【作者】 李岚；

【导师】 王小龙；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2021， 硕士

【摘要】 毛乳头细胞(dermal papilla cells,DPCs)是位于毛囊底部的特殊的成纤维细胞团,绒山羊绒毛生长和周期性发育机制与毛乳头细胞的增殖密切相关,这一过程受到多个基因的调控。研究表明Hoxc13基因在控制毛发形成中具有重要作用,其主要在真皮乳头细胞中表达,经过该基因过表达或者缺陷处理的小鼠都显示异常的毛发生长。目前利用转录组等多种方法鉴定了大量与绒山羊绒毛生长和周期性发育相关的候选基因,但所筛选的基因数目庞大,且差异较大,这为从中选择少数关键基因而进行后续的功能研究带来了不便。因此,需要在筛选方法上进行创新。基于CRISPR的筛选系统可实现多靶点的基因组编辑,同时在基因功能的鉴定方面被大规模的应用,包括对细胞耐药性、细胞增殖等相关基因的筛选。因此,本研究利用CRISPR聚焦型文库,通过在陕北白绒山羊DPCs中实现基因敲除,进而鉴定DPCs增殖的必需基因(essential genes)。试验过程及具体结果如下:(1)设计并构建CRISPR聚焦型文库。选择本实验室前期所鉴定的以及NCBI上已公布的绒山羊皮肤和毛囊差异表达基因,共822个基因用于文库的构建。该文库包含4910条sg RNA,其中40条为非靶向山羊基因组的阴性对照sg RNA,另外4870条sg RNA分别靶向822个蛋白编码基因。通过高通量测序技术对文库质量进行评估,发现文库中85%的sg RNA具有最佳的GC含量;97.85%的sg RNA靶向所有Ref Seq同工型共有的外显子;86%的sg RNA靶向基因内mRNA的5’端。以上结果表明CRISPR聚焦型文库具有高度的编辑活性,可用于高质量筛选。(2)毛乳头细胞的鉴定。显微镜下观察细胞,可见该细胞具有凝集性生长特性、呈扁平状和多角形等特征;对细胞进行免疫荧光染色,发现α-SMA和Vimentin毛乳头细胞分子标记物免疫荧光呈阳性表达,表明该细胞为DPCs。(3)毛乳头细胞增殖必需基因的筛选。在MOI值为0.5的条件下对DPCs进行慢病毒文库感染,48 h后使用嘌呤霉素进行筛选,而后转入正常的细胞培养,分别在药筛后第0天(D0)、第9天(D9)和第15天(D15)收集存活的细胞用于高通量测序。利用高通量测序及生物信息学技术筛选DPCs增殖的必需基因,最终共获得57个候选基因。结合GO分析和KEGG通路分析发现这些候选基因与细胞生长、细胞周期和细胞凋亡有关。(4)必需基因的功能验证。在排名前10的候选基因中选择SBDS、HJURP以及FNDC5进行功能验证。利用siRNA干扰SBDS、HJURP以及FNDC5基因在DPCs中的表达,通过CCK8增殖试验、Ed U和流式细胞仪检测细胞周期技术等检测其对DPCs增殖的影响。结果表明干扰SBDS基因和HJURP基因的表达抑制了DPCs的增殖;干扰FNDC5基因的表达促进了DPCs的增殖。本试验以陕北白绒山羊背部皮肤为原材料分离纯化DPCs,根据实验室前期以及NCBI上已公布的与皮肤毛囊生长有关的差异基因构建CRISPR聚焦型敲除文库,利用慢病毒将CRISPR文库导入DPCs,进而实现基因敲除,随后基于阴性筛选策略利用高通量测序技术以及生物信息学技术筛选并鉴定DPCs增殖过程中的必需基因。本论文为进一步挖掘和鉴定在绒山羊中具有重要研究和育种价值的关键基因提供了新的研究方法,同时为动物功能基因组学研究提供了参考。更多还原

【Abstract】 Dermal papilla cells(DPCs)are special fibroblasts,which exist at the bottom of hair follicles.The mechanism of cashmere growth and periodic development in cashmere goats is highly related to the proliferation of DPCs,and this process is regulated by several genes.Studies have shown that Hoxc13 gene has an important role in controlling hair formation,and it is mainly expressed in DPCs.Mice treated with Hoxc13 overexpression or defect show abnormal hair growth.At present,a large number of candidate genes related to cashmere fiber growth and periodic development have been identified by transcriptomic or other methods,however,the number of gene list is large,bringing inconvenience for the selection key regulatory genes for the following functional studies.Therefore,it is necessary to innovate in the screening method.CRISPR-based screening system can achieve multi-target genome editing,and has been widely used in functional genomics,including the screening of genes related to cell resistance and cell proliferation.Therefore,in this study,we used CRISPR focused library to achieve gene knockout in Shanbei White Cashmere goat,and then identified the essential genes for the proliferation of DPCs.The test process and specific results are as follows:(1)Design and construction of a focused CRISPR library.A total of 822 differentially expressed genes in cashmere goat skin and hair follicles identified in our laboratory and published on NCBI were used to construct the library.The library contains 4,910 sg RNA,of which 40 are negative control sg RNA.and the other 4,870 sg RNA target 822 protein-coding genes respectively.The quality of the library was evaluated by high-throughput sequencing technology,and it was found that 85% of the sg RNA in the library had the best GC content;97.85% of sg RNA targeted exons common to all Ref Seq isotypes;86% of sg RNA targeted the 5 ’mRNA.These results show that the CRISPR focused library has high editing activity and can be used for genetic screening.(2)Identification of DPCs derived from cashmere goats.The cells,observed under the microscope showed that the cells had the characteristics of agglutinative growth,flattening and polygonal;immunofluorescence staining showed that α-SMA and Vimentin dermal papilla cell molecular markers were positive,indicating the DPCs can be used for CRISPR screening.(3)Screening of essential genes for proliferation of DPCs.DPCs was infected with lentivirus library under the condition of MOI 0.5,after 48 hours,puromycin was used for screening.The surviving cells were collected on the 0th day(D0),9th day(D9)and 15 th day(D15)for high-throughput sequencing.The essential genes for DPCs proliferation were screened by high-throughput sequencing and bioinformatics techniques,and a total of 57 candidate genes were obtained.Combined with GO analysis and KEGG analysis,it was found that these genes were mainly enriched to cell growth,cell cycle and apoptosis.(4)Functional verification of essential genes.Among the top 10 essential genes,SBDS,HJURP and FNDC5,were selected as candidate genes for functional identification.siRNA was used to interfere the expression of the SBDS,HJURP and FNDC5 genes in DPCs,and the effect on the proliferation of DPCs was detected by CCK8 proliferation test,EDU and flow cytometry.The results showed that interfering with the expression of SBDS gene and HJURP gene inhibited the proliferation of DPCs,while interfering with the expression of FNDC5 gene promoted the proliferation of DPCs.In this study,DPCs was isolated and purified from the back skin of Shanbei White Cashmere goat.A total of 822 differentially expressed genes in cashmere goat skin and hair follicles identified in our laboratory and published data on NCBI were used to construct the library.we used this library to achieve gene knockout in Shanbei White Cashmere goat,and then identified the essential genes for the proliferation of DPCs.Based on the negative screening strategy,high-throughput sequencing and bioinformatics techniques were used to screen and identify the essential genes underlying proliferation in DPCs.This study provides a new method for further mining and identification of key genes,which have important research and breeding value in cashmere goats,and further provides a reference approach for the study of functional genomics in livestock.更多还原