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

为了为开发新的基因靶向方法奠定基础，我们研究了基因敲入的效率和特异性标记表位标签的使用。通过使用这种方法，感兴趣的基因可以通过抗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。还对候选表位标签进行了详细的表征1。一种新的内毒素诱导基因MAIL的研究。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着上调，并且该基因产物特异性定位于细胞核。我们克隆并测序了MAIL的基因组DNA。也进行了MAIL基因的靶向破坏。几丁质酶编码基因的研究。由于几丁质酶基因的组织特异性，我们选择该基因作为靶基因。对几丁质酶基因进行克隆、测序和表达谱分析，为基因靶向研究奠定基础。其他靶基因的研究。诱导基因，如B-13和组织特异性基因，如葡萄糖转运蛋白，被研究作为靶点。表位标签分析。研究了汉坦病毒候选表位标签的抗原特异性和细胞功能。开发敲入法。我们尝试了基因敲入的两步靶向法。然而，没有获得预期的重组体。需要进一步的研究来澄清失败的原因。

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.