【作者】 李东；

【导师】 李碧春； 吴信生；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2017， 博士

【摘要】 生殖细胞可以把生物的遗传信息传递给下一代,维持生命的传递,对生殖细胞分化研究具有重要意义。原始生殖细胞（Primordial germ cells,PGCs）是所有生殖细胞的始祖细胞,能够在性腺中分化为精原干细胞（Sperrmatogonial stem cells,SSCs）或卵原干细胞（Ovary stem cells,OSCs）,进而进一步发育分化为精子或卵子。PGCs的发生受到许多内源性调控因子和细胞外基质等外源因素的调控,这些因子通过不同的信号通路,构成复杂的调控网络,直接或间接地调控PGCs的形成。目前,研究者们通过体外分离培养、体外诱导、构建转基因细胞等方多种方式对PGCs的生长和分化进行了大量的研究,然而关于调控PGCs发生过程的具体调控网络分子机制却知之甚少,因此PGCs发生过程机制的探究成为近年来备受关注的焦点。随着PGCs研究的深入,研究者们已经探究发现存在一些关键性的基因、信号通路、生长因子以及表观遗传修饰因素在此过程中起到重要的调控作用,随着这些因素对于PGCs发生起到了一定的促进作用,但是并没有真正从意义上提供提高PGCs生成效率,进而无法满足相关的科研需求,因此亟需对从新的领域对PGCs的生成机制进行创新性地探究,深入了解和认识PGCs的产生过程,从而获取大量的PGCs。为了进一步探究调控PGCs发生及分化的作用机制,从根本上解决PGCs生成效率低下的问题,本研究基于本实验室前期对鸡胚胎干细胞（Embryonic stem cells,ESCs）、PGCs和SSCs的RNA-Seq测序结果中筛选到PGCs特异表达的新基因LOC418414（Genbank登录号:XM₄16629.3）的基础上,根据其表达位置命名为C1EIP,对其在鸡ESCs向PGCs分化过程中从体内和体外两个水平上进行系统的功能和机制验证,深入了解家鸡ESCs向PGCs分化过程的调控机制,为有效提高ESCs向PGCs诱导分化效率提供理论依据和参考,并且为鸡ESCs向PGCs分化其他类似关键功能基因的功能及机制研究提供理论基础。研究结果如下:1.为了有效确定候选特异基因C1EIP在的生物功能以及其真核细胞中的定位,为后期功能及机制研究奠定基础,对C1EIP进行生物信息学分析显示,C1EIP主要与鸟类同源性较高,但同源蛋白为未知蛋白,未发现特异的结构域。我们结合NCBI数据库提供的C1EIP编码序列,利用RT-PCR扩增C1EIP编码区,分别构建重组表达载体pEGFP-C1EIP 和 pcDNA3.0-C1EIP。以 pEGFP-C1EIP 和 pcDNA3.0-C1EIP 转染 DF1 细胞,确定C1EIP的定位,并以RT-PCR和Western Blot检测C1EIP在真核细胞中转录和翻译的情况;同时以PEI包裹pcDNA3.0-C1EIP载体,肌肉注射小鼠后采血制备抗小鼠血清,以IFA和Western Blot检测抗体效价。酶切及测序结果均显示pEGFP-C1EIP和pcDNA3.0-C1EIP载体构建成功。pEGFP-C1EIP转染DF1细胞后,C1EIP表达于细胞质中,说明C1EIP定位于细胞质中,且能够启动转录和翻译;以1μg:1.5μg的比例将PEI包裹pcDNA3.0-C1EIP产物注射小鼠肌肉制备抗小鼠血清,IFA检测最佳效价为1:25,并以此效价进行Westen Blot检测显示能够识别到C1EIP蛋白。2.为了探究C1EIP在鸡PGCs生成过程中生物学功能探究,我们针对C1EIP编码区设计3个shRNA靶位点和1个阴性对照位点,构建慢病毒干扰载体,并进行慢病毒包裹,以qRT-PCR检测慢病毒干扰载体的干扰效率。在体外,以慢病毒干扰载体和过表达载体分别转染2代ESCs细胞,结合RA诱导剂诱导,观察ESCs形态变化,并以qRT-PCR、细胞免疫化学和流式细胞分析检测PGCs生成效率;在体内,以慢病毒RNA干扰载体侵染鸡胚,摸索出最佳的侵染效率,侵染后的鸡胚以qRT-PCR、PAS切片染色、免疫组化以及流式细胞分析等方法检测PGCs生成效率。结果显示成功构建慢病毒干扰载体C1EIP-shl、C1EIP-sh2 和 C1EIP-sh3,干扰效率分别为 80.39%、87.80%和 38.36%。在体外,正常RA诱导在第2d出现小类胚体（Embryoid Body,EB）,在第4d时类胚体增多、变大,第6d类胚体的边缘开始出现裂口,并且伴随有少量的细胞从类胚体内部释放,第8d时类胚体严重破裂,大量细胞释放,在第10-12d类胚体基本上裂解完全,出现类精原干细胞（Spermatogonial stem cells-like,SSCs-like）,并聚团成葡萄串状克隆;过表达组中,在第2d出现大的类胚体,第4d类胚体边缘开始出现小缺口,第6d类胚体大部分破裂并释放大量内部细胞,第8d出现类SSCs细胞,第10-12d出现典型的呈葡萄串状的SSCs克隆;在敲低中,第2-6d细胞未出现类胚体,第8d开始出现小的类胚体,然而培养至12d类胚体的数量、大小以及形态并未出现明显的变化;qRT-PCR结果显示过表达C1EIP后,全能性基因sox2表达量总体上由1.00±0.04持续降低至0.14±0.01,而cvh、c-kit、Stra8、Dazl、integrin α6和integrin β1表达量自始至终都逐步上升至2.14±0.03、2.79±0.05、2.26±0.06、2.73±0.03 和 2.59±0.05,目的基因 C1EIPP 表达量在第 4d 上升至3.99±0.06,在第12d时由下降至2.87±0.07;敲低C1EIP后,sox2表达量在第0-12d中没有显著的变化,cvh、c-kit和C1EIP表达量分别由1.00±0.02、1.00±0.01和1.00±0.02稳定持续下降至 0.55±0.01、0.70±0.01 和 2.87±0.07,Stra8、Dazl、integrin α6和integrin β1表达量由 1.00±0.04、1.00±0.04、1.00±0.03 和 1.00±0.04 分别先下降至 0.53±0.04、0.45±0.05、0.86±0.03 和 0.76±0.02,然后再略微上升至 0.59±0.01、0.80±0.04、0.88±0.04、0.93±0.08;细胞免疫化学结果显示相对于RA诱导组,过表达组显著增加CVH和C-KIT的表达,而敲低组抑制CVH和C-KIT的表达。流式细胞分析结果显示,RA诱导组中CVH+细胞比例为3.4%,在过表达C1EIP后,CVH+细胞比例显著上调至4.6%,然而敲低C1EIP使得CVH+细胞降低至2.9%。在体内,鸡胚注射慢病毒载体的最佳条件为钝端注射10μL 106TU/mL慢病毒干扰载体+90μL DMEM,注射后能够稳定表达egfp且鸡胚能够激发绿色荧光。qRT-PCR结果显示各个基因的表达量在Blank组和sh-Ctrl组之间没有显著的变化,而在敲低C1EIP后,全能性基因sox2的表达量下降趋势相对于其他分组有所减缓,而cvh、c-kit、stra8、Dazl、integrin α6和integrin β1等生殖相关基因以及目的基因C1EIP的大幅度下降到0.20±0.07、0.43±0.08、0.45±0.06、0.51±0.07、0.35±0.07、0.46±0.04和0.22±0.03;PAS结果显示敲低C1EIP能够显著降低PGCs的产生。免疫组织化学结果显示空白组和阴性对照组中生殖嵴中高度表达CVH和C-KIT,且表达部位几乎充满整个生殖嵴中,然而在敲低组中,CVH和C-KIT蛋白表达明显降低,并且表达的部位仅仅局限于生殖嵴的边缘部分。流式细胞分选结果显示,相对于空白组和对照组（4.6%和4.3%）,敲低C1EIP使得CVH标记的PGCs比例明显下调至3.1%。3.为了系统有效地阐明C1EIP表达的转录水平调控机制,我们结合NCBI和UCSC数据库中查询C1EIP转录起始位点上游启动子2000bp左右的序列,扩增C1EIP启动子长片段,克隆至pEGFP-N1载体中,置换CMV启动子,构建重组表达载体pC1EIP-EGFP,转染至DF1细胞中检测C1EIP启动子长片段的启动活性;通过缺失片段克隆技术,构建C1EIP启动子不同缺失片段载体PGL3-P1～PGL3-P10,转染DF1细胞后以双荧光素酶报告系统检测各个缺失片段的启动子活性,筛查出C1EIP启动子核心调控区域;在此区域以生物信息学预测关键转录因子结合位点,并对各个转录因子结合位点分别进行定点缺失,构建缺失载体,同样以双荧光素酶报告系统检测转录因子结合位点缺失对启动子活性的影响;最后分别 10μmol/L、1μmol/L 和 4mmol/L 的 5-Aza-2’-deoxycytidine（5-Azadc）、Trichostatin A（TSA）和 valproic acid（VPA）处理,检测 DNA 甲基化和组蛋白乙酰化对C1EIP启动子活性以及C1EIP在ESCs、PGCs和SSCs中表达变化的影响。酶切及测序结果均表明pC1EIP-EGFP构建正确,转染DF1细胞后能够激发绿色荧光,定性地说明扩增的C1EIP启动子长片段具有启动活性;同时启动子各个缺失片段载体酶切和测序结果也表明载体构建成功,双荧光素酶检测启动子活性结果显示P4-P6间,即-1025～-912bp为C1EIP启动子的核心活性区域;在此区域内关键的转录因子结合位点包括MEISS1、MAFG::NFE2L1、STAT3、HLTF和Hand1::Tcf3 其中只有STAT3起到正向调控作用;最后发现在DNA甲基化抑制5-Azadc以及组蛋白乙酰化抑制剂TSA和VPA处理后,C1EIP 启动子活性由 10.82±0.54 上升至 18.49±0.72、33.27±0.83 和 38.11±0.53,同时5-Azadc、TSA和VPA处理后,C1EIP在鸡ESCs、PGCs和SSCs中表达量分别由1.00±0.23、5.73±0.54、0.94±0.23 变化为 1.43±0.42、10.60±0.26、2.83±0.18 和2.64±0.23、9.89±0.37、3.25±0.16 以及 2.99±0.43、8.33±0.56、2.94±0.29。4.为了探究C1EIP调控PGCs生成的机制,我们通过构建C1EIP原核表达载体,诱导其在大肠杆菌中表达,以GST pull-down挖掘C1EIP互作蛋白,构建融合表达载体pcDNA3.0-ENO1-HA,以体外免疫共沉淀技术验证ENO-HA和C1EIP-EGFP间互作关系。结合在线数据库,以生物信息学分析技术探究互作基因ENO1的关联基因,以qRT-PCR验证其在PGCs生成过程中的表达变化情况。通过构建Myc启动子的双荧光素酶报告载体PGL3-Myc-pro以及重组表达载体pcDNA3.0-MBP-1,同时为了消除同源家族基因的干扰,我们还构建了ENO2的重组表达载体pcDNA3.0-ENO2,结合之前构建完成的pcDNA3.0-ENO1-HA载体,共转染DF1细胞,以双荧光素酶报告系统检测Myc启动子活性变化情况,并根据所得结果和查找相关文献,绘制出C1EIP和ENO1/MBP-1互作介导Notch信号通路调控PGCs生成的模式图。GST pull-down结果显示C1EIP与ENO1之间存在互作,且体外免疫共沉淀验证C1EIP之与ENO1间的互作关系。通过Uniport和PSORT Ⅱ数据库以及共表达网络分析发现ENO1定位于细胞质中,与Myc相互关联,Myc下游基因为Notch1。qRT-PCR结果显示C1EIP和ENO1在鸡ESCs向SSCs分化过程中表达趋势保持一致,并且与Myc和Noth1表达趋势相反。酶切和测序结果显示双荧光素酶报告载体PGL3-Myc-pro以及重组表达载体pcDNA3.0-MBP-1和pcDNA3.0-ENO2均构建成功,且双荧光素酶检测结果显示MBP-1对Myc启动子活性存在抑制作用,而ENO1和ENO2对其没有影响。最后根据以上结果和相关文献报道绘制C1EIP和MBP-1互作介导Notch信号通路调控PGCs生成的模式图,结果显示C1EIP可能作为锚定蛋白将ENO1蛋白固定于细胞质中,当ENO1发生磷酸化或去磷酸化后,C1EIP将其释放,并易位入核,在易位过程中丢失前面96个氨基酸残基,形成MBP-1蛋白,从而结合至Myc启动子,抑制其转录,进而阻断Notch信号通路的信号传导,最终反向促进鸡ESCs向PGCs方向分化。更多还原

【Abstract】 Germ cell has an irreplaceable role in passage of the genetic information to the next generation and sustain life.Primordial germ cells（PGCs）are the ancestor cells of all germ cells can differentiate into spermatogonial stem cells（SSCs）or ovarian stem cells（OSCs）in the gonads,and further developed into sperm or ovum.The occurrence of PGCs is regulated by many exogenous factors such as endogenous regulatory factors and extracellular matrix.These factors form a complex regulatory network through different signaling pathways,directly or indirectly regulate the formation of PGCs.Currently,the researchers have done a lot of research on the growth and differentiation of PGCs by in-vitro isolation,in-vitro induction,construction of transgenic cells and so on.However,little is known about the molecular mechanism of regulation of PGCs,Therefore,the mechanism of PGCs has become the focus of attention in recent years.With the deepening of PGCs research,researchers have explored the discovery that there are key genes,signaling pathways,growth factors,and epigenetic factors that play important regulatory roles in this process.Although these factors have contributed to the occurrence of PGCs,there is no real sense to improve the efficiency of PGCs formation and thus can not meet the relevant research needs.Therefore,it is urgent to innovate the mechanism of PGCs from new fields to explore and understand the generation process of PGCs in order to obtain a large number of PGCs.Therefore,in order to further explore the mechanism of regulating the occurrence and differentiation of PGCs and fundamentally solved the problem of PGCs generation efficiency,based on RNA-Seq performed in chicken Embryonic stem cells（ESCs）,PGCs and SSCs transcriptome,we screened significantly different and PGCs’ specific expressed gene,C1EIP（Genbank Accession:XM₄16629.3）in differentiation process of ESCs to SSCs and systematically explored its function and regulation mechanism in the process of differentiation of male germ cells from the two levels of in vivo and in vitro.The aim of this study is to understand the mechanism of differentiation of chicken ESCs into PGCs,provide a theoretical basis and reference for the effective differentiation of ESCs to PGCs and a theoretical basis for the study of the function and mechanism of other similar key functional genes for the differentiation of chicken ESCs into PGCs.The results are as follows:1.In order to effectively determine the biological function of the candidate specific gene C1EIP and its localization in eukaryotic cells and lay the foundation for the study of late function and mechanism,we constructed bioinformatics analysis and the results showed the C1EIP had a major high homology with birds,but also was unknown homologous proteins and was not found in specific domains of gene function and so require further study.we constructed the recombinant expression vectors pEGFP-C1EIP and pcDNA3.0-C1EIP by RT-PCR with amplification of C1EIP CDS squence provided by NCBI database.DF1 cells were transfected with pEGFP-C1EIP and pcDNA3.0-C1EIP to determine the localization of C1EIP and RT-PCR and Western Blot were used to detect the transcription and translation of C1EIP in eukaryotic cells.At the same time,the pcDNA3.0-C1EIP vector was encapsulated with PEI and injected into the mice intramuscular to prepare anti-mouse serum whose titer was detected by IFA and Western Blot.The results showed that the digestion and sequencing results indicated the pEGFP-C1EIP and pcDNA3.0-C1EIP vectors were successfully constructed.egfp expressed in the cytoplasm after transfection of DF1 cells with pEGFP-C1EIP,indicating that C1EIP is localized in eukaryotic cytoplasm and is capable of initiating transcription and translation.Anti-mouse serum was prepared by encapsulating pcDNA3.0-C1EIP product with PEI at a ratio of 1|μg:1.5μg.The IFA detection titer was 1:25 and the Westen Blot assay showed that the C1EIP protein was recognized.2.In order to explore the biological function of C1EIP in the process of chicken PGCs formation,we desigined 3 shRNA target sites and 1 negative control site for the C1EIP,and then.lentiviral interference vector was constructed and lentivirus was encapsulated.qRT-PCR was used to detect the interference efficiency of lentivirus interference vectors.In vitro,the 2nd generation ESCs were transfected with the lentiviral interference vector and overexpression vector respectively,and then induced by RA.ESC morphological changes were observed every 2 days,and the efficiency of PGCs formation was detected by qRT-PCR,cellular immunochemistry and flow cytometry.In vivo,the chick embryos were infected with the lentivirus interference vector,and the best infection efficiency was explored.Infected chick embryos were used to detect PGCs production efficiency by qRT-PCR,PAS staining,immunohistochemistry and flow cytometry analysis.The results showed that the lentiviral vectors C1EIP-sh1,C1EIP-sh2 and C1EIP-sh3 were successfully constructed and the interference efficiency was 80.39%,87.80%and 38.36%,respectively.In vitro,the normal RA induced the Embryoid Bodies（EBs）on the 2nd day.At the 4th day,the EBs became more and larger,and the edge of the EBs of the 6th day began to crack,and a small amount of cells were released.At the 8th day,the EBs were seriously ruptured and a large number of cells were released.At the 10th and 12th day,the EBs were completely cleaved and the SSCs-like cells appeared and grew together into grape strings.In overexpression group,large EBs appeared on the 2nd day,and small gaps appeared on the edge of the EBs at the 4th day.Most of the EBs on the 6th day were ruptured and released with a large number of internal cells.The SSCs-like cells were appeared on the 8th day and the typical grape string of SSCs clones were appeared on the 10th to 12th day.In knock-down group,the Ebs did not appear on the 2nd to 6th day.Until to the 8th day,the small EBs appeared.However,the number,size and morphology of the Ebs cultured to 12d did not show any significant changes.qRT-PCR results showed that overexpression of C1EIP resulted in the expression of sox2 was consistently decreased from 1.00±0.04 to 0.14 ± 0.01,and the expression of cvh,c-kit,stra8,Dazl,integrin a6 and integrin β1 gradually increased to 2.14±0.03,2.79±0.05,2.26±0.06,2.73±0.03 and 2.59±0.05.The expression level of C1EIP was increased to 3.99 ± 0.06 on the 4th day and decreased to 2.87 ±0.07 at the 12th day.while the expression at the 8th to 12th day remained essentially unchanged,and the expression levels of c-kit,stra8,Dazl,integrin a6 and integrin β1 were gradually increased to 2.14±0.03,2.79±0.05,2.26 ± 0.06,2.73 ± 0.03 and 2.59 ± 0.05.However,after knocking down C1EIP,the expression of sox2 did not change significantly until to the 12th day,and the expression of cvh,c-kit and C1EIP decreased from 1.00±0.04,1.00±0.04,1.00±0.03 and 1.00±0.04 to 0.53±0.04,0.45±0.05,0.86±0.03 and 0.76±0.02.stra8,Dazl,integrin α6 and integrin β1 expression firstly decreased from 1.00±0.04,1.00±0.04,1.00±0.03 and 1.00±0.04 to 0.53± 0.04,0.45 ± 0.05,0.86 ± 0.03 and 0.76 ± 0.02,and then slightly increased to 0.59±0.01,0.80 ± 0.04,0.88 ± 0.04 and 0.93 ± 0.08.The results of immunocytochemistry showed that the expression of CVH and C-KIT was significantly increased in the overexpression group compared with the RA-induced group,while the knockdown group inhibited the expression of CVH and C-KIT.Flow cytometry analysis results showed that the proportion of CVH+ cells in the RA-induced group was 3.4%,and the percentage of CVH+ cells was significantly up-regulated to 4.6%after overexpression of C1EIP,however,knockdown of C1EIP reduced CVH+cells to 2.9%.In vivo,the best conditions for chicken embryos to inject lentiviral vectors were blunt-ended 10 μL 106 TU/mL lentiviral vector + 90 μL DMEM.After injection,EGFP could be detected by PCR in chicken embryos and green fluorescence could be seen.The results of qRT-PCR showed that there was no significant change in the expression level of each gene between the blank group and the sh-Ctrl group.The decrease of the expression of sox2 in knock-down group were lower than that in other groups,and the expression of cvh,c-kit,Stra8,Dazl,integrinα6,integrin β1 and C1EIP decreased significantly to 0.20 ± 0.07,0.43±0.08,0.45 ± 0.06,0.51±0.07,0.35 ± 0.07,0.04 and 0.22±0.03.PAS results show that knock down C1EIP can significantly reduce PGCs production.Immunohistochemical results showed that CVH and C-KIT were highly expressed in the reproductive ridge of the blank group and the negative control group,and the expression site was almost filled with the whole reproductive ridge.However,the expression of CVH and C-KIT was significantly decreased in the knock-down group,and the site of expression is confined to the edge of the reproductive ridge.The results of flow cytometry showed that the knockdown of C1EIP resulted in a significant reduction in cvh-labeled PGCs to 3.1%relative to the blank and control groups（4.6%and 4.3%）.3.In order to systematically elucidate the transcriptional regulation mechanism of C1EIP expression,we screened about 2000bp sequence upstream of C1EIP transcription start site provided in NCBI and UCSC database,the long fragment of C1EIP promoter was amplificated and cloned into pEGFP-N1 vector for replacing the CMV promoter to constructe recombinant expression vector pC1EIP-EGFP,which was transfected into DF1 cells to detecte the promoter activity of the long fragment of the C1EIP promoter.C1EIP promoter with different deletion fragment vectors PGL3-P1-PGL3-P10 were constructed with deletion fragment cloning technique.After transfection of DF1 cells,the promoter activity of each deletion fragment was detected by double luciferase reporter system,and the core regulatory region of C1EIP promoter was screened.In the core regulatory region,bioinformatics was used to predict the key transcription factor binding sites.The deletion vectors of the transcription factor binding sites were constructed and the effect of deletion of transcription factor binding sites on the activity of the promoter was also examined by double luciferase reporter system.Finally,The effects of methylation and acetylation on the promoter activity of C1EIP and C1EIP expression in ESCs,PGCs and SSCs were detected with lOpmol/L 5-Aza-2’-deoxycytidine（5-Azadc）,1μmol/L Trichostatin A（TSA）and 4mmol/L Valproic acid（VPA）treatment.The results of digestion and sequencing showed that pC1EIP-EGFP was constructed correctly and transfected into DF1 cells to stimulate the green fluorescence.It was qualitatively indicated that the extended fragment of the amplified C1EIP promoter had the activity of initiation.Simultaneously,the results of restriction enzyme digestion and sequencing of the deletion fragment of the promoter also indicated that the vector was constructed successfully.Double luciferase assay showed that-1025～-912bp was the core active region of the C1EIP promoter and the key transcription factor binding sites in this region include MEIS1,MAFG::NFE2L1,STAT3,HLTF and Hand1::Tcf3,in which only STAT3 plays a positive regulatory role.Finally,It was found that the promoter activity of C1EIP increased from 10.82±0.54 to 18.49 ± 0.72,33.27±0.83 and 38.11±0.53 after methylation inhibition of 5-Azadc and acetylation inhibitor TSA and VPA.The expression of C1EIP in chicken ESCs,PGCs and SSCs was changed from 1.00±0.23,5.73± 0.54 and 0.94 ±0.23 to 1.43 ± 0.42,10.60±0.26,2.83 ± 0.18 and 2.64 ± 0.23,9.89±0.37,3.25±0.16 and 2.99±0.43,8.33 ± 0.56,2.94 ± 0.29 after treatment with 5-Azadc,TSA and VPA,respectively.4.In order to explore the mechanism of C1EIP regulation of PGCs,we constructed C1EIP prokaryotic expression vector and induced it to express in E.Coli and digged out C1EIP interaction protein by GST pull-down.The fusion expression vector pcDNA3.0-ENO1-HA was constructed and used for in vitro Co-immunoprecipitation to verify the interaction between ENO-HA and C1EIP-EGFP.The interaction protein of ENO1 were explored by bioinformatics analysis,and then their expression changes in the process of PGCs formation was confirmed by qRT-PCR.Myc promoter double luciferase reporter vector PGL3-Myc-pro and recombinant expression vectors pcDNA3,0-MBP-1.At the same time,in order to eliminate the interference of homologous family genes,we also constructed recombinant expression vector pcDNA3.0-ENO2.pcDNA3.0-ENO1-HA,pcDNA3.0-MBP-1 and pcDNA3.0-ENO2 were co-transfected with PGL3-Myc-pro respectively and the activity of Myc promoter was detected by double luciferase reporter system.Simultaneously,based on the results obtained and the related literature,the pattern of PGCs generated by C1EIP and ENO1/MBP-1 mediated Notch signaling pathway was drawn.GST pull-down results showed that there was interaction between ClEIP and ENO1,and in vitro Co-IP was used to verify the interaction between C1EIP and ENO1.Through the Uniport and PSORT II database and the co-expression of network analysis,we found that ENO1 located in the cytoplasm and is associated with Myc,whose downstream gene is Notch1.The results of qRT-PCR showed that C1EIP and ENO1 were consistent in the differentiation of chicken ESCs into SSCs and were opposite to those of Myc and Notch1.The results of digestion and sequencing showed that the double luciferase reporter vector PGL3-Myc-pro and the recombinant expression vectors pcDNA3.0-MBP-1 and pcDNA3.0-ENO2 were all constructed successfully,and the double luciferase results showed that MBP-1 had an inhibitory effect on the activity of Myc promoter,while ENO1 and EN02 had no effect on it.Finally,according to the above results and related literature,we draw the pattern that C1EIP interactd with ENO1/MBP-1 to mediate Notch signaling to regulate PGCs generation.As an anchor protein,C1EIP immobilized ENO1 in the cytoplasm.When ENO1 is phosphorylated or dephosphorylated and translocated into the nucleus and lost the previous process of 96 amino acid residues to form MBP-1 protein,which combined to Myc promoter,which inhibited Myc transcription and blocks the signal transduction of Notch signaling pathway,eventually promoted the differentiation of chicken ESCs into PGCs.更多还原