采用基因导入法培育3种被毛稀疏小鼠
发布时间：2018-10-17 12:43
【摘要】：基因导入法是培育小鼠动物模型的有效手段,本文分别以微卫星标记及斑秃表型为选择标准,通过反复交配的策略,将目标基因组片段导入新的遗传背景中,获得了新的被毛稀疏小鼠。 1以微卫星为标记培育2种Plcd1修饰基因导入系小鼠 前期工作发现在C57BL/6J(简称B6)小鼠基因组上有4个位点对DBA/2J背景的snthr-1Bao小鼠被毛疏密程度有显著的调节作用。本实验首先在4个具有调节效应的基因组区域内选择微卫星为标记,在第2号染色体上32-68cM范围内选择D2Mit156, D2Mit249, D2Mit62;第5号染色体上24-84cM范围内选择D5Mit356, D5it314, D5Mit409;第742.7-52.7cM范围内选择D15Mit71,D15Mit29,D15Mit171。随后将C57BL/6J小鼠与snthr-1Bao小鼠配种,并通过微卫星标记在后代小鼠中选择带有C57BL/6J相应基因组片段的后代,互交其后代,并将互交后得到的后代继续与snthr-1Bao小鼠回交配种,经过连续6次回交,最后互交及选择,获得了2种分别携带C57BL/6J小鼠第2号、第15号染色体具有调节效应基因组片段的snthr-1Bao稀毛小鼠,并证实来自C57BL/6J小鼠第2号染色体上的修饰位点可加重斑秃症状、导致被毛减少,第15号染色体上的修饰位点可减轻斑秃症状、导致被毛增多。而第5号、第7号染色体上的修饰基因在导入过程中小鼠未能成功繁殖后代。本实验为相关基因的相互作用机制研究及化妆品评价提供新的模型。 2一种C57BL/6J背景斑秃基因导入系小鼠的培育 斑秃样小鼠AAtj是在昆明种小鼠生产繁殖过程中被发现培育而成的一个突变品系,突变基因呈单基因隐性遗传。该小鼠幼年时被毛正常,随着年龄增长,背部毛发局灶性稀疏,并缓慢进展,最后几乎全身无毛,这与人类疾病斑秃的临床症状较为相似。由于该斑秃样小鼠遗传背景复杂,为了定位、鉴定斑秃突变基因及研究资料的对比,需要“纯化”其遗传背景。本实验将AAtj斑秃样小鼠与C57BL/6J、鼠交配繁殖得到F1代小鼠。F1代含有突变基因但无斑秃表型,F1进行互交得到F2,F2中纯合子即表现为斑秃,再将F2代的斑秃样小鼠与C57BL/6J小鼠交配,得到F3代,F3代互交得到F4代小鼠,如此反复,一直进行到第8代,取F8代中的斑秃样小鼠互交,保种。建立了同源导入近交系斑秃样小鼠。在此基础上对F6代2月龄斑秃基因导入系小鼠的主要脏器、免疫器官及不同发育阶段的皮肤组织(从初生至8周,每周一次)行组织病理学检查,发现斑秃突变小鼠4周龄以后毛囊数量逐渐减少,皮肤厚度较野生型显著增加,免疫组化显示皮肤毛囊周围CD8阳性细胞浸润。本实验纯化了斑秃模型小鼠的遗传背景,为模型价值评估及对突变基因的定位鉴定奠定了基础。
[Abstract]:Gene introduction method is an effective method to breed mouse animal model. In this paper, microsatellite markers and alopecia areata phenotypes were used as the selection criteria, and the target genomic fragments were introduced into the new genetic background by repeated mating strategy. A new hairy sparse mouse was obtained. 1 two Plcd1 modified genes were cultured by microsatellite markers in mice with DBA/2J background. The results showed that there were four loci in the genome of C57BL/6J (B6) mice. The degree of hair density in snthr-1Bao mice was significantly regulated. In this study, microsatellites were first selected as markers in four genomic regions with regulatory effects, D2Mit156, D2Mit249, D2Mit62 in the 32-68cM range of chromosome 2, D5Mit356, D5it314, D5Mit409 in 24-84cM on chromosome 5, and D15Mit71-D15Mit29D15Mit171in the range of 742.7-52.7cM. Then the C57BL/6J mice were bred with snthr-1Bao mice, and the offspring with the corresponding genomic fragment of C57BL/6J were selected from the progeny mice by microsatellite markers, and their offspring were crossbred with each other, and the offspring obtained from the cross-breeding were continued to mate with the snthr-1Bao mice. After 6 consecutive backcrosses, finally, two snthr-1Bao thinly hairy mice carrying C57BL/6J mice with chromosomes 2 and 15 with regulatory effect genomes were obtained. The modified site on chromosome 2 of C57BL/6J mice can aggravate the symptoms of alopecia areata and reduce the coat hair. The modified site on chromosome 15 can alleviate the symptoms of alopecia areata and lead to the increase of coat hair. However, the modified gene on chromosome 5 and 7 failed to reproduce successfully. This study provides a new model for the study of the interaction mechanism of related genes and the evaluation of cosmetics. (2) A C57BL/6J background alopecia areata gene transfer line mice breeding alopecia areata mice AAtj is in Kunming mice A mutant strain discovered and bred during production and reproduction, The mutant gene is a single gene recessive inheritance. The hair coat of the mouse was normal in infancy. With the age, the hair on the back was focal sparse and developed slowly. Finally, the hair was almost hairless, which was similar to the clinical symptoms of human disease alopecia areata. Because of the complex genetic background of the alopecia areata mice, the genetic background of alopecia areata needs to be "purified" in order to identify the mutant gene and compare the research data. In this experiment, AAtj alopecia area-like mice were mated with C57BL / 6J, and F1 generation mice were bred. F1 generation contained mutant gene but had no alopecia areata phenotype. The F _ (1) F _ (2) F _ 2 homozygote appeared as alopecia areata, and the F _ (2) generation alopecia areata mice were mated with C57BL/6J mice. F 3 generation, F 3 generation cross get F 4 generation mice, so repeatedly, until the eighth generation, take the F 8 generation of alopecia areata like mice cross, keep the species. The homologous introduced inbred alopecia areata mice were established. On this basis, histopathological examination was performed on the main organs, immune organs and skin tissues (from birth to 8 weeks, once a week) of 2-month-old alopecia areata transgenic mice in F6 generation. It was found that the number of hair follicles gradually decreased and the thickness of skin increased significantly after 4 weeks of age in alopecia areata mutant mice. Immunohistochemical staining showed CD8 positive cell infiltration around the hair follicles. The genetic background of alopecia areata model mice was purified, which laid a foundation for evaluation of model value and identification of mutant genes.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332
本文编号：2276696
[Abstract]:Gene introduction method is an effective method to breed mouse animal model. In this paper, microsatellite markers and alopecia areata phenotypes were used as the selection criteria, and the target genomic fragments were introduced into the new genetic background by repeated mating strategy. A new hairy sparse mouse was obtained. 1 two Plcd1 modified genes were cultured by microsatellite markers in mice with DBA/2J background. The results showed that there were four loci in the genome of C57BL/6J (B6) mice. The degree of hair density in snthr-1Bao mice was significantly regulated. In this study, microsatellites were first selected as markers in four genomic regions with regulatory effects, D2Mit156, D2Mit249, D2Mit62 in the 32-68cM range of chromosome 2, D5Mit356, D5it314, D5Mit409 in 24-84cM on chromosome 5, and D15Mit71-D15Mit29D15Mit171in the range of 742.7-52.7cM. Then the C57BL/6J mice were bred with snthr-1Bao mice, and the offspring with the corresponding genomic fragment of C57BL/6J were selected from the progeny mice by microsatellite markers, and their offspring were crossbred with each other, and the offspring obtained from the cross-breeding were continued to mate with the snthr-1Bao mice. After 6 consecutive backcrosses, finally, two snthr-1Bao thinly hairy mice carrying C57BL/6J mice with chromosomes 2 and 15 with regulatory effect genomes were obtained. The modified site on chromosome 2 of C57BL/6J mice can aggravate the symptoms of alopecia areata and reduce the coat hair. The modified site on chromosome 15 can alleviate the symptoms of alopecia areata and lead to the increase of coat hair. However, the modified gene on chromosome 5 and 7 failed to reproduce successfully. This study provides a new model for the study of the interaction mechanism of related genes and the evaluation of cosmetics. (2) A C57BL/6J background alopecia areata gene transfer line mice breeding alopecia areata mice AAtj is in Kunming mice A mutant strain discovered and bred during production and reproduction, The mutant gene is a single gene recessive inheritance. The hair coat of the mouse was normal in infancy. With the age, the hair on the back was focal sparse and developed slowly. Finally, the hair was almost hairless, which was similar to the clinical symptoms of human disease alopecia areata. Because of the complex genetic background of the alopecia areata mice, the genetic background of alopecia areata needs to be "purified" in order to identify the mutant gene and compare the research data. In this experiment, AAtj alopecia area-like mice were mated with C57BL / 6J, and F1 generation mice were bred. F1 generation contained mutant gene but had no alopecia areata phenotype. The F _ (1) F _ (2) F _ 2 homozygote appeared as alopecia areata, and the F _ (2) generation alopecia areata mice were mated with C57BL/6J mice. F 3 generation, F 3 generation cross get F 4 generation mice, so repeatedly, until the eighth generation, take the F 8 generation of alopecia areata like mice cross, keep the species. The homologous introduced inbred alopecia areata mice were established. On this basis, histopathological examination was performed on the main organs, immune organs and skin tissues (from birth to 8 weeks, once a week) of 2-month-old alopecia areata transgenic mice in F6 generation. It was found that the number of hair follicles gradually decreased and the thickness of skin increased significantly after 4 weeks of age in alopecia areata mutant mice. Immunohistochemical staining showed CD8 positive cell infiltration around the hair follicles. The genetic background of alopecia areata model mice was purified, which laid a foundation for evaluation of model value and identification of mutant genes.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332
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