| Co-Investigator(Kenkyū-buntansha) |
BALLING Rudo ドイツ哺乳類遺伝学研究所, 所長
IMAI Kenji Institute of Mammalian Genetics, GSF,Researcher, 研究員
ARTZT Karen テキサス大学オースチン校, 動物学部, 教授
HRABE de Ang ドイツ哺乳類遺伝学研究所, 研究員
ABE Kuniya KUMAMOTO UNIVERSITY,Associate Professor, 医学部, 助教授 (40240915)
KAREN Artzt University of Texas at Austin, Professor
MARTIN Hrabe de Angelis Institute of Mammalian Genetics, GSF,Researcher
RUDOLF Balling Institute of Mammalian Genetics, GSF,Director
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| Research Abstract |
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
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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
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