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KIS inhibitory sequences cloning in retroviral vector and stable retroviral producing lineage establishment

Grant number: 15/22778-9
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): December 01, 2015
Effective date (End): November 30, 2017
Field of knowledge:Biological Sciences - Genetics
Principal Investigator:Leticia Fröhlich Archangelo
Grantee:Laissa Mayara da Silva Paz
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:14/01458-3 - Defining the functional role of the splicing factor regulator (KIS) during leukemogenesis using a murine bone marrow transplantion model, AP.JP

Abstract

Somatic mutations in genes related to splicing, including SF1 and SF3b1 mutations, were recently described in hematologic diseases, consolidating defects in splicing machinery as a new mechanism in leukemogenesis. Not only mutations, but also the aberrant expression of splicing factors and changes in the phosphorylation pattern of these proteins, may affect the operation and efficiency of splicing and possibly lead to carcinogenesis. KIS is a serine / threonine kinase that controls the cell cycle and activity of splicing factors. Through its UHM domain, KIS interacts with splicing factors SF1 and SF3b1. Through its kinase activity, KIS phosphorylates these factors. Our hypothesis is that, as observed for the kinase SRPK2, increased or decreased KIS' expression can lead to aberrant phosphorylation of its substrates (SF1 and / or SF3b, in this case) and thus help the leukemogenic process. The Jovem Pesquisador project (FAPESP 14/01458-3 process) has as one of its goals investigate KIS' leukemogenic potential through its overexpression in progenitor cells from mouse bone marrow. Recent data from our group shows that, unlike in humans, the murine progenitor bone marrow cells have extremely high expression of endogenous KIS. In this context, we sight the need not only to overexpress KIS (in progress), but also to inhibit its expression in hematopoietic progenitor cells, that will be used in a model of bone marrow transplantation in mouse. This project will focus on designing shRNAs targeting the murine orthologue of KIS, cloning the oligonucleotides into the biscistronic retroviral vector MIG, and produce a cell line capable of stable production of retrovirus particles. (AU)

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