engleski

Analysis of expression and function of three mouse genes modified by gene trap method

Gene trap method is a random approach, which enables molecular tagging of mouse genes, with their subsequent expression pattern and functional analysis. Genetic modification was done with a nonhomologous DNA construct via embryonic stem cells. Construct consisted of splice acceptor followed with promoterless lacZ and neoR genes, and the polyA signal at the end. If the construct was introduced within a transcribed gene, splice acceptor was active and lacZ and neoR acted as the last exons in the “ trapped” gene. NeoR gave resistance to neomycine and enabled selection of the gene trap events in the embryonic stem cells, while lacZ coded for beta-galactosidase, which could be easily monitored in tissues by simple histochemical staining. Three gene trap modifications and corresponding mouse lines carrying these modifications were selected from the gene trap screen. They were called H15, lobel and kosenic. H15 mutation corresponds to an insertion of the gene trap vector within the gene coding for nucleolar protein 1 (Nol1), a gene implicated to be important in cell proliferation. Gene is expressed in all organs and tissues during embryo development, and it is strongly present in the cell nucleolus. Homozygous embryos die during embryo development around blastocyst stage. Lobel is a mutation within Signal transducing adapter molecule 2 (Stam2), which is involved in endosome mediated cell signaling pathway. It is expressed in the developing and adult brain and heart. Homozygous mice are alive and fertile. Kosenic was until recently thought to be a novel mouse gene, but the new data obtained from the sequencing of the mouse genome revealed it as a mouse homologue of the human Krüppel-like factor 8 (Klf8). This is a transcription factor with unknown function. It is located on X chromosome and expressed in the subset of cells in the developing heart, and in the developing and adult brain. Hemizygous males, which lack a non-mutated copy of the gene are alive and fertile, although congenic strain on C57Bl/6 inbred background was not achieved, as the mice failed to breed after several generations.

gene trap

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