Generation of specific antibodies against Ki-1/57 regulatory protein for functiona...
| Grant number: | 14/21700-3 |
| Support type: | Regular Research Grants |
| Duration: | June 01, 2015 - August 31, 2017 |
| Field of knowledge: | Biological Sciences - Biochemistry - Molecular Biology |
| Principal Investigator: | Jörg Kobarg |
| Grantee: | Jörg Kobarg |
| Home Institution: | Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil |
Abstract
Ki-1/57 was discovered through the cross-reactivity of the monoclonal antibody Ki-1, the first antibody which specifically detects the cells of the Hodgkin lymphome. Preliminary studies on this protein revealed several characteristics that are common to onco-proteins. Yeast-two hybrid experiments identified interacting proteins involved in the control of transcription and RNA metabolism. Several of them are functionally related to or interact with p53 protein family members. P53 is a transcription factor and tumor suppressor and an important regulator of the cellular stress response. Ki-1/57 interacts with several proteins involved in pre-mRNA splicing, colocalizes with cytoplasmic stress granules and is sumoylated. Microarray data, which we published recently, offered a new perspective for the function of Ki-1/57 and its involvement in mechanisms of the cellular stress response, since its ectopic over-expression led to the repression of the expression of genes involved in cell proliferation and cell death. These new informations, together with the sub-cellular localization and the protein interaction profile of Ki-1/57, suggest that it acts under certain conditions at the control of gene expression, possibly at various levels , from transcription to translation. In order to analyze in more depth the possible functions of Ki-1/57 we want to perform the overexpression of Ki-1/57 and p53 simultaneously, and analyze the cell cycle and stress responses. Since Ki-1/57 is related to cancer we also want to study its expression in normal and cancer tissues. In order to better understand the role of Ki-1/57 and its paralog CGI-55 in the context of expression regulation, we want to identify and characterize the profile of mRNA that are targets of these proteins and map the interaction sites through cross-linking immunoprecipitation followed by next generation sequencing (CLIP-seq).Furthermore, we are in advanced stages of the generation of knock out mice for the gene of Ki-1/57. We already obtained mosaic knockout animals, by using the new sistem called CRISPR/Cas9. Furthermore we already have heterozygous embryonic stem cells (ES) with a classcical gene knock out for Ki-1/57, that have been generated by homologous recombination. We are already analyzing the role of Ki-1/57 in pluripotent stem cells in the course of neuronal diferentiation and of the cardiomyocyte diferentiation. First injections of the Ki-1/57 knock out stem cells into blastocysts generated quimeric animals. The most of the work that is lying ahead involves the histological, horphological, physiological and functional analysis of the Ki-1/57 knock out mice in comparison to wild type animals. (AU)