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

利用表位标签开发新的基因打靶方法的研究

基本信息

批准号：
11556064
负责人：
MORIMATSU Masami
金额：
$ 7.23万
依托单位：
Iwate University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11556064/
关键词：
epitope-tag gene targeting interaction of molecules cellular localization 遺伝子機能解析マウス

项目摘要

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.

为了建立开发新基因靶向方法的碱基，我们研究了基因敲入的效率和使用称为表位标记的特定标记的效率。通过使用这种方法，应通过针对表位标签的抗体来分析感兴趣的基因。我们选择了一些用于靶向实验的基因。还进行了表位标签的候选者的详细表征1。对新型内毒素诱导基因的研究作为靶标。邮政为靶向实验，因为已知该基因的转录被内毒素刺激显着上调，并且该基因产物被特异性地定位于核中。我们克隆并测序了邮件的基因组DNA。还进行了靶向邮件基因的靶向破坏。2。编码几丁质酶的基因的研究。由于几丁质酶基因的组织特异性，我们选择该基因作为靶标。我们克隆，测序和分析了几丁质酶基因的表达模式，并建立了基因靶向的基础3。研究了其他靶基因的研究。表位标签的分析。检查了汉塔病毒蛋白的候选表位标记的抗原特异性和细胞功能。5。开发敲门方法。我们尝试了基因敲入的两步靶向方法。但是，未获得预期的重组。需要研究以阐明失败的原因。