| Mus musculus is one of the laboratory animals which are used widely in biomedical field. With the development of modern biomedicine, all kinds of animal models are needed in studying gene functions, discovering mechanism of human diseases and developing new type medicines. Because of its similarity to humanity in genomic sequences, biochemical metabolism and physiological mechanism, mouse is the ideal model for human diseases. There are many means to gain mouse models such as spontaneous, induced mutations, gene trap, transgenic mice and target gene. The scant hair mouse snthr-1Bao was obtained in 2002 by ENU induced mutagenesis which background strain was DBA/2. The snthr-1Bao mouse showed recessive heredity and scant hair. The mutant gene of snthr-1Bao mouse had been primary mapped on chromosome 9 about 71cM from the centromere. Mapping and cloning the mutant gene can help to discover the mechanism of hair shedding and corresponding human disease by comparative research. This is a new strategy for studying human disease and exploiting new remedial targets. We phenotyped snthr-1Bao mouse, finely mapped the mutant gene and primary mapped the modified genes of snthr-1Bao mouse which showed the characters of quantitative trait locis (QTLs).1. Biological characters of scant hair mouse snthr-1BaoWe observed the characters of growth, reproduction, anatomy, morphology and blood physiology of snthr-1Bao mouse and DBA/2 control mouse. We weighed, sized and compared the two strains mice from new born to 12 weeks old. 25 snthr-1Bao mice and 25 DBA/2 mice were executed after weighted, and necropsied to observer morphology of all organs. Each heart, liver, spleen, lung, kidney, brain, lymphoma, thymus, adrenal gland and germen was taken out, weighted, and compared the coefficient respectively. The results showed that snthr-1Bao mouse grew slower than DBA/2 mouse. The litter size of snthr-1Bao mouse was smaller than that of DBA/2 mouse (p﹤0.01). There were no morphological differences of other organs between snthr-1Bao mouse and DBA/2 mouse. But there were some differences (p﹤0.05 or p﹤0.01) in weight and coefficient of liver, spleen and ovary between two strains. Especially both testicles'weight and coefficient of snthr-1Bao mouse were lighter than DBA/2's (p﹤0.01). In addition, there were differences between snthr-1Bao mouse and DBA/2 mouse in the number of leukocyte, neutrophil leukocyte, eosinophils and basophil (p﹤0.01). So the mutation of snthr-1Bao mouse not only affects its coat but also affects its growth, some organs'weight and coefficient, reproduction and the ingredients of blood cells.2. Pathological study and skin development of snthr-1Bao mouseTo observe the pathological changes of each organ of snthr-1Bao mouse and the development of skin, heart, liver, spleen, lung, kidney, brain, lymphoma, thymus, testicle, ovary, intestine and lymphoma of intestine of 2 months old of DBA/2 control mouse and snthr-1Bao mouse were taken out respectively. The dorsal skin tissues of snthr-1Bao mouse and DBA/2 mouse were taken out when they were embryonic 18.5 days, new born, 1 week, two weeks, 1 month and 2 months old. These tissues were fixed, dehydrated, embedded and cut according to the routine pathological methods. The results indicated that all organs of snthr-1Bao mouse had no pathological change but the skin. There was no difference in the skin between snthr-1Bao mouse and DBA/2 mouse when they were younger than 1 week old. In this period, some hair follicles began developing, subcutaneous fat and sebaceous gland appeared but the structure of skin was normal. In the age from 1 week to 2 weeks, the epidermis and the dermis of snthr-1Bao mouse became thick. The epidermis and hair follicle were keratosic obviously. The number of sebaceous gland and fibroblasts increased companied with irregular arrangement of some hair roots which were curled and could not permeate the epidermis. When snthr-1Bao grew up, the dermatic pathological changes became severe. At 1 month old, the acanthocytes of snthr-1Bao mouse were incrassate. The epidermis and hair follicle were keratosic obviously. Some hair was curled and cannot permeate the epidermis, but some hair still developed well. The number of fibroblast was increased distinctly, and its arrangement was disorder. The skin becomes thin and the number of sebaceous gland is increased when mouse grows up. But compared with DBA/2 control mouse, the skin of snthr-1Bao mouse was still incrassate obviously and the number of sebaceous gland was getting more and more. So the dermatic pathological changes of snthr-1Bao mouse begin from postnatal 1 week old to 2 weeks old, and becomes more acutely as it grows up. The primary pathological changes include epidermal incrassation and hyperkeratosis and the structural deformation of some hairs. The following pathological changes include hairs curling in the skin, damnifing of the skin, local noninfective inflammation and scarring.3. Fine mapping of the mutant gene of snthr-1Bao mouse and Plcd1 being of the candidate geneThe mutant gene of snthr-1Bao mouse was mapped on the chromosome 9 about 71cM from the centromere by using 145 F2 mice [(C57BL/6J×DBA/2)F1 intercross]. To finely locate the mutant gene of snthr-1Bao mouse, more F2 mice and genomic markers nearby the mutant gene were required. 1100 F2 scant hair mice from about 4500 F2 mice were selected and genotyped. Around bilateral of D9Mit18, 16 STSs, 2 microsetallites, 35 SSRs and 3 SNPs were chosen and tested their polymorphism. The results were that D9Mit151, SSR NC0000756044061095, two SNPs rs8254361 and rs30195705 showed polymorphism between DBA/2 mouse and C57BL/6J mouse. After detecting 1100 F2 mice, there were 10 crossovers between the mutant gene and D9Mit18, 28 crossovers between the mutant gene and D9Mit151, 3 crossovers between the mutant gene and rs8254361 and NC0000756044061095 and 15 crossovers between the mutant gene and rs30195705. The mutant gene located in the region of 117762Kb to 119129Kb(1.3Mb) from the centromere on the chromosome 9. Checking the Esseml database, there were 15 genes in this region, and 10 of them were expressed in the mouse's skin. They were Itga9,Golga4,Azi2,Dlec1,Oxsr1,2010110K16RiK,Ctdspl,Vill,Plcd1,Acaa1b. Expressing levels of these 10 genes were detected by FQ-PCR. Itga9 was found over expressed, but no mutation was found after sequencing. Azi2 and Plcd1 were low expressed in the skin. It was reported that the phonotype of the gene Plcd1 knockout mouse obtained by Yoshikazu shared the same phenotype with snthr-1Bao mouse. Plcd1 is believed to be the strong candidate gene of snthr-1Bao based on biological characters, fine mapping, expressing level detecting and relative papers. This work provides important groundwork to clone the scant hair gene.4. Quantitative trait locus (QTLs) mapping of snthr-1Bao modify geneThe scant hair phenotype of F2 snthr-1Bao mouse, the offspring of (C57BL/6J×DBA/2)F1 intercross, was found showing three different kinds of phenotypes: more hair, less hair and the midst. We guessed that there were some modify genes which were introduced from C57BL/6J to F2 mouse and regulated the functions of mutant gene. Based on such a hypothesis, a new strategy was designed to map the QTLs of correlative modify genes. 35 microsetallites on 19 euchromosomes, 70 F2 mice with more hair and 57 F2 mice with less hair were used to genomic scan. After genotyping each F2 mouse, software R/qtl1.04-53 was used to calculate the possible linkage between the markers and the potential modify genes by the models of linear model regression and logistic regression separately. 63% was considered as threshold for suggestive QTLs if the score of LOD was exceed 2 in the same time. D4Mit17,D11Mit258,D12Mit136,D14Mit50 and D15Mit34 were found to be the candidate QTLs of modify gene. Then single QTL effect was analyzed by R/qtl1.04-53. The results were that B6 type D4Mit17 and D15Mit34 were interrelated to the F2 phenotype of more hair and B6 type D11Mit258,D12Mit136 and D14Mit50 were interrelated to the phenotype of less hair. Based on these results, the synergistic or antagonistic effects of the five candidate QTLs were tested by the same software. After three times calculations, D11Mit258,D14Mit50 and D15Mit34 were found to have association effect with each other. The statistical significance was pretty notable and the LOD score exceeded 2.3 up to 4.0. The conclusions are that D15Mit34, D11Mit258 and D14Mit50 are the modify locis of snthr-1Bao. Gene nearby D15Mit34 from B6 is responsible for more hair in F2 mouse. Genes nearby D11Mit258 and D14Mit50 from B6 are responsible for less hair of F2 mouse. These genes, with snthr-1Bao mutant gene together, affect with each other in the same molecular pathway to regulate the development of skin and hair. This work provides a key for studying the molecular mechanism of snthr-1Bao mouse and exploring new medicinal targets.
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