyp51 and de novo synthesis of cholesterol in the development of the embryo and foetus and showed which organs, tissues and processes in the event of failure of CYP51 were mostly affected. The second experiment revealed the role of Cyp51 and certain intermediates (MAS sterols) on the development of germ cells and hence fertility. This knowledge is very useful in the interpretation of other biosynthetic routes where there is the same phenomenon. The third experiment confirmed the hypothesis on the importance of de novo cholesterol biosynthesis in the immune response. Our publication in the premier journal of CELL Metabolism is the first ever demonstrating that the intermediates of cholesterol biosynthesis can be direct ligands of endogenous genes (in this case a gene RORgama t) with important biological function. This binding is essential for signalling and normal lymphocyte development and, consequently, the functioning of the immune system. These results have changed the current view of the involvement of cholesterol biosynthesis in development processes (eg, immune system function, maturation of gametes), and highlighted the functional role of cholesterol biosynthesis intermediates (MAS) in comparison with the final product of this biosynthetic pathway (cholesterol). The development of genetic resources. An very important product of this project was development of unique genetic resources for future studies of cholesterol homeostasis. As described in Section 3 we have already established links with a number of interested parties from Europe to use genetic resources of this project. Our transgenic mouse model have since been used in numerous studies around the world and also in a FP7 project to study the mechanism and treatment of non-alcoholic fatty liver disease. This is a complex disorder that occurs when 30 million Europeans and may progress to a fatal form of non-alcoholic steatosis. All three transgenic animal models developed during the project will be useful for future studies related to cholesterol homeostasis, which will in the long term contribute to improving the quality of life of people and animals. The development of new technologies: In the past four years, our research group introduced targeted modification of genes in mice in Slovenia. We have established a technology of embryonic stem cells and laboratory breeding of transgenic mice. Transgenic mouse lines used in this project is the first genetically modified mouse model, which was developed in Slovenia without the participation of foreign scientists. We believe that the project has contributed significantly to the further development of the technology of transgenic animal models in Slovenia and the use of these models in the field of functional genomics.
Significance for the country
The benefit to the health and quality of life for humans and animals The project J4-4306 was conceived as a fundamental, and therefore did not include partners and sponsors from industry. We believe, however, that many of the results provide a good basis for planning application projects in the search for new medicines to lower blood cholesterol, infertility treatment and a reduction in immune response and inflammatory diseases. In the future, we intend to establish contact with the relevant companies in Slovenia should they be interested in the transfer of knowledge acquired within this project in the development of new products, especially in the field of biomedicine and health of animals. The results of the first experiment will serve as a basis for applied research associated with the role of cholesterol biosynthesis in the development of humans and animals. One of the possible applications is diagnostics to identify syndromes of cholesterol homeostasis associated with impairments in Cyp51 function. Despite knowledge of the fatal consequences of genetic mutations (eg hypercholesterolemia due to mutations in the LDLR pigs) we often pay too little attention to the importance of research for health and quality of life of animals. Our study has brought new tools (biomarkers) and knowledge which will make it possible to investigate the effects of Cyp51 and cholesterol biosynthesis in the embryonic development of animals (prenatal death) and abnormalities in adult animals. The second experiment has produced new information on the role of Cyp51 and MAS in regulating the maturation of male germ cells. They can be a good basis for applied studies, which will contribute to the interpretation of unexplained causes of male infertility and their treatment. Our third pilot experiment clearly showed that subjects with deffective cholesterol homeostasis have impaired responsiveness of the immune system. Further studies can be applied to investigate if such cases can be treated with diet or pharmacological therapy. Promotion of Slovenian Science The main goal of our project was to contribute knowledge and approaches to better health and quality of life of people and animals, which is actually the main priority of a developed society. The use of new in vivo transgenic models in combination with the latest technologies ("omics") is the most promising research approach to functional genomics. With this project Slovenia joined the global trends in this area since we were unknown in this field so far. That the present project will contribute to the greater visibility of Slovenia testify the impact of our publications. In addition, there is a high interest in transgenic models that have been developed within this project. Our lines of mice were sent around the world to academic and industrial institutions in virtually every continent except Africa. Due to our increased workload in the distribution of these genetic resources, we will now get help from the largest company in the field of breeding and sales of laboratory animals, The Jackson Laboratory in the USA. This company has assumed all the costs of transports and cryopreservation, that is, freezing embryos and sperm of our lines as a safety measure and deposited them in their gene bank of important lines. Training of staff Indirect importance of the project is also in recruiting and training new employees who participated in the project. Within this project two doctoral studies were completed and a number of graduates in the first and second stage of Bologna studies trained. Both doctoral candidates as a result of excellent publications got a job – unfortunately they are now both employed due mainly to the current crisis in employment of young researchers in Slovenia.
Most important scientific results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
Most important socioeconomically and culturally relevant results
Annual report
2011,
2012,
2013,
final report,
complete report on dLib.si
