【作者】 李文静；

【导师】 肖磊；

【作者基本信息】 浙江大学 ， 动物遗传育种与繁殖， 2019， 博士

【摘要】 (1)利用TALENs/CRISPR技术制备NLRP3基因修饰猪背景:转基因抗病猪、高品质猪等拥有巨大的经济前景和社会效益。因为猪的胚胎干细胞系尚未建立,所以常用的方法是将体细胞基因修饰与体细胞核移植技术相结合产生克隆猪。但是由于体细胞的扩增代数有限,且体细胞打靶效率较低,定点基因修饰猪的推广受到限制。ZFN、TALENs和CRISPR技术的相继诞生使得基因定点敲除变得更为高效。人类NLRP3(NACHT,LRR and PYD domains-containing protein 3)基因第260位氨基酸发生错意突变(R260W)会导致NLRP3炎症小体的激活而产生自身炎症性疾病。小鼠、猪在人类NLRP3 R260W基因致病突变位点的是保守的,小鼠对应氨基酸突变位点为R258W,猪对应氨基酸突变位点为R259W。在小鼠R258W突变的模型中,NLRP3炎症小体的活化并未表现出严重的炎症反应,而是导致其抗病力增强。提示我们通过在猪上模拟NLRP3基因的相同突变可能制备出抗病能力增强的克隆猪。此外,我们拟同时制备NLRP3基因敲除猪模型,通过对比NLRP3基因敲除猪和定点突变猪的各项生理指标差异,共同作为NLRP3炎性体相关疾病机理的大型动物模型。方法:我们分别利用TALENs、CRISPR基因修饰技术,在猪成纤维细胞上分别筛选出基因编辑活性较高的TALENs组合以及gRNA位点,然后分别将TALENs、CRISPR基因修饰技术与同源重组技术相结合,用于制备NLRP3基因修饰的猪成纤维细胞系。然后再结合体细胞核移植技术(SCNT),将重组胚胎体外培养后移植到受体母猪中,用于制备NLRP3敲除/定点修饰的转基因猪。结果:我们构建出针对猪NLRP3基因的TALENs打靶载体,以及用作同源重组模板的单链DNA(ssDNA),并筛选出活性最高的TALENs组合;同时构建了针对猪NLRP3基因的CRISPR基因编辑系统打靶载体,以及相应的ssDNA。我们利用CRISPR/Cas9基因修饰技术,获得了 10株NLRP3敲除的猪成纤维细胞系。我们利用CRISPR/Cpf1基因修饰技术结合同源重组技术,获得了 3株NLRP3定点突变的猪成纤维细胞系。我们将获得的NLRP3敲除/定点突变的猪成纤维细胞作为供体细胞,通过体细胞核移植技术和胚胎移植技术,成功获得了 28头NLRP3R259W纯合突变的克隆猪,1头NLRP3基因双拷贝敲除13bp的克隆猪。结论:我们模拟人自然发生的NLRP3基因突变,结合TALENs/CRISPR基因定点敲除技术、同源重组技术以及体细胞核移植技术,成功制备了 NLRP3基因修饰的克隆猪,以供进一步的研究。(2)RYR2突变的iPSCs建立和初步分化背景:诱导多能干细胞的出现为疾病模型的建立提供了新的思路。由于可以从患者自身的很多易得的细胞来源(例如尿液、皮肤成纤维细胞)诱导,且具有与胚胎干细胞相似的多能性,因此hiPSCs可用于建立个性化疾病模型,且不存在伦理问题,大大拓展了精准医学的范围。儿茶酚胺多形性室性心动过速Ⅰ型(CPVT1)是一种由兰尼碱受体2型基因(RYR2)单基因突变导致的遗传性心脏病。RYR2基因突变导致了心脏功能的紊乱,以应激诱发的室性心律失常为特征,每年会导致大量年轻人猝死。因此,建立RYR2单基因突变导致的遗传性心脏病iPSCs并将其分化成心肌细胞,对于研究疾病机理,进行药物筛选等方面具有重要意义。方法:本研究利用慢病毒系统将Yamanaka四因子(Oct4、Sox2、Klf4和Nanog)整合到儿茶酚胺多形性室性心动过速I型(CPVT1)患者的皮肤成纤维细胞中,将其诱导成为iPSCs,并通过形态,表面抗原,基因表达,多能细胞特异性基因的表观遗传状态等方面验证其多能性。然后,再将iPSCs在体外定向分化成心肌细胞,并鉴定心肌细胞的特异性标志物的表达情况,从而初步建立RYR2单基因突变导致的遗传性心脏病模型。结果:我们鉴定了一名儿茶酚胺多形性室性心动过速I型(CPVT1)患者的基因型特征,从该患者皮肤成纤维细胞诱导出了 2株hiPSCs。通过对获得的hiPSCs干细胞标记物染色,可见其表现出很强的碱性磷酸酶活性,且表达Oct4和Nanog以及胚胎干细胞细胞特异性表面抗原,包括阶段特异性胚胎抗原-3(SSEA3),阶段特异性胚胎抗原-4(SSEA4),肿瘤相关抗原TRA-1-60,TRA-1-81。通过对hiPSCs的Oct4基因启动子区域进行了去甲基化程度分析,确认2株hiPSCs,即iPS-2和iPS-4的Oct4基因启动子区域均被很大程度地去甲基化。通过实时荧光定量PCR的方法,发现hiPSCs中多能性基因的表达量显著高于成纤维细胞(P<0.01)。通过采用RT-PCR方法,发现hiPSCs分化得到的拟胚体(EB)的三个胚层标志基因的表达量是hiPSCs的5～50倍,证明hiPSCs具有在体外分化成三个胚层的潜能。通过将hiPSCs接种非肥胖糖尿病/重症联合免疫缺陷(NOD-SCID)小鼠获得的畸胎瘤切片进行HE染色,证明hiPSCs具有在体内分化成三个胚层的潜力。我们还通过联重复技术(STR)检测确定了 iPS细胞为单一细胞来源,不存在交叉污染现象。以上证明我们诱导得到的hiPSCs具有很高的多能性。然后我们对RYR2 iPSCs进行了定向诱导分化,分化11天后可以观察到细胞有自发的跳动簇。我们采用RT-PCR检测发现跳动的细胞中心肌发育相关的早期基因的表达量为hiPSCs的20～40倍。结论:我们成功诱导了儿茶酚胺多形性室性心动过速I型(CPVT1)患者特异性iPS细胞系2株,并证明其具有很强的多能性。我们初步将iPS细胞向心肌细胞定向分化,可以观察到心肌细胞有节律收缩现象,并高表达心肌特异性标志物。但由于本研究处于初步探索阶段,心肌分化方案尚需完善,功能性验证实验尚未涉及,有待进行进一步研究。更多还原

【Abstract】 (1)Generation of NLRP3 gene modified pigs using TALENs/CRISPR technologyBackground:Genetically modified disease-resistant pigs and high-quality pigs have enormous economic prospects and social benefits.Since pig embryonic stem cell lines have not yet been established,a common method to produce cloned pigs is to combine somatic genetic modification with somatic cell nuclear transfer technology.However,due to the limited expansion ability of somatic cells and the low efficiency of somatic cell gene targeting,the fixed-point genetically modified pigs is difficult to obtain.Fortunately,the development of gene editing technologies,such as ZFN,TALENs,and CRISPR,has made gene-based modifications more efficient.A genetic mutation(R262W)on 262th amino acid of the human NLRP3(NACHT,LRR and PYD domains-containing protein 3)gene causes activation of NLRP3 inflammatory bodies and produces an autoinflammatory disease.Mice,pigs and humans are conserved in the NLRP3 mutation site.In transgenic mice that mimic human R262W mutations,NLRP3 inflammatory body activation did not show a severe inflammatory response,but increased enhancement that leading to increased disease resistance.It is suggested that we can prepare cloned pigs with enhanced disease resistance by simulating the same mutation of the NLRP3 gene in pigs.In addition,we intend to simultaneously produce the NLRP3 knockout pigs,and compare the physiological indicators of NLRP3 knockout pigs with fixed-point mutant pigs,and build large animal models to study NLRP3 inflammasome-related disease mechanism.Method:We built TALENs and CRISPR gene modification vector separately and screen ones with high gene editing activity on porcine fibroblasts.Then we use the gene modification technology and homologous recombination technology to prepare NLRP3 gene-modified porcine fibroblast cell lines.Next,the somatic cell nuclear transfer technology(SCNT)is operated and the recombinant embryos are cultured in vitro and transplanted into recipient sows to produce of NLRP3 knockout or site-directed transgenic pigs.RESULTS:We constructed a TALENs targeting vector targeting the porcine NLRP3 gene,as well as single-stranded DNA(ssDNA)used as a template for homologous recombination,and screened for the most efficient TALENs combination.At the same time,a CRISPR gene editing system,including targeting vector for porcine NLRP3 gene and corresponding ssDNA,were constructed.Using the CRISPR/Cas9 gene modification technology,we obtained 10 NLRP3 knockout porcine fibroblast cell lines.Using the CRISPR/Cpf1 gene modification technology combined with homologous recombination technology,we obtained three porcine fibroblast cell lines with NLRP3 point-fixed mutagenesis.Conclusion:We mimic the human naturally occurring NLRP3 gene mutation,and combine TALENs/CRISPR gene modification technology with homologous recombination technology and somatic cell nuclear transfer technology,obtaining NLRP3 genetically modified pigs successfully.(2)Establishment and preliminary differentiation of RYR2 mutant iPSCsBackground:The emergence of induced pluripotent stem cells provides new ideas for the establishment of disease models.Since it can be induced from many of the patient’s own readily available cell sources(eg,urine;dermal fibroblasts)and has similar pluripotency to embryonic stem cells,hiPSCs can be used to create personalized disease models without ethical issues,greatly expanding the scope of precision medicine.Catecholamine polymorphic ventricular tachycardia type I(CPVT1)is a hereditary heart disease caused by a single gene mutation in the ryanodine receptor type 2 gene(RYR2).Mutations in the RYR2 gene cause a disturbance in cardiac function,characterized by stress-induced ventricular arrhythmias,which lead to a large number of young people to die each year.Therefore,the establishment of iPSCs of patients with hereditary heart disease caused by RYR2 single gene mutation and differentiation them into cardiomyocytes is of great significance for the study of disease mechanisms and drug screening.Methods:We use a lentiviral system to integrate Yamanaka s four factors(Oct4^Sox2,Klf4,and Nanog)into skin fibroblasts from patients with catecholamine polymorphic ventricular tachycardia type I(CPVT1)and induced them into iPSCs.Then verify the hiPSCs pluripotency by morphology,surface antigen,gene expression,and epigenetic status of pluripotent cell-specific genes.Then,iPSCs were differentiated into cardiomyocytes in vitro,and the expression of specific markers of cardiomyocytes was identified,thereby establishing a genetic heart disease model caused by RYR2 single gene mutation.Results:We identified the genotypic characteristics of a catecholamine polymorphic ventricular tachycardia type I(CPVT1)patient,and induced two hiPSCs from the patient’s skin fibroblasts.By staining stem cell markers,it can be seen that hiPSCs exhibit strong alkaline phosphatase activity and express Oct4 and Nanog as well as embryonic stem cell-specific surface antigens,including stage-specific embryonic antigen-3(SSEA3),stage-specific embryonic antigen-4(SSEA4),tumor associated antigen(TRA-1-60)and tumor associated antigen(TRA-1-81).By analyzing the demethylation degree of Oct4 gene’s promoter region of hiPSCs,it was confirmed that the promoter regions of both two hiPSCs,iPS-2 and iPS-4,were largely demethylated.The expression of pluripotency genes in hiPSCs was significantly higher than that in fibroblasts by real-time fluorescent quantitative PCR(P<0.01).The expression levels of three germ layer marker genes of embryoid bodies(EB)differentiated from hiPSCs were 5 to 50 times that of hiPSCs by RT-PCR,which proved that hiPSCs we obtained have the potential to differentiate into three germ layers in vitro.HE staining of teratoma sections obtained by inoculation of non-obese diabetic/severe combined immunodeficiency(NOD-SCID)mice with hiPSCs demonstrated that hiPSCs have the potential to differentiate into three germ layers in vivo.We also determined that iPS cells were a single cell source by tandem repeat technique(STR)detection,and there was no cross-contamination.The above proves that the hiPSCs we induced are highly pluripotent.Then we differentiated iPSCs into cardiomyocytes,and spontaneous cell pulsation clusters were observed after 11 days of differentiation.Using RT-PCR,we found that the expression of early genes related to myocardial development in beating cell clusters was 20-40 times higher than that of hiPSCs.Conclusion:We successfully induced two specific iPS cell lines of catecholamine polymorphic ventricular tachycardia type I(CPVT1)and demonstrated that they have strong pluripotency.We initially differentiated iPS cells into cardiomyocytes,and observed myocardial cells with rhythmic contraction and high expression of myocardial specific markers.However,due to the preliminary exploration stage of this study,the myocardial differentiation program needs to be improved,and the functional verification experiment has not been involved,and further research is needed.更多还原