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Transgenic animal model showing genomic instabilities like human hypervariable minisatellite and its applications in forensic genetics.

显示基因组不稳定性（如人类高变小卫星）的转基因动物模型及其在法医遗传学中的应用。

基本信息

批准号：
18390205
负责人：
TAMAKI Keiji
金额：
$ 10.56万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390205/
关键词：
minisatellite Medaka genome animal model

项目摘要

Since Sir Alec Jeffreys' initial discovery of tandemly repeated DNA units varying amongst different individuals in 1984, a growing body of evidence has come to describe the element of human hypervariable minisatellites. Decades of active research dedicated solely to these tandem repetitive units have produced a vast amount of insight into their underlying molecular basis; however, the genomic dynamics of minisatellites at meiosis in germ cells has long remained an elusive issue. In the present study, Chyzias latipes (or Medaka) were selected to examine genomic instability in minisatellites owing to the inherent difficulty of human testing.Information retrieved from the Medaka Genome Database identified 30,504 VNTR (variable number of tandem repeats) sequences within the8chromosomes comprising the medaka genome. These results are loosely comparable to that of the human genome. Conversely, the number of expected hypervariable minisatellites-using HistoryR and GC content as an index謡as su … More bstantially lower in medaka. To initiate the study, 174 minisatellite loci were selected on 4 medaka chromosomes. Of these, 31 loci were interrogated for polymorphisms via polymerase chain reaction (PCR) using DNA derived from Oryzias latipes and Cab as templates. Preliminary screening efforts revealed multiple alleles at four of these loci, for which sequences were subsequently determined.Next, trans-minisatellite medaka were established by introducing the short allele of human minisatellite B6.7 into fertilized medaka eggs. 22 individual fish survived to adulthood (GO), of which the presence of B6.7 was only confirmed in a small fraction (n=4). In the following generation (F1), hundreds of fish were screened, but the search ended in zero positive confirmations of B6.7 alleles. Consequently, the scope of the screening was reconstituted to include the flanking sequences of the human alleles, and B6.7 was reinserted into a new batch of fertilized medaka eggs. This produced 25 fish (GO), in which one was positive for the B6.7 allele. This fish was crossed with non-injected GO fish to establish the F1 generation. In total, 325 F1 fish were born and B6.7 was retained in the genome of 4 of these fish. These 4 fish were back-crossed to produce the F2 generation. The retention rate of B6.7 in F2 fish drastically increased to 45%. Following this, F2 were further back-crossed to establish the F3 generation. At present, F3 fish are in the process of being self-mated (F3 with F3) in order to establish B6.7 homozygotes. While our preliminary results showed 112/257 eggs positive for B6.7 in heterozygous (F2) fish, no length variance, nor mutations, were noted in these alleles. Less

自从1984年亚历克·杰弗里斯爵士首次发现不同个体之间存在不同的重复DNA单位以来，越来越多的证据来描述人类高可变小卫星的元素。几十年来，专门致力于这些串联重复单位的活跃研究已经对其潜在的分子基础产生了大量的见解；然而，生殖细胞减数分裂时的小卫星的基因组动力学长期以来一直是一个难以捉摸的问题。在本研究中，由于人类测试的固有困难，本研究选择青竹(或Medaka)来检测微小卫星的基因组不稳定性。从Medaka基因组数据库检索的信息在组成Medaka基因组的8条染色体中鉴定了30,504个VNTR(可变数目的串联重复序列)序列。这些结果与人类基因组的结果大致相似。相反，预期的超变量小卫星数量-使用历史R和GC含量作为索引謡作为su…更明显的是，在青竹中更低。为了启动研究，在青竹的4条染色体上选择了174个小卫星座位。其中，以水稻和Cab的DNA为模板，通过聚合酶链式反应(PCR)检测了31个基因座的多态性。初步的筛选工作在其中4个座位上发现了多个等位基因，并对其进行了序列测定。其次，将人类小卫星B6.7的短等位基因导入青竹受精卵中，建立了跨小卫星青竹。22条个体鱼存活到成体(GO)，其中B6.7的存在仅被证实为一小部分(n=4)。在下一代(F1)中，对数百条鱼进行了筛选，但搜索结果为B6.7等位基因零阳性确认。因此，重新调整了筛选范围，将人类等位基因的侧翼序列包括在内，并将B6.7重新插入到新一批受精卵中。这产生了25条鱼(GO)，其中一条对B6.7等位基因呈阳性。该鱼与未注射的围棋鱼杂交，建立F1代。总共出生了325条F1鱼，其中4条在基因组中保留了B6.7。这4条鱼回交产生F2代。B6.7在F2鱼体内的存留率大幅提高到45%。随后，进一步回交F2，建立F3代。目前，F3代鱼类正在进行自交(F3代与F3代)以建立B6.7纯合子。而我们的初步结果显示，在杂合子(F2)鱼中，有112/257个卵呈B6.7阳性，这些等位基因没有发现长度变异或突变。较少