engleski

Characterization of a gene trap integration within a gene expressed during embryo development

Gene trap method is a powerful method to identify new genes and analyze their function during mammalian embryonic development. Due to the random insertion of the gene trap vector in the genome of embryonic stem cells, any, presumably novel, gene can be tagged, and the corresponding mouse line carrying the gene trap mutation produced. The inserted vector contains a selectable marker (neoR) for mutation, a reporter gene (lacZ) that reflects endogenous gene expression and a polyA site that disables the 3´ part of the endogenous gene. The gene trap method simultaneously enables cloning of the mutated genes, determination of its expression pattern, and by phenotype analysis it gives insight in the function of the mutated gene. Using a gene trap method we produced a mouse line with an insertional mutation in a gene that was named lobel. The specific structure of the gene trap vector enabled us to determine its sequence and to define the expression pattern. We obtained the coding sequence of lobel cDNA isolated from mouse embryonic stem cells. Analysis of cDNA from embryonic stem cells, adult liver and brain showed alternative splicing of lobel. Expression pattern analysis performed by X-gal staining showed that lobel is expressed in the neural tube and in the heart of the mouse embryo, and in the brain of the adult mice. The suggested function of lobel is in the signal transduction after growth factor-receptor complex internalization. We confirmed that the lobel gene is present in the mouse genome in a single copy. The gene trap vector insertion is unique and it is located in the first part of the lobel gene. We assumed that the insertion of the gene trap vector into the lobel gene caused loss of function of the Lobel protein. However, the homozygous mice with the gene trap mutation in both alleles were fertile and showed no macroscopic malformations. Our future efforts are directed to the analysis of the phenotype of the lobel mutant mice.

gene trap; mouse; development

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