Gene targeting in murine embryonic stem cells for generating a Ki-1/57 knockout mice

• Grant number: 13/04492-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: August 01, 2013
• End date: November 30, 2013
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Jörg Kobarg
• Grantee: Ângela Saito
• Supervisor: Richard Robert Behringer
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Institution abroad: University of Texas MD Anderson Cancer Center (MD Anderson), United States
• Associated to the scholarship: 10/15760-2 - Functional characterization of the human regulatory protein Ki-1/57: cellular and in vivo model studies, BP.DD

Abstract

Ki-1/57 is a regulatory protein, intrinsically unstructured, located in the nucleus and cytoplasm. It is encoded by the gene Habp4, located on chromosome 13 in mice. Several post-translational modifications regulate the function of Ki-1/57 such as phosphorylation by PKC, methylation by PRMT1 and SUMOylation. Yeast Two-hybrid assays and immunoprecipitation from mammalian cells extracts showed that it interacts with several regulatory proteins such as RACK1, p53, CHD3, SFRS9, hnRNPQ, CIRP, and RPL38 FXR1. It is involved in the events of pre-mRNA splicing and regulation of the translational machinery. Due to its pleiotropic role, it is very interesting to investigate the biological role of Ki-1/57 in animal model using gene knockout in mice. The aim of this project is to perform in the laboratory of Dr. Richard Behringer from University of Texas M. D. Anderson Cancer Center the step of gene targeting aiming to disrupt the Habp4 gene in mice embryonic stem cells. The targeting vector was designed and built by the PhD student and will be used to replace, by homologous recombination, the first 5 exons of the gene for a cassette containing lacZ-neo-loxP. Recombinant clones will be selected, frozen, genomic DNA extracted for confirmation by Southern blot. The student will have a great opportunity to learn the details of the techniques with specialists to perform this step of the PhD project faster than if it were held in Brazil. (AU)