Final Report Summary - RABPSTEM (Comparative Analysis of Rabbit and Mouse Pluripotency in Embryos and Stem Cells)
The scientific aim of RabPStem was to generate novel comparative information on the regulation of pluripotency in rabbit. The importance of the pluripotency genes in understanding development and health was clearly demonstrated before. In RabPStem we aimed to isolate, sequence and analyse the main pluripotency genes in rabbit as such information was lacking. We intended to explore the differences in the expression signatures between LIF- and FGF-dependent pluripotent stem cells, and to compare it with existing mouse data. Furthermore, characterization of pluripotent rabbit ES and iPS cells would provide valuable informations to be used for further gene targeting to create animal models of human genetic diseases.
The key pluripotency genes from rabbit (Oct4, Nanog, Klf4, c-Myc) were isolated, based on the sequences of other species published so far, and its expression pattern was examined by immunohistochemistry. Sequences of each individual rabbit pluripotency genes were compared to mouse and human sequences from different databases and conserved regions were identified and evolutionary relevancies were established. Immuneprecipitation was used to analyse interactions among pluripotency genes. Direct and indirect interactions have been examined between Oct4 and its suspected targeted genes. In vitro differentiation was performed to analyse the functionality of pluripotency.
The key pluripotency genes from rabbit (Oct4, Nanog, Klf4, c-Myc) were isolated and identified. The rabbit Oct4, C-Myc, Sox2 and Nanog showed higher degree of homology with human than with mouse sequences in nucleotide and amino acid level, only the Klf4 exposed the contrary manner. Whole of the gene expression patterns were examined by immunohistochemistry and successfully detected with commercially available antibodies. Interactions have been examined between Oct4 and its suspected targeted genes by immuneprecipitation. We have found that the Oct4 and Sox2 could interact with FGF receptor, Rex1 and UTF1 and create a complex similar to that in mouse, however, in case of LIF receptor, FBXO15 and OPN these connections were not detected by Western Blot. In this phenomenon the rabbit pluripotent cells are similar to human ESCs. We have demonstrated that rabbit ES and iPS cells are able to differentiate towards neural and cardiac lineage.
RabPStem was expected to understand better pluripotency in rabbit. With the newly isolated rabbit pluripotency genes better iPS cells might be created than before, and it would generate a new platform for further applications of rabbit biotechnology. Our founding helped to highlight the mechanisms of the regulation of pluripotency in non-rodent mammals, with a significant importance to human medical research including the use of human pluripotent stem cell lines, as well.
Socio-economic aspects:
The ageing European population is prone to various diseases, causing immense societal and economic problems. The rabbit is a very relevant animal model for the study of a wide range of human diseases. However, the lack of embryonic stem cell technology that would make it possible to generate genetically modified animals by gene targeting, as it was developed in the mouse 20 years ago, severely hampers the full exploitation of the rabbit model. The RabPStem project was aimed to overcome this technological barrier. The isolated and characterized rabbit pluripotency genes provide us to go further to be in integrating efforts in stem cell biology of rabbit. Furthermore, directly applicable outcomes of the project are expected from better rabbit stem cells to generate relevant human disease models in this species.
Contact details: Scientist-in Charge: Prof Andras Dinnyes, BioTalentum Ltd, 2100 Godollo, Aulich Lajos u.26. Hungary. Tel: +36 20 510 9632, Email address: andras.dinnyes@biotalentum.hu
The key pluripotency genes from rabbit (Oct4, Nanog, Klf4, c-Myc) were isolated, based on the sequences of other species published so far, and its expression pattern was examined by immunohistochemistry. Sequences of each individual rabbit pluripotency genes were compared to mouse and human sequences from different databases and conserved regions were identified and evolutionary relevancies were established. Immuneprecipitation was used to analyse interactions among pluripotency genes. Direct and indirect interactions have been examined between Oct4 and its suspected targeted genes. In vitro differentiation was performed to analyse the functionality of pluripotency.
The key pluripotency genes from rabbit (Oct4, Nanog, Klf4, c-Myc) were isolated and identified. The rabbit Oct4, C-Myc, Sox2 and Nanog showed higher degree of homology with human than with mouse sequences in nucleotide and amino acid level, only the Klf4 exposed the contrary manner. Whole of the gene expression patterns were examined by immunohistochemistry and successfully detected with commercially available antibodies. Interactions have been examined between Oct4 and its suspected targeted genes by immuneprecipitation. We have found that the Oct4 and Sox2 could interact with FGF receptor, Rex1 and UTF1 and create a complex similar to that in mouse, however, in case of LIF receptor, FBXO15 and OPN these connections were not detected by Western Blot. In this phenomenon the rabbit pluripotent cells are similar to human ESCs. We have demonstrated that rabbit ES and iPS cells are able to differentiate towards neural and cardiac lineage.
RabPStem was expected to understand better pluripotency in rabbit. With the newly isolated rabbit pluripotency genes better iPS cells might be created than before, and it would generate a new platform for further applications of rabbit biotechnology. Our founding helped to highlight the mechanisms of the regulation of pluripotency in non-rodent mammals, with a significant importance to human medical research including the use of human pluripotent stem cell lines, as well.
Socio-economic aspects:
The ageing European population is prone to various diseases, causing immense societal and economic problems. The rabbit is a very relevant animal model for the study of a wide range of human diseases. However, the lack of embryonic stem cell technology that would make it possible to generate genetically modified animals by gene targeting, as it was developed in the mouse 20 years ago, severely hampers the full exploitation of the rabbit model. The RabPStem project was aimed to overcome this technological barrier. The isolated and characterized rabbit pluripotency genes provide us to go further to be in integrating efforts in stem cell biology of rabbit. Furthermore, directly applicable outcomes of the project are expected from better rabbit stem cells to generate relevant human disease models in this species.
Contact details: Scientist-in Charge: Prof Andras Dinnyes, BioTalentum Ltd, 2100 Godollo, Aulich Lajos u.26. Hungary. Tel: +36 20 510 9632, Email address: andras.dinnyes@biotalentum.hu