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Analysis of TALEN-induced mutations in the IL7R gene

Grant number: 14/25151-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2015
Effective date (End): December 31, 2015
Field of knowledge:Health Sciences - Medicine
Principal Investigator:José Andrés Yunes
Grantee:Leonardo Granato Pissinato
Home Institution: Centro Infantil de Investigações Hematológicas Dr Domingos A Boldrini (CIB). Campinas , SP, Brazil

Abstract

Acute lymphoblastic leukemia (T-ALL) of T cell represents 10% to 15% of cases of childhood ALL, and is caused by uncontrolled proliferation of T lymphocytes precursors. Nine percent of T-ALL have mutations in the extracellular juxtamembrane portion of the IL-7 receptor (IL7R) gene, caused by short insertion/deletions that ultimately add a cysteine residue. Presence of cystein was shown to lead to constitutive JAK/STAT signaling , contributing to leukemogenesis. Mutations are concentrated in a small stretch (20pb) of exon 6 of the IL7R gene. In this project we intend to initiate a study on the molecular events that cause these mutations. The hypothesis to be investigated is whether breaks in double-stranded DNA (DSBs) in this region of the IL7R gene, predisposes to the insertion of cysteine codon and how often does that occurs. To this end, we intend to induce DSBs in exon 6 of IL7R in HEK293T mammalian cell line, using TALENs (binding proteins associated with DNA nucleases), analyzing both the type and the frequency of mutations resulting. The experiments will be repeated, depending on the results obtained, in lymphocytes cell lines, with TdT activity, for comparison of frequencies of cysteine-codons mutations. Futurely, we intend to investigate if the knockdown of repair machinery proteins accentuate the occurrence of cysteine codon insertions.