DEVELOPMENT OF INTRODUCTION OF TRANSGENE INTO MEDAKA EGGS AND MANUPILATION OF GENE EXPRESION

将转基因导入青鳉鱼卵并进行基因表达操作的开发

• 批准号: 11236208
• 负责人: KINOSHITA Masato
• 金额: $ 28.29万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11236208/
• 关键词: MEDAKA; TRANSGENIC; CRE; loxp; INSULATOR; GREEN FLUORESCENT PROTEIN; RED FLUORESCENT PROTEIN; Cre; LoxP; GFP; 発現制御; vasa; トランスジェニック; RFP; トランス ジェニック; パーティクルデリバリー; Vasa

In this study, to elevate the usefulness of medaka as a model, I tried to adapt and improve the transgenic methods in medaka.1)Gene induction with particle gun system: Microinjection method is a most popular one for the introduction foreign gene into fish eggs. But this method requires special skill. To develop more easy method, I adapted particle gun method in medaka transgenesis and optimized parameters. The key of this method was that bombardment should be carried out before chorion hardened. Transgenic medaka established this method revealed that transgene was transmitted stablely over 5th generation. 2)Effective selection marker of germ line transmitters : One of the most complicated works in establishing transgenic strains is the selection of the individual which harboring transgene in germ cells. We found that vasa-GFP vector was a good indicator of germ line transmitter, which told us induction of transgene in germ cell before hatch. We also found that germ cell specific cis element locates in 3'-UTR of vasa transcript. 3) Regulation of transgene expression : To prevent transgene expression from the effect of enhancer existing near inserted chromosomal position, I showed the effectiveness of sea-urchin insulator in medaka transgenesis. For the conditional gene expression, Cre-LoxP system was available in medaka. Additionally, about 200 bp sequence including loxp sequence showed internal ribosomal entry activity. 4)Transgenic medaka strain with fluorescent label: Some transgenic medaka strains with tissue specific fluorescent label were established. They had green or red fluorescent in skeletal muscle, green fluorescent in germ cells, green fluorescence in lever, green or red fluorescence in almost all tissues and green fluorescence in nerve cells

本研究以青鳉为研究对象，对青鳉转基因方法进行了改进和完善，以提高青鳉作为转基因模式的实用性。1）基因枪系统的基因诱导：显微注射法是目前将外源基因导入鱼卵的最常用的方法。但这种方法需要特殊的技巧。为了使转基因方法更简便易行，本研究采用了粒子枪法对青鳉进行转基因，并对参数进行了优化。该方法的关键是轰击应在绒毛膜硬化之前进行。建立的转基因青鳉转基因方法表明，转基因青鳉在第5代以上稳定遗传。2)生殖系传递基因的有效选择标记：建立转基因株系最复杂的工作之一是选择在生殖细胞中携带转基因的个体。我们发现vasa-GFP载体是一个很好的生殖系传递物的指示剂，它告诉我们在孵化前生殖细胞中的转基因诱导。我们还发现生殖细胞特异的顺式元件位于vasa转录本的3 '-UTR。3)转基因表达的调控：为了防止转基因表达受到插入染色体位置附近存在的增强子的影响，我证明了海胆绝缘子在青鳉转基因中的有效性。对于青鳉的条件基因表达，Cre-LoxP系统是可行的。另外，包括loxp序列在内的约200 bp序列显示了内部核糖体进入活性。4)荧光标记转基因青鳉品系：建立了组织特异性荧光标记转基因青鳉品系。骨骼肌呈绿色或红色荧光，生殖细胞呈绿色荧光，肝脏呈绿色荧光，几乎所有组织均呈绿色或红色荧光，神经细胞呈绿色荧光

项目成果

Kinoshita, M.Yamauchi, M., Sasanuma, M., Ishikawa, Y., Osada, T., Inokue, K., Wakamatus, Y., Ozato, K.: "A transgene and its expressiion profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method."Zool. Sci.. 20.

Kinoshita, M.Yamauchi, M.、Sasanuma, M.、Ishikawa, Y.、Osada, T.、Inokue, K.、Wakamatus, Y.、Ozato, K.：“转基因及其表达谱被稳定地传递到

Yamauchi, M., Kinoshita, M., Sasanuma, M., Tsuji, S., Terada, M., Morimyo, M., Ishikawa, Y.: "Introduction of a foreign gene into medakafish using the particle gun method."J. Exp. Zool.. 287. 285-293 (2000)

Yamauchi, M.、Kinoshita, M.、Sasanuma, M.、Tsuji, S.、Terada, M.、Morimyo, M.、Ishikawa, Y.：“使用粒子枪方法将外源基因引入青鳉鱼。”

Kinoshita, M.: "A transgene and its expression profile are stably transmitted to offspring in transgenic medaka generated by the particle gun method."Zool.Sci.. 20. 869-875 (2003)

Kinoshita, M.：“转基因及其表达谱在通过粒子枪方法产生的转基因青鳉中稳定地传递给后代。”Zool.Sci.. 20. 869-875 (2003)

田中実: "遺伝子機能解析のためのポストゲノム実験動物メダカ(生きたまま内蔵(細胞)を見られる「透明・遺伝子導入」メダカ)"生物物理. 43. 93-96 (2003)

Minoru Tanaka：“用于基因功能分析的后基因组实验动物青鳉（“透明/基因转移”青鳉，其内部器官（细胞）在活着时可以看到）” 43. 93-96 (2003)。

田中実: "メダカの細胞ひとつひとつを光らせて生殖腺形成機構を解明する一生殖細胞系列特異的にRNA翻訳制御を指示するエレメントがわかった"化学と生物. 41. 278-280 (2003)

Minoru Tanaka：“通过使每个青鳉细胞发光来阐明性腺形成的机制 - 指导种系特异性 RNA 翻译控制的元素的识别”，化学与生物学，41. 278-280 (2003)。