engleski

Gene trap modification of mouse gene Nol1 (Nucleolar protein 1)

Important goals in gene analysis are to assess gene function and to determine spatial and temporal expression pattern. Gene trap method is a random approach, which enables molecular tagging of mouse genes with possibility to analyze both, function and expression pattern. Embryonic stem (ES) cells were genetically modified by a nonhomologous DNA vector containing a splice acceptor and fused promoterless lacZ and neoR genes. The vector was integrated randomly within the genome, and the inserted lacZ and neoR genes were active only if the vector was within a transcribed endogenous gene. That modification yielded a few benefits: 1) modified ES cells were resistant to neomycin, and could be selected in vitro, 2) expression of lacZ gene mirrored the expression of endogenous gene mutated by gene trap vector, hence its spatial and temporal expression pattern could be monitored, 3) the gene trap vector tagged the mutated genes, what facilitated their identification by 5’ rapid amplification of cDNA ends (RACE), and 4) the insertion was likely to disturb the endogenous gene, that offered possibility to get insight in gene function by analyzing the phenotype of the corresponding mice, obtained from ES cells. Nucleolar protein 1 (Nol1) is a marker of nucleolus in highly proliferated cells. It is present in majority of malignant tissues (lung, breast, prostate, colon, and brain), but also in non-malignant, highly proliferative tissues. High expression of Nol1 in different carcinoma is a marker of poor clinical prognosis. Nol1 expression is regulated during cell cycle: its protein synthesis starts at late G1, and peaks in S phase of cell cycle. We obtained the gene trap insertion within Nol1 gene and we used the mice carrying this mutation to investigate expression and function of Nol1 gene. The expression of Nol1 gene during development was determined by whole mount histochemical staining by beta-galactosidase substrate X-gal. Staining was found all over the embryo and in all investigated developmental stages. The strongest staining was found between stages E8.5 and E12.5. Although all tissues expressed Nol1, it was not present in all cells. At the cellular level, majority of staining was found within the nucleolus. In order to get insight in the function of Nol1, intercross of heterozygous mice carrying gene trap insertion was performed. Using the PCR method we demonstrated that homozygous mice were not present among newborn animals. Therefore we assumed that homozygous phenotype is lethal.

gene trap; Nol1

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