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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

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 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