Generation and characterization of KO lymphocyte cell line for Zn transporters

Curs 2015-2016La deficiencia en zinc puede ser debida a una malnutrición (afecta a >17% de la población mundial) o como consecuencia a enfermedades y al envejecimiento. Ésta deficiencia provoca retraso del crecimiento, problemas neurológicos y un mayor riesgo de infecciones, causado por la baja eficiencia del sistema inmunitario. Existe la suplementación de zinc, pero las bases biológicas de este efecto preventivo no son conocidas. A pesar del importante papel que juega el zinc en el sistema inmunitario, no se sabe en profundidad su forma de entrada, sobre todo a lo que se refiere a los linfocitos T, donde se ha visto que éstos son vulnerables a la deficiencia del zinc, afectando a la maduración, la activación, la diferenciación y envejecimiento. Este TFG es parte de un gran proyecto. En la primera fase, se caracterizaron los transportadores de zinc durante la maduración, activación, diferenciación y envejecimiento de los linfocitos T en diferentes condiciones fisiológicas. De los 14 candidatos, se seleccionaron los 4 transportadores más representativos: ZIP6, ZIP8, ZIP10 i ZIP14.Zinc has been the focus of considerable interest during the last two decades. This interest has been awakened by the impact of zinc deficiency in human health. Individuals at high risk of zinc deficiency include: infants, adolescents, women at reproductive age and the aged. A deficiency of this metal causes impairment in body growth, neurological disorder and immunosuppression, among others 1,2. Zinc functions have been organized into 3 categories: catalytic, structural and regulatory 3. There are several transporters and zinc binding proteins that operate coordinately in order to maintain zinc homeostasis. Although it is known the impact of zinc in the immune system, it is not clear how this element enters the immune system cells. This lack of knowledge is even greater in T lymphocytes, where it is described that zinc deficiency alters T cell maturation, influences Th subsets polarization and impairs cytokine production 4. The aim of the project is to identify the gate of zinc entry in T cells in order to have a better understanding of zinc transporters in T cells. The main objective of this final project and the second phase of the zinc project leaded by Dr. Rubén Vicente Garcia was to develop a tool by depleting Zip6, Zip8, Zip10 and Zip14 of the acute T cell leukemia Jurkat cell line using the revolutionary tool CRISPR-CAS9 technique. This tool would help us to study how the absence of these zinc transporters affects zinc kinetics and T cell function as activation, proliferation and differentiation