Identification of genes responsible for developmental mutant mice

鉴定负责发育突变小鼠的基因

• 批准号: 09044325
• 负责人: YAMAMURA Ken-ichi
• 金额: $ 4.16万
• 依托单位: KUMAMOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044325/
• 关键词: BAC; mutant mouse; development; transgenic mouse; positional cloning; ボンショナルクローニング; YAC; 胚発生; ゲノム解析

Transgenic techonology has been very useful and successful for delineating functions of any genes of interest in vivo. However, there are several technical limitations in conventional transgenic techniques. Since introns and essential regulatory sequences required for correct in vivo expression are omitted in the constructs, transgene expression is sometimes different from the expression of corresponding endogenous gene. So-called 'postion-effect' that affect foreign gene expression depending on its chromosomal integration site also conpromise the transgene expression. In addition, the maximum size of transgene is only 40-50 kb, due to constraints on the insert length that can be cloned in phage or cosmid vectors. These limitations can be overcome by using cloning systems which accomodate submegabase DNA such as YAC (yeast artificial chromosome) or BAC (bacterial artificial chromosome) in the generation of transgenic mice.On the other hand, more than a thousand mouse mutants have been … More identified and maintained over the past century and the number will drastically increase in near future by several 'large scale' mutagenesis projects operating in international mouse communities. However, the task of isolating the responsible gene for any of these mutantions is still labor intensive and so limits the usufullness of thisexperimental resources. Again, the ability to introduce large size DNA into the mouse germ lines should help rectify this situation. It should be possible to define the location of the responsible gene on a large DNA fragment by introducing YACs or BACs into mutant mice and examining the functional rescue of the mutant phenotype.Toward this end, we have established a series of techniques to introduce large size DNA into mouse germ lines. During these processes, we found that YAC clones contain cloning artifacts including chimerism and deletions, we have switched to use BAC which contain less DNA rearrangements.We have produced more than fifty founder transgenic mice carrying BAC clones derived from the t-complex region of Chr. 17, tail kinks (tk) locus and PaxI locus. We have identified and characterized qkL gene, a candidate for the classical neurological mutation, quaking. We introduced a transgenic line harboring a 160-kb BAC that contains the whole qkI locus and crossed the strain with the mutant animals. quaking is a recessive mutantion with tremors due to hypomyelination in CNS.Introduction of the qkl containing BAC successfully corrected the mutant phenotype ; homozygous mutant with the BAC do not show tremor phenotype and myelination occurred normaly. We have also rescued brachynry mutation which affect notochord development and tail morphology. These results thus demonstrated the usufullness of the BAC transgenesis particulary in identification and functional analysis of mutated genes in mice. Less

转基因技术在描述任何感兴趣的基因在体内的功能方面已经非常有用和成功。然而，传统的转基因技术有几个技术限制。由于在体内正确表达所需的内含子和必要的调控序列被省略，转基因表达有时不同于相应的内源基因的表达。根据外源基因的染色体整合部位影响外源基因表达的所谓“位置效应”也保证了转基因的表达。此外，由于可在噬菌体或粘粒载体中克隆的插入长度的限制，转基因的最大大小仅为40-50kb。这些限制可以通过在转基因小鼠的产生中使用包括亚百万碱基DNA的克隆系统来克服，例如酵母人工染色体或细菌人工染色体。另一方面，已经有一千多个小鼠突变体被…在过去的一个世纪里，更多的老鼠被识别和维护，在不久的将来，通过在国际老鼠社区运作的几个‘大规模’突变项目，这个数字将急剧增加。然而，分离这些突变的负责基因的任务仍然是劳动密集型的，因此限制了这种实验资源的实用性。同样，将大尺寸DNA引入小鼠生殖系的能力应该有助于纠正这种情况。通过将YAC或BAC导入突变小鼠并检测突变表型的功能挽救，应该可以确定负责基因在大DNA片段上的位置。为此，我们建立了一系列将大DNA导入小鼠生殖系的技术。在这些过程中，我们发现YAC克隆含有嵌合体和缺失的克隆伪迹，我们改用DNA重排较少的BAC，我们已经产生了50多只携带来自ChR t-复合区的BAC克隆的方正转基因小鼠。17、尾部扭结(Tk)基因和Paxi基因。我们已经鉴定并鉴定了经典神经突变的候选基因QKL颤抖。我们引入了一个含有160kb BAC的转基因品系，该品系包含整个qkI基因座，并将该品系与突变动物进行杂交。震颤是一种隐性突变，由于中枢神经系统髓鞘过少而伴有震颤。引入含有BAC的QKL成功地纠正了突变表型；带有BAC的纯合子突变体不显示震颤表型，髓鞘形成正常发生。我们还挽救了影响脊索发育和尾巴形态的短臂突变。这些结果证明了BAC转基因特异性在小鼠突变基因的鉴定和功能分析中的应用。较少

项目成果

Pusch C,Hustert E,Pfeifer D,Sudbeck P,Kist R,Roe B,Wang Z,Balling R,Blin N,Scherer G: "The SOX10/sox10 gene from human and mouse : sequence, expression, and transactivation by the encoded HMG domain transcription factor" Hum Genet. 103. 115-123 (1998)

Pusch C、Hustert E、Pfeifer D、Sudbeck P、Kist R、Roe B、Wang Z、Balling R、Blin N、Scherer G：“来自人类和小鼠的 SOX10/sox10 基因：序列、表达和编码的反式激活

A,Kikuti.et al.: "cDNA Cloning,Northern hybridization and mapping Shigenari,A.,Kawata,H.,Ikemura,T.analysis of a putative GDS(guanine nucleotide dissociation,Kimura M.,and Inoko,H.stimulator of G proteins)-related protein gene at the centromeric ends of t

A，Kikuti. 等人：“cDNA 克隆、Northern 杂交和作图 Shigenari,A.、Kawata,H.、Ikemura,T. 假定 GDS 的分析（鸟嘌呤核苷酸解离、Kimura M. 和 Inoko,H.stimulator）

Matsuki,Y.,Kaname,T.,Suematsu,S.,Yamaguchi,Y.,Abe,K.and Yamamura,K.: "Mouse K-glypican gene,Gpc4,maps to chromosome X." Genomics. 54. 358-359 (1998)

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前田 浩: "SMANCS研究の最近のあゆみ" 癌と化学療法. 25. 1-9 (1998)

Hiroshi Maeda：“SMANCS 研究的最新进展”癌症与化疗 25. 1-9 (1998)。

Wilm, B.,Dahl, E.,Peters, H.,Balling, R.,Imai. K.: "Targeted disruption of Pax1 defines its null phenotype and proves haploinsufficiency" Proc. Natl. Acad. Sci. USA. 95. 8692-8697 (1998)

威尔姆，B.，达尔，E.，彼得斯，H.，鲍林，R.，今井。