Study for the development of new gene targeting method utilizing epitope tags

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To establish the bases for developing new methods of gene targeting, we investigated the efficiency of gene knock-in and the use of specific markers called epitope-tags. By using this approach, gene of interest is to be analyzed by antibodies against epitope-tags. We selected some genes for targeting experiments. Detailed characterization of candidates for epitope-tags was also carried out.1. Investigation of a novel endotoxin-inducible gene, MAIL, as a target.MAIL is suitable for targeting experiment because it is known that transcription of the gene is markedly up-regulated by endotoxin stimulation and that the gene product is specifically localized in the nucleus. We cloned and sequenced the genomic DNA of MAIL. Targeted disruption of MAIL gene was also performed.2. Investigation of genes encoding chitinase as a target.Because of tissue-specificity of chitinase gene, we choose this gene as a target. We cloned, sequenced, and analyzed expression pattern of chitinase gene, and established the basis for gene targeting.3. Investigation of other target genes.Inducible genes, such as B-13, and tissue-specific genes, such as glucose transporters, were investigated as targets.4. Analysis of epitope-tags.Antigenic specificity and cellular function of candidate epitope-tags from hantavirus proteins were examined.5. Developing knock-in method.We tried two-step targeting method for gene knock-in. However, expected recombinants were not obtained.Further research is needed to clarify the cause of the failure.