ENU-induced Mutagenesis In Mice And Mapping And Cloning Several Mutation Genes

As the accomplishment of the Human Genome Project, life sciences have entered post-genome era to systematically study gene functions on a large scale. Because of its similarity to humanity in genomic sequences, biochemical metabolism and physiological mechanism, Mus musculus is the ideal model animal in the study of functional genome. "Phenotype-driven" is one of the major strategies to study functional gene at present. The researchers induce mutation in mice with ENU (N-ethyl-N-nitrosourea ) to produce new strains, then map and clone the mutant gene through deep research of mutant, and get the functional informations of the mutation gene. It is a relatively promising shortcut to establish the animal model of human diseases and to study gene functions. We have carried out work in this area and have got 14 kinds of mutant mice through screening and genetic trials using approximate 10000 mice, and have successfully mapped 6 kinds of white spot genes after screening whole genome with microsatellite marks. At last, we have cloned 4 kinds of white spot mutant genes by position-candidate method, and identified the base change of 2 kinds mutation mice. Our work is divided into four parts.1. Obtaining 14 Kinds Mutants by ENU-induced Mutagenesis in Mice136 male C57BL/6(B6) mice (GO) at 8-10-week-old were injected intraperitoneally with ENU(100mg/kg) once a week for 2-4 times. After recovering its reproduction ability, every male mouse was mated with the females of the same strain, then we screened the mutant according to the visible phenotype and blood-biochemistry-item in the offspring (G1). We got 262 mutation mice after screening 4009 Gl mice, and mutagenesis rate was about 6.5%. Mutants included 124 with eye abnormity, 25 with tooth abnormity, 42 with white spot, 18 dwarfs, 20 with blood-biochemistry-item abnormity, and 33 others. We made mutation mice back-cross with the mice of the same strain, and there werel93 Gl mice successfully passed suchheredity test, in which 14 showed dominant genetic.The following were their number and major characters: B6-24 white spot, B6-136, B6-312, B6-980, B6-1244, B6-1866 and B6-2952, their coat on claws ,tail and venter were white in varying degrees, B6-3499 were cornea cascade, and all of them showed single gene dominant heredity, with penetrance of 100%. B6-24 showed iris defection, and also single gene dominant heredity, with penetrance about 85%. B6-343, B6-1610 and B6-2340 showed cornea cascade, B6-126 was dwarf, B6-29 tail was curly, and the genetic mode of these mice had not been confirmed. 7 kinds of white spot mutation mice, B6-24, 4 kinds of cornea cascade mice, B6-126 and B6-29 tail were good models for human piebald disease, human iris defection, inborn cornea degeneration, dwarf and the study of development respectively. These models would be used not only to study the pathologic mechanisms but also to study relevant gene functions.2. Establishment of 39 Microsatellites Marks System Distributed on Mouse Genome and Detection for 6 Mouse StrainsWe selected 39 microsatellites, distributed averagely on mouse genome and showed polymorphism between B6 and DBA(D2),and they were DlMit372, D1Mit84, DlMit273, D2Mit6, D2Mitl7, D2Mit528, D3Mit268, D3Mit15, D4Mit17, D4Mit54, D5Mit352, D5Mitl68, D6Mit274, D6Mit339, D7Mit246, D7Mit333, D8Mit4, D8Mit320, D9Mit325, D9Mit243, D10Mit3, D10Mit73, D11Mit163, DllMit258, D12Mitl36, D12Mit17, D13Mitl8, D13Mit262, D14Mit50, D14Mit205, D15Mit226, D15Mit34, D16Mitl89, D16Mit100, D17Mit33, D17Mit39, D18Mit52, D19Mitl28 and D19Mit33.Their PCR conditions were explored by a gradient method. Genetic background of 6 strains (total 8 groups) including B6,BALB/c,DBA,CBA,FVB,ICR from our Lab. and B6, BALB/c from Shanghai, were detected with these microsatellites. DNA samples were extracted from 10 mice tails of every group, then mixed as a DNA pool and amplified by microsatellites. The productions were used to compare the inbred coefficient of every strain after being electrophoresed by 12%PAGE. As a result, there were large differences of PCR c...