Analysis of localization and physiological function which uses H-Ras protein myc tagging mice

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To investigate the in vivo functions and functional redundancy of the three ras genes, H-ras, N-ras and K-ras, we generated mice deficient in each of these genes. The development of H-ras homozygous mutant mice appeared to be normal. However, no one can imagine the subtle abnormality in H-ras deficient mice. Thus, it is necessary to examine the expression pattern of H-Ras proteins. In case of this kind of studies, specific antibodies against H-Ras proteins for immunohistochemical analyzes were indispensable. Thus, we intended to add a 10-amino acid epitope tag derived from human c-Myc to the C-terminal ends of H-Ras(H-Ras-myc-tag)for specific marker antigen and express them in mice by gene targeting.By two-steps of gene targeting in ES cells, we introduced H-ras-myc-tag into the mouse H-ras locus. Chimeric mice were generated by microinjection of the targeted ES cells into blastocysts. Chimeric males were mated to confirm the germline transmission of the targeted allele. Since the purpose of this project was to generate mice which express the Myc-tagged H-Ras proteins and obtain the offspring of these mice, this purpose has been successful.In situ hybridization analysis of H-, K-ras mRNA were carried out during the mouse embryonic development and mice which lack two or three ras genes simultaneously were generated. These study showed the existence of functional cooperation between the three Ras proteins in mouse development.We also intend to examine the intracellular distribution of H-Ras proteins in each organ by immunostaining the mice expreesing the Myc-tagged H-Ras proteins with an anti-Myc antibody. It is likely these analyzes will give an insight into the Ras mediated signal transduction pathway under the physiological conditions.