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

为了为开发新的基因打靶方法奠定基础，我们研究了基因敲入的效率和称为表位标签的特定标记的使用。通过使用这种方法，感兴趣的基因将通过针对表位标签的抗体进行分析。我们选择了一些基因进行靶向实验。还对表位标签的候选者进行了详细的表征。以一种新的内毒素诱导基因MAIL为靶点进行研究。MAIL适合于靶向实验，因为已知该基因的转录在内毒素刺激下显著上调，并且基因产物特异性地定位于细胞核。我们对MAIL的基因组DNA进行了克隆和测序。本实验还进行了MAIL基因的靶向阻断。以几丁质酶编码基因为靶点，由于几丁质酶基因的组织特异性，我们选择该基因作为靶点。克隆、测序并分析了几丁质酶基因的表达模式，为基因打靶奠定了基础。其他靶基因的研究：以B-13等诱导基因和葡萄糖转运蛋白等组织特异性基因为靶点进行研究。表位标签分析：检测汉坦病毒蛋白候选表位标签的抗原性和细胞功能。发展了基因敲入方法。我们尝试了两步打靶的基因敲入方法。然而，没有得到预期的重组体，需要进一步的研究来阐明失败的原因。

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.