發(fā)布時間：2018-10-17 12:43

【摘要】：基因?qū)敕ㄊ桥嘤∈髣游锬Ｐ偷挠行侄?本文分別以微衛(wèi)星標記及斑禿表型為選擇標準,通過反復(fù)交配的策略,將目標基因組片段導(dǎo)入新的遺傳背景中,獲得了新的被毛稀疏小鼠。 1以微衛(wèi)星為標記培育2種Plcd1修飾基因?qū)胂敌∈?前期工作發(fā)現(xiàn)在C57BL/6J(簡稱B6)小鼠基因組上有4個位點對DBA/2J背景的snthr-1Bao小鼠被毛疏密程度有顯著的調(diào)節(jié)作用。本實驗首先在4個具有調(diào)節(jié)效應(yīng)的基因組區(qū)域內(nèi)選擇微衛(wèi)星為標記,在第2號染色體上32-68cM范圍內(nèi)選擇D2Mit156, D2Mit249, D2Mit62;第5號染色體上24-84cM范圍內(nèi)選擇D5Mit356, D5it314, D5Mit409;第742.7-52.7cM范圍內(nèi)選擇D15Mit71,D15Mit29,D15Mit171。隨后將C57BL/6J小鼠與snthr-1Bao小鼠配種,并通過微衛(wèi)星標記在后代小鼠中選擇帶有C57BL/6J相應(yīng)基因組片段的后代,互交其后代,并將互交后得到的后代繼續(xù)與snthr-1Bao小鼠回交配種,經(jīng)過連續(xù)6次回交,最后互交及選擇,獲得了2種分別攜帶C57BL/6J小鼠第2號、第15號染色體具有調(diào)節(jié)效應(yīng)基因組片段的snthr-1Bao稀毛小鼠,并證實來自C57BL/6J小鼠第2號染色體上的修飾位點可加重斑禿癥狀、導(dǎo)致被毛減少,第15號染色體上的修飾位點可減輕斑禿癥狀、導(dǎo)致被毛增多。而第5號、第7號染色體上的修飾基因在導(dǎo)入過程中小鼠未能成功繁殖后代。本實驗為相關(guān)基因的相互作用機制研究及化妝品評價提供新的模型。 2一種C57BL/6J背景斑禿基因?qū)胂敌∈蟮呐嘤?斑禿樣小鼠AAtj是在昆明種小鼠生產(chǎn)繁殖過程中被發(fā)現(xiàn)培育而成的一個突變品系,突變基因呈單基因隱性遺傳。該小鼠幼年時被毛正常,隨著年齡增長,背部毛發(fā)局灶性稀疏,并緩慢進展,最后幾乎全身無毛,這與人類疾病斑禿的臨床癥狀較為相似。由于該斑禿樣小鼠遺傳背景復(fù)雜,為了定位、鑒定斑禿突變基因及研究資料的對比,需要“純化”其遺傳背景。本實驗將AAtj斑禿樣小鼠與C57BL/6J、鼠交配繁殖得到F1代小鼠。F1代含有突變基因但無斑禿表型,F1進行互交得到F2,F2中純合子即表現(xiàn)為斑禿,再將F2代的斑禿樣小鼠與C57BL/6J小鼠交配,得到F3代,F3代互交得到F4代小鼠,如此反復(fù),一直進行到第8代,取F8代中的斑禿樣小鼠互交,保種。建立了同源導(dǎo)入近交系斑禿樣小鼠。在此基礎(chǔ)上對F6代2月齡斑禿基因?qū)胂敌∈蟮闹饕K器、免疫器官及不同發(fā)育階段的皮膚組織(從初生至8周,每周一次)行組織病理學(xué)檢查,發(fā)現(xiàn)斑禿突變小鼠4周齡以后毛囊數(shù)量逐漸減少,皮膚厚度較野生型顯著增加,免疫組化顯示皮膚毛囊周圍CD8陽性細胞浸潤。本實驗純化了斑禿模型小鼠的遺傳背景,為模型價值評估及對突變基因的定位鑒定奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R-332

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