Generación y caracterización de modelos murinos deficientes en los genes RAS
AdvisorGuerra, María del Carmen
EntityUAM. Departamento de Biología Molecular; Centro Nacional de Investigaciones Oncológicas (CNIO)
SubjectsOncogenes-Tesis doctorales; Biología y Biomedicina / Biología
NoteTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 12-02-2009
Our main goal is to understand the role of the mammalian Ras proteins in general, and of K-Ras in particular. Previous studies have shown that K-Ras gene is the only member of the Ras family that is essential for normal mouse development. Embryos lacking this gene die between days E12-14 because of liver defects and anemia. (Johnson et al.,1997; Koera et al., 1997).To determine the physiological function of K-Ras gene in adult mouse, we have generated a conditional strain of mice that were crossed with an inducible Cre-ERT mouse model (Guerra et al., 2003), in order to inactivate the K-Ras gene upon 4-OH tamoxifen treatment. Ablation of K-Ras gene in K-Ras(lox/lox);RERT(ert/ert) mice leads to leathality within 12-14 months due to unkown reasons, showing that K-Ras is an essential gene that cannot be compensated by any other member of the Ras family in adulthood. We have also study the role of K-Ras in vitro in mouse embryonic fibroblasts (MEFs) derived from these mice. This defective MEFs proliferate normally and they can be immortalized similar to the wild-type MEFs. These results indicate that the abscence of K-Ras gene is dispensable for the normal behaviour of MEFs suggesting overlaping cellular roles among the different members of the Ras family. In order to evaluate the distinct contributions of Ras signalling in normal cells, we have generated compound [H-Ras(-/-); N-Ras(-/-); K-Ras(lox/lox); RERT(ert/ert)] mice by crossing K-Ras condicional mutant mice with double knockout animals lacking the H-Ras and N-Ras loci (kindly provided by E.Santos, CIC, CSIC, Salamanca, Spain). Treatment of young (p21) or adult (p60) [H-Ras(-/-); N-Ras(-/-); K-Ras(lox/lox); RERT(ert/ert)] animals with 4OHT results in complete lethality within 3 to 5 weeks because of a dramatic weight loss and severe cachexia. Detailed pathological analysis of these mice revealed conserved architecture of organs and tissues although most of them appeared smaller, particularly the thymus. It has been also observed considerable atrophy of white and brown adipose tissue and the skin was also affected with multiple ulcerations and crusts over the snout and limbs. We have also generated as well cells lcking the three members of the three members of the ras gene family. Compound [H-Ras(-/-); N-Ras(-/-); K-Ras(lox/lox); RERT(ert/ert)] MEFs grow well in culture and become immortalized with normal kinetics. Exposure to 4OHT results in loss of the floxed allele alter 2-3 passages. “Rasless” cells immediatly cease proliferation and acquire a quiescent “senescente-like” phenotype that can be maintained for various passages. These observations demonstrate that Ras proteins are essential for survival of adult mice. Moreover, they provide genetic evidence that Ras are a unique class of proteins whose activity cannot be compensated by any other members of the Ras superfamily of small GTPases.
Files in this item
"Texto de la Tesis Doctoral"
Google Scholar:Dhawahir Scala, Alma
This item appears in the following Collection(s)
Showing items related by title, author, creator and subject.
Galán Antoñanzas, Javier
Generacion y caracterizacion de modelos murinos para el estudio del papel del oncogen H-Ras en la tumorogenesis y en el sindrome Costello Jiménez Schuhmacher, Alberto
Estudio de las funciones fisiológicas y patológicas de la proteína tau mediante el uso de modelos murinos deficientes en tau Gómez de Barreda Santiago, Elena