Academic journal article Genetics

Identification of a Novel Point Mutation of Mouse Proto-Oncogene C-Kit through N-Ethyl-N-Nitrosourea Mutagenesis

Academic journal article Genetics

Identification of a Novel Point Mutation of Mouse Proto-Oncogene C-Kit through N-Ethyl-N-Nitrosourea Mutagenesis

Article excerpt

ABSTRACT

Manipulation of the mouse genome has emerged as an important approach for studying gene function and establishing human disease models. In this study, the mouse mutants were generated through N-ethyl-N-nitrosourea (ENU)-induced mutagenesis in C57BL/6J mice. The screening for dominant mutations yielded several mice with fur color abnormalities. One of them causes a phenotype similar to that shown by dominant-white spotting (W) allele mutants. This strain was named Wads because the homozygous mutant mice are white color, anemic, deaf, and sterile. The new mutation was mapped to 42 cM on chromosome five, where proto-oncogene c-kit resides. Sequence analysis of c-kit cDNA from Wads^sup m/m^ revealed a unique T-to-C transition mutation that resulted in Phe-to-Ser substitution at amino acid 856 within a highly conserved tyrosine kinase domain. Compared with other c-kit mutants, Wads may present a novel loss-of-function or hypomorphic mutation. In addition to the examination of adult phenotypes in hearing loss, anemia, and mast cell deficiency, we also detected some early developmental defects during germ cell differentiation in the testis and ovary of neonatal Wads^sup m/m^ mice. Therefore, the Wads mutant may serve as a new disease model of human piebaldism, anemia, deafness, sterility, and mast cell diseases.

N-TETHYL-A^NITROSOUREA (ENU)-induced mutagenesis has become a powerful tool for the study of gene functions and generation of human disease models recently (NoLAN el al. 2000; HRABE DE ANGELIS et al. 2000; HERRON et al. 2002). The growing mouse mutant archives provide a rich resource for identifying the novel disease-related genes, deciphering pathogenic mechanisms, and developing new therapies and new drugs (BALLING 2001; BROWN and HARDISTY 2003). Recently, we established >40 lines of mutant mice by screening the ENU-induced dominant mutants in C57BL/6J mice (HE et al 2003). Among them, 11 lines displayed an interesting white spotted coat. One of these lines, Wads, displayed similar phenotypes to the mouse W/c-kit strain.

Mutations at the mouse W/c-kit locus on chromosome five can lead to pleiotropic developmental defects, including sterility, coat color abnormalities, severe macrocytic anemia, loss of interstitial cells of Cajal (ICC), and mast cell deficiency (GEISSLER et al. 1981; CHABOT et al. 1988; HUIZINGA et al. 1995; TSUJIMURA 1996). A total of 76 mutant alleles at this locus have been accumulated in mouse up to 1997 (see the Mouse Genome Database, http://www.informatics.jax.org/searches/mlc.cgiP10603). Human c-kit mutations were also identified in patients with piebaldism, mastocytosis, gastrointestinal stromal tumors (GISTs), acute myeloid leukemia, and germ cell tumors (see online Mendelian Inheritance in Man, OMIM, http://www.ncbi.nlm.nih.gov/entrez/dispomim. cgi?id= 164920). Encoded by c-kit, KIT is a type III receptor tyrosine kinase that binds to stem cell factor (SCF), which is the product of the mouse Sl locus (WITTER 1990). Ligand binding activates KIT through dimerization and autophosphorylation (HELDIN 1995). The phophorylated KIT then further activates downstream pathways in a variety of cell types (PRICE et al. 1998; TIMOKHINA et al 1998; LINNEKIN 1999; Hou et al 2000). Unfortunately, mechanisms by which the different KIT mutations cause various diseases are largely unknown.

In this study, we reported the preliminary phenotypic analysis of the Wads mouse, including hearing ability, hematopoiesis, mast cell development, and germ cell differentiation. The Wads mutation was also mapped and cloned. The novel point mutation at nucleotide 2567 of the c-kit cDNA results in a substitution of Phe to Ser at KIT amino acid 856 (F856S).

MATERIALS AND METHODS

Animals: C57BL/6J mice were obtained from Shanghai Laboratory Animal Center (Shanghai, China). CAST/Ei mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained under specific pathogen-free environment. …

Search by... Author
Show... All Results Primary Sources Peer-reviewed

Oops!

An unknown error has occurred. Please click the button below to reload the page. If the problem persists, please try again in a little while.