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

为了建立新的基因打靶方法的基础，我们研究了基因敲入的效率和使用称为表位标签的特异性标记。通过使用这种方法，通过针对表位标签的抗体来分析感兴趣的基因。我们选择了一些基因进行靶向实验。还对表位标签的候选物进行了详细的表征。一种新的内毒素诱导基因MAIL作为靶点的研究MAIL适合于靶向实验，因为已知该基因的转录被内毒素刺激显著上调，并且该基因产物特异性地定位于细胞核中。对MAIL基因组DNA进行了克隆和测序。对MAIL基因进行了靶向破坏.以几丁质酶基因为靶基因的研究由于几丁质酶基因具有组织特异性，因此我们选择该基因为靶基因。对几丁质酶基因进行了克隆、测序和表达谱分析，为基因打靶奠定了基础.其他靶基因的研究：以诱导性基因如B-13和组织特异性基因如葡萄糖转运蛋白为靶基因进行研究.表位标签的分析：检测汉坦病毒蛋白候选表位标签的抗原特异性和细胞功能.基因敲入方法的研究：尝试了两步打靶法进行基因敲入。但未获得预期的重组子，其失败原因有待进一步研究。

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.