CN113186189A - 一种用于敲除猪NPHS2基因的gRNA及其相关应用 - Google Patents
一种用于敲除猪NPHS2基因的gRNA及其相关应用 Download PDFInfo
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- CN113186189A CN113186189A CN202110546496.2A CN202110546496A CN113186189A CN 113186189 A CN113186189 A CN 113186189A CN 202110546496 A CN202110546496 A CN 202110546496A CN 113186189 A CN113186189 A CN 113186189A
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Abstract
本发明提供了一种用于敲除猪NPHS2基因的gRNA及其相关应用。所述gRNA的核苷酸序列如SEQ ID No.2所示。本发明的gRNA可用于敲除猪的NPHS2基因,从而构建激素抵抗性肾病综合征猪模型,为研究激素抵抗性肾病综合征提供理想的动物模型。
Description
技术领域
本发明属于生物技术领域,具体而言,涉及一种用于敲除猪NPHS2基因的gRNA及其相关应用。
背景技术
原发性肾病综合征(primary nephritic syndrome,PNS)是以大量蛋白尿(>3.5g/d)、低白蛋白血症(<30g/L)、高脂血症和不同程度水肿为特征的临床综合征。临床上10%-20%PNS患者糖皮质激素治疗无效,KDIGO指南将儿童经单纯激素治疗8周或成人经单纯激素治疗16周后仍不缓解的PNS患者定义为激素抵抗性肾病综合征(steroid-resistantnephritic syndrome,SRNS),此类患者多较快进展至终末期肾病,严重时威胁生命。
近年来,SRNS的发病机制已进入基因组学水平研究。目前的基因学分析显示,30%-40%的SRNS患者与足细胞蛋白相关基因变异有关。其中NPHS2基因突变最早发现于家族性常染色体隐性遗传SRNS,此后相继发现也存在于成人散发SRNS及迟发性局灶节段性肾小球硬化患者,目前,该基因突变已被认为是SRNS发病最重要的机制之一。
NPHS2基因编码的podocin蛋白,是脂质筏相关蛋白Stomatin家族的成员,位于足细胞裂孔膜(slit membrane,SD),呈发卡样结构插入细胞膜。Podocin在肾脏中特异表达于足细胞,通过C-末端与Nephrin和CD2AP连接形成复合物,形成SD骨架并保持肾小球滤过屏障的正常结构和功能。Podocin富含大量的神经鞘脂以及胆固醇,同时还能增强Nephrin所引导的信号转导,该信号的级联及转导在维持足细胞形态和SD结构完整性方面起着重要作用。NPHS2基因突变或缺失时,podocin表达量下降,SD复合体结构失衡,足细胞结构破坏,出现大量蛋白尿。但当NPHS2基因突变或缺失时,SD复合体间三种蛋白到底以何种方式和途径相互作用,其与足细胞骨架蛋白间到底通过哪条信号传导通路进行影响,这些机制仍未明确。对于上述机制的进一步研究将有助于临床上SRNS患者治疗方式的改进及生存率的提高。
猪和人类在解剖学,生理学和遗传学上高度相近,并且已成为生物医药研究领域中的重要动物模型。许多遗传修饰的猪疾病模型已经在囊性纤维化和神经退行性疾病以及移植等领域得到了应用。但由于缺乏可用的胚胎干细胞,无法使用传统的基因打靶方法建立基因敲除猪。
因此,需要开发一种高效的构建NPHS2敲除的猪疾病模型的方法,为SRNS的治疗提供良好的研究基础。
发明内容
本发明的一个目的在于提供一种用于敲除猪NPHS2基因的gRNA。
本发明的另一个目的在于提供含有所述gRNA的打靶载体。
本发明的另一个目的在于提供所述gRNA或者所述打靶载体在制备用于敲除猪NPHS2基因的试剂中的应用。
本发明的另一个目的在于提供一种制备猪NPHS2基因敲除的细胞或制备NPHS2基因敲除的激素抵抗性肾病综合征猪模型的方法。
本发明还提供了一种用于敲除猪NPHS2基因的试剂盒。
一方面,本发明提供了一种gRNA,其核苷酸序列如SEQ ID No.2所示。
本发明还提供了一种用于敲除NPHS2基因的打靶载体,其中,所述打靶载体含有本发明所述的gRNA。
根据本发明的具体实施方案,本发明中,所述打靶载体的构建方法包括:
合成5’端磷酸化寡核苷酸链的SEQ ID No.2所示的靶点序列;将靶点序列在初始载体骨架上进行克隆,得到打靶载体。
根据本发明的具体实施方案,本发明中,所述初始载体包括pX330。
本发明还提供了所述gRNA或者所述打靶载体在制备用于敲除猪NPHS2基因的试剂中的应用。
本发明还提供了一种制备猪NPHS2基因敲除的细胞的方法,包括:
将本发明所提供的打靶载体与Cas9 mRNA转染猪的体细胞系,筛选获得NPHS2基因敲除的体细胞。
根据本发明的具体实施方案,本发明中,所述体细胞系包括成纤维细胞系。
进一步,本发明还提供了一种制备NPHS2基因敲除的激素抵抗性肾病综合征猪模型的方法,该方法包括:利用核移植技术,将去核卵母细胞与本发明中获得的NPHS2基因敲除的体细胞融合,构建重组卵母细胞;待重组卵母细胞发育成囊胚后,将囊胚移植到同期发情的母猪子宫内,制备基因敲除猪。
根据本发明的具体实施方案,本发明所述制备NPHS2基因敲除的激素抵抗性肾病综合征猪模型的方法,其中,所述方法还包括将本发明所述的gRNA与Cas9 mRNA注射到自然受精的受精卵中,将注射后的胚胎移植到同期发情的母猪子宫内,制备基因敲除猪。
此外,本发明还提供了一种用于敲除猪NPHS2基因的试剂盒,其中,所述试剂盒包括本发明所述的gRNA或本发明中的打靶载体。
本发明成功构建了基于CRISPR/Cas9系统的敲除猪NPHS2基因的gRNA和打靶载体,成功构建了与NPHS2基因相关的激素抵抗性肾病综合征基因编辑大动物模型。模型动物已出生稳定的F1代,尿蛋白结果阳性,肾脏PAS染色呈符合疾病的病理表现,免疫荧光证实了NPHS2基因在小型猪肾脏表达,Western blot结果显示基因敲除组肾脏中podocin蛋白的表达远低于正常对照组,说明已成功模拟了激素抵抗性肾病综合征模型,为此疾病的发病机制提供了良好的平台,并为未来多种肾脏疾病的研究指明了一个新的方向。
附图说明
图1为三条gRNA cas9体外酶切检测结果。
图2为gRNA打靶效率检测结果。
图3为基因敲除成功F0代尿蛋白肌酐比结果;与对照组相比,模型组P/C明显升高(*P<0.05)。
图4为肾组织PAS染色光镜下观察结果。
图5A显示Western Blot检测结果;图5B显示podocin的半定量分析结果;CON为正常对照组;模型组与正常对照组相比podocin蛋白表达显著下降,*P<0.05。
图6为免疫荧光检测对照组和基因敲除组中podocin蛋白的表达;Podocin表达分布在肾脏足细胞内,模型组与正常对照组相比,podocin蛋白表达量显著下降。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现对本发明的技术方案进行以下详细说明,但不能理解为对本发明的可实施范围的限定。
实施例中未详细说明的方法,按照所属领域中的常规方法进行,或按照仪器厂商建议的操作条件进行。
各实施例中所用材料和统计学处理方法
(一)实验动物
本发明所使用实验动物为实验用广西巴马小型猪,由北方大动物中心提供且饲养。给予自由饮水,每日投食2次,饲养室温为15-25℃,相对湿度50%-80%,每日光照约12h,持续空气交换。雌性实验猪正常性成熟、具有规律的发情周期,具备代孕要求。
(二)主要试剂、耗材与仪器
主要细胞培养试剂:FBS(Gibc公司),DMEM(Gibco公司),0.25%Trypsin-EDTA(Gibco公司),G418(Gibco公司),DPBS(Gibco公司),DMSO(Sigma公司),1×Penn/Strepsolution(Gibco公司)。培养液的试剂(Sigma公司)用于配置胚胎培养液。以上试剂均分开单独使用。
主要分子实验试剂:Surveyor突变检测试剂盒(Transgenomic公司),小鼠抗猪CD3(Abcam公司),小鼠抗猪CD79α(Abcam公司),DH5α(Takara公司),质粒小提试剂盒(天根生物技术有限公司),BbsⅠ(Fermentas公司),BsaⅠ等限制性内切酶(NEB公司),pX330质粒骨架(Addgene plasmid公司),pMD18-T vector(Takara公司),BCA蛋白测定试剂盒(Pierce公司),PCR产物胶回收试剂盒(MACHEREY-NAGEL公司),ELISA试剂盒(Abnova公司),质粒中提试剂盒(QIAGEN公司),SDS-PAGE电泳预染蛋白Marker(Bio Labs公司),ECL WesternBlotting Detection Reagents(Thermo公司)。
主要耗材:细胞培养板/皿(Corning公司),克隆环(Gibco公司),1.5mLEP管(Corning公司),15mL、50mL离心管(Corning公司),细胞冻存管(Corning公司)。
主要仪器:酶标仪:BioTek美国;细胞核转染仪:Lonza美国;体视显微镜:Nikon日本;流式细胞仪:BD美国;细胞融合仪:Cryologic澳大利亚。
(三)统计学处理
使用SPSS 21.0软件进行统计分析,所有定量数据采用均数±标准差(x±s)表示,以P<0.05认为具有统计学差异。
实施例1、gRNA的设计及打靶载体的构建
1.gRNA的设计
在Ensembl中经过检索获得基因Sus scrofa(pig)种属的NPHS2的序列,Gene ID为ENSSSCG00000015526,其具有八个外显子。
根据Ensemble中NPHS2的序列信息,针对以下外显子(SEQ ID No.1)设计gRNA。
SEQ ID No.1:
GCATCCCGGACCGCGGTCAGCTCCCGGAGCCCGGCTGCTCTAAGGATGGAGAAGAGGGCTCGGAGCTCCTCCAGAGAGGCCCATCGGAGAGGCGGCGGGTCCCCGCACAAGGAGAACAAGAAGCCGAACGCCCAGAAGGGCGGCAGAGGCCGCGGGCGCCGGGAGCAGCCGGCCGTCGGGCGCGCAGAGAGCCCAGCGGAGCCCCGACCGCCGGCAGCCACCGTGGTGGACGTGGATGAAGTCCGGGGCTCTGGCGAGGAGGGCACCGAAGTGGTGGCGCTGCTGGAGAGTGAGCGGCCAGAGGAAG
根据cas9靶点的设计原则:5’端是G,3’端是PAM序列(NGG),在目的基因上筛查靶点位置,设计gRNA。本实施例中,所设计的gRNA包括三条,分别为:
gRNA1:CTCTCTGCGCGCCCGACGGCCGG(SEQ ID No.2)。
gRNA2:GTGGACGTGGATGAAGTCCGGGG(SEQ ID No.3)。
gRNA3:TGGGCTCTCTGCGCGCCCGACGG(SEQ ID No.4)。
2.构建CRISPR/Cas9打靶载体
(1)采用pX330质粒(Addgene plasmid公司)作为表达hSpCas9及gRNA的骨架质粒;
(2)对5’端磷酸化寡核苷酸链的靶点序列进行合成,再将靶点序列在pX330的骨架载体上进行克隆,具体步骤如下:
a)利用限制性内切酶(Bbs I)对pX330质粒(1μg)进行处理:温度37℃,时间30分钟。pX330(1μg)+Fast Digest Bbs I(1μL,Thermo)+Fast AP(1μL,Thermo)+10X FastDigest Buffer(2μL)+dd H2O(15μL)→总量(20μL);
b)利用琼脂糖胶分离酶切的pX330质粒,用胶回收的酶切产物再通过试剂盒纯化;
c)对5’端磷酸化寡核苷酸链的靶点序列进行退火,
退火程序如下:37℃30分钟→95℃5分钟→此后以每分钟下降5℃的速度降至25℃;
d)启动连接反应:室温反应10分钟,
Bbs I(1μL)消化pX330(50ng,由步骤a)中获得)+磷酸化的退火后的寡聚体(1μL,1:250稀释,由步骤c)中获得)+2X快速连接缓冲液(5μL,NEB)+dd H2O(1μL)→subtotal(10μL)+快速连接酶(1μL,NEB)→总量(11μL);
e)应用Plasmid Safe核酸外切酶,处理连接体系进而消除错误链接质粒:连接反应产物(11μL,由步骤d)获得)+10X质粒安全缓冲液(1.5μL)+10mM ATP(1.5μL)+质粒安全核酸外切酶(1μL)→总量(15μL)→37℃反应30分钟;
f)进行转化;
g)进行小提质粒,进行测序,以明确是否成功构建打靶质粒。
3.SURVEYOR核酸酶检测法筛选设计的3条gRNA:
(1)将cas9质粒转染293T细胞,设置温度37℃,CO2培养箱孵育72小时,收集细胞后应用试剂盒(基因组提取试剂盒,天根)提取细胞基因组。
(2)于cas9质粒的打靶位点前后分别设计引物,利用PCR技术扩增靶点位置序列;用PCR产物/胶回收试剂盒MN对PCR产物进行纯化、回收;其中,引物序列如下:
5’-CATCGCGCAATGCACTGAC-3’(SEQ ID No.5);
5’-GGCTCAGCTGGGCCACAATG-3’(SEQ ID No.6)。
(3)纯化的PCR产物进行重新退火:
a)反应体系:纯化的PCR产物(400ng)+10X Taq聚合酶Buffer(2μL)+Fast AP(1μL,Thermo)+dd H2O(3μL)→总量(20μL);
b)重新退火反应程序:95℃10分钟→95℃至85℃(以每秒下降2℃的速度进行)→85℃至25℃(以每秒下降0.25°的速度进行)→25℃保持1分钟。
(4)SURVEYOR核酸酶进行酶切:
重新退火PCR产物(400ng)+1/10体积的0.15M氯化镁+10X Taq聚合酶Buffer(最终浓度1×)+增强剂S(1μL)+核酸酶(1μL)→42℃,60分钟+1/10体积终止液。
(5)使用PAGE胶进行鉴定:
使用15%的PAGE胶,将酶切的PCR产物进行分离,将电压设定在125V,观察到溴酚蓝跑离胶体后,进行染胶,20分钟,使用凝胶成像系统进行拍照,以此观察是否已经存在切开的短片段,最后利用灰度值对Cas9体外酶切效率进行初步测定。
本实施例中,按照上述操作,使用限制性内切酶BbsI对gRNA载体质粒进行酶切线性化,并切胶回收检测,检测结果参见图1,结果显示,gRNA1合格,含有酶切位点,gRNA3虽然也含有酶切位点,但其酶切位点条带灰度值较低,不如gRNA1合适。
4.体外验证Cas9质粒的打靶效率
为验证gRNA1打靶效率,将gRNA1和Cas9 mRNA注射到MII期卵母细胞中,然后进行孤雌激活。共获得20个囊胚,其中19个囊胚genotyping成功(#18囊胚PCR失败):均有uncut条带,即均被打靶,gRNA1打靶效率为100%(见图2)。
实施例2、通过核移植构建NPHS2基因敲除猪
1.应用cas9质粒及上一步筛选出来的gRNA1对原代的猪胎儿的成纤维细胞进行转染
复苏原代猪的胎儿成纤维细胞(来自中国人民解放军总医院),并将其置于直径为60毫米的细胞培养皿中,养至细胞长满皿底90%左右(细胞培养液:DMEM+20%FBS+1×Penn或Strep),使用胰酶消化细胞后,将其转移至离心管中,进行离心5分钟,300g速度,最后收集细胞,细胞数大概约1.5×106。
使用核转染仪(Lonza公司)对细胞进行核转染:
(1)按照4.5:1的比例将两种核转染试剂混合,并在混合后将其恢复至室温:NucleofectorTM Solution(82μL)+supplement(18μL)→核转反应液(100μL)。
(2)将步骤(1)的100μL反应液按照5:1的比例将需共转染的打靶质粒Cas9和gRNA1加入,用时加入表达G418抗性的质粒(TD tomato),将两者轻轻混匀。
(3)使用DPBS清洗液对细胞进行清洗后,离心,将100μL的预混反应液(步骤(2)中获得)加入沉淀中,缓慢重悬,过程中注意尽量避免气泡的产生。
(4)将步骤(3)得到的体系,向电转杯内转移,过程中注意动作的轻柔,避免气泡的产生;将电转杯放置于杯槽中,选择转染程序,接着,于六孔板中用完全培养基培养(含20%血清)对电击转染的细胞进行培养,间隔24小时后换液。
2.对基因打靶的细胞克隆进行筛选和鉴定
将转染了Cas9打靶质粒的细胞至于六孔板中,待细胞长满六孔板孔后,对细胞进行消化,再以1×105/10mL的细胞数铺在培养皿上,加浓度为800μg/mL的G418进行筛选,每3至5天换培养液,生长至10天到14天,即可获得具有G418抗性的单细胞克隆。挑取上述细胞继续培养(24孔板),长满后进行传代(12孔板或6孔板),同时留取部分24孔板中细胞,用裂解液(见表1)裂解,条件设置为55℃60分钟,95℃10分钟。对单细胞克隆的PCR产物测序,设计PCR引物,模板参照基因组裂解产物。在测序峰图上,双峰或测序序列比对突变的样本,将进行下一步转化,将PCR产物连接于T载体,选择15个至20个进行测序,根据结果判断该细胞是否满足目的基因敲除。
对最终被确定的细胞克隆进行冻存,具体需根据其细胞数量和状态决定。
表1 NP-40裂解液成分
成分 | 浓度/体积 |
NP-40 | 0.35μL |
10×Buffer | 10mL |
K蛋白 | 1mg/mL |
终体积 | 10mL |
3.体细胞的核移植技术(SCNT)
(1)收集卵母细胞及其体外成熟
收集母猪的卵巢(6个月以上性成熟者),放入0.9%的无菌生理盐水中,整个过程注意无菌操作,盐水温度始终保持在25至30℃之间,对卵泡中未成熟卵子进行人工抽取,挑选其中的优质卵子,并将其于培养基中培养至发育成熟。具体操作步骤如下:
a)使用一次性无菌注射器从卵巢中提取卵泡,一般多为抽出直径3-8毫米的卵泡,收集至15毫升的洁净离心管中。
b)将离心管静置,卵泡液沉淀20分钟后,弃去上清液,用TL-hepes洗涤液(配方见表2)重新悬浮,在重新沉淀,重复进行3遍。
c)最后,将上述步骤洗涤过的卵泡加入到洗卵液中,静置重悬后,置入培养皿中(10厘米)。然后使用立体显微镜,用抽拉的无菌毛细管玻璃管完全包裹卵丘。最后将2层以上包裹致密的细胞层挑选出,同时均匀细胞质的卵丘-卵母细胞复合物(COCs)。
d)将卵母细胞成熟液IVM(配方见表3)预先放置于二氧化碳培养箱中,进行预先平衡,将从平衡过成熟液中拣出的COCs进行三遍过洗,然后以每孔100个左右的密度,将其移入四孔板的卵母细胞成熟液中(四孔板每孔700μL IVM,覆300μL矿物油)。
e)将四孔板移入培养箱中继续培养42-44小时(38.5℃、5%CO2),直至成熟。
表2猪卵母细胞清洗液液配方
成分 | 1L |
氯化钠 | 6.6633g |
氯化钾 | 0.2386g |
KH<sub>2</sub>PO<sub>4</sub> | 0.0408g |
小苏打 | 0.1680g |
乳酸钠 | 1.868mL |
6水氯化镁 | 0.1016g |
羟乙基哌嗪乙硫磺酸 | 2.3830g |
盘尼西林G | 0.0650g |
酚红 | 0.0100g |
2水氯化钙 | 0.2940g |
聚乙烯醇 | 0.1000g |
山梨糖醇 | 2.1860g |
庆大霉素 | 0.0250g |
丙酮酸 | 0.0220g |
调整pH值处于7.2-7.4之间。
表3卵母细胞成熟液配方
成分 | 50mL |
TCM-199 | 50mL |
PVA | 0.0500g |
D-葡萄糖 | 0.0500g |
丙酮酸钠 | 0.2750g |
半胱氨酸 | 0.57mM/L |
黄体化激素 | 0.5mg/mL |
卵泡刺激素 | 0.5mg/mL |
表皮生长因子 | 10ng/mL |
链霉素 | 0.5mg/mL |
盘尼西林G | 75mg/mL |
调整pH值处于7.2-7.4之间
(2)体细胞核移植(SCNT)和重组胚胎的形成
该操作需在体视显微镜下操作完成,步骤如下:
a)排卵丘:使用透明质酸酶溶液(具体配方见表4),将于体外成熟了40-42小时的卵母细胞进行消化,以去除粒细胞。掌握消化时间,目的为尽可能除去卵母细胞的透明带以外的颗粒细胞,但也不可损坏卵母细胞,还应避免时间过长。
b)挑极体:在上一步中已去除了粒细胞的卵母细胞,将其转移至体外操作(具体配方见表5),洗涤3次,然后转移到50μL覆矿物油的操作液微滴中。选择卵细胞时,应挑选具有完整细胞膜的,卵周间隙也应清晰,且已经排出了第一极体,符合以上条件的卵母细胞挑选后备用。
c)去核:使用显微进行操纵,使用显微操纵器去除卵母细胞核带的部分细胞质,注意防止染色会对卵母细胞产生的不利影响,去核的完整性要保持。将去核的卵母细胞转移到50μL的矿物质操作液微滴中,等待下一步的体细胞移植。
d)注细胞:对改造的克隆细胞进行挑选,挑选适当大小的体细胞注入到卵母细胞间隙中。最后,将卵母细胞转移至猪卵母细胞发育液中(具体配方见表6),等待两者的融合。
e)电击融合:用卵母细胞激活液中洗涤注入体细胞的卵母细胞3次(具体配方见表7),并沿电极条垂直线排列的方向排列卵母细胞和体细胞在激活液覆盖的0.5毫米融合槽中的两个平行电极中调节融合仪器参数,并施加140v/mm的直流电电击30微秒。融合后,将其转移至预先平衡的猪卵母细胞发育溶液中,进行洗涤3次。
f)挑选重构胚:将上一步洗涤后的细胞,转移至50μL的覆矿物油PZM-3中。30分钟后,可开始对体细胞与卵母细胞膜融合的重构胚胎进行选择,该胚胎具有发育为新个体的潜在能力。
g)重组卵母细胞的密度为每孔50-80个,对重构的卵母细胞进行转移至四孔板中,每孔含PZM-3覆盖300μL矿物油及700μL猪卵母细胞发育溶液。将四孔板置于培养箱中(38.5℃、5%CO2)培养约5天,可见囊胚形成。
表4透明质酸酶溶液
成分 | 浓度/体积 |
DPBS | 100mL |
透明质酸酶 | 0.100g |
调整pH值处于7.2-7.4之间
表5卵母细胞体外操作液配方
调整pH值处于7.2-7.4之间
表6猪卵母细胞发育液配方
成分 | 1L |
氯化钠 | 108.0mM |
氯化钾 | 10.0mM |
KH<sub>2</sub>PO<sub>4</sub> | 0.35mM |
小苏打 | 24.08mM |
丙酮酸钠 | 0.18mM |
乳酸钙5水 | 1.8mM |
L-谷胱氨酸 | 1.8mM |
亚牛磺酸 | 4.8mM |
BME氨基酸溶液 | 20.0mL |
MEM非必须氨基酸溶液 | 10.0mL |
MgSO<sub>4</sub>·7H<sub>2</sub>O | 0.38mM |
庆大霉素 | 0.05g |
脂肪酸BSA | 3.0g |
消毒水 | 定容至1L |
调整pH值处于7.2-7.4之间
表7猪卵母细胞激活液
成分 | 1L |
甘露醇 | 0.28mol |
氯化钙 | 1.0mmol |
氯化镁 | 0.1mmol |
羟乙基哌嗪乙硫磺酸 | 0.5mmol |
消毒水 | 定容至1L |
调整pH值处于7.2-7.4之间
4.囊胚的子宫移植—构建NPHS2基因敲除猪
通过养殖场内的工作人员的观察,掌握代孕猪每天的发情状况,一段时间后选定发情期规律以及处于合适发情期周期的健康母猪作为受体猪。麻醉受体猪后,于下腹部正中做切口,逐层分离找到子宫,并将约100个重建的囊胚(37℃恒温保温箱携带)移植到子宫角中。手术完成后,将受体猪单独饲养于定位栏上。一个月后,应用B超检测受体猪孕况。如果检测到孕猪,需密切监视其妊娠状况,直到胎儿出生。共进行冲胚和胚胎移植13次,其中5头受体母猪怀孕到期。(见表8)
表8胚胎移植及受孕情况
移植胚胎数量 | 受体母猪号 | B超 |
12枚 | 1339604 | 未怀孕 |
13枚 | 1309701 | 未怀孕 |
9枚 | 1225801 | 未怀孕 |
7枚 | 1224504 | 未怀孕 |
9枚 | 1443401 | 已怀孕 |
21枚 | 1312803 | 已怀孕 |
9枚 | 1303101 | 未怀孕 |
IVF embryos,210枚 | 1304303 | 已怀孕 |
14枚 | 1235305 | 已怀孕 |
IVF embryos,186枚 | 08 | 未怀孕 |
11枚 | 1237201 | 未怀孕 |
21枚 | YJ160624 | 未怀孕 |
21枚 | 1309701 | 已怀孕 |
F0代出生情况:
共生产5窝F0代小猪,13只,其中存活7只,出生后死亡4只,死胎2只(见表9)。
表9F0代出生情况
窝次 | 性别 | 基因型 | 母猪号 | 来源 | |
1629 | ♀ | +/- | 1443401 | 巴马猪,冲胚注射 | 存活 |
1629 | ♂ | +/- | 1443401 | 巴马猪,冲胚注射 | 存活 |
1629 | ♂ | -/- | 1443401 | 巴马猪,冲胚注射 | 存活 |
1629 | ♂ | -/- | 1443401 | 巴马猪,冲胚注射 | 存活 |
1630 | ♀ | +/+ | 1312803 | 巴马猪,冲胚注射 | 存活 |
1630 | ♀ | -/- | 1312803 | 巴马猪,冲胚注射 | 死亡(30天) |
1630 | ♂ | +/- | 1312803 | 巴马猪,冲胚注射 | 死亡(42天) |
16186 | ♀ | -/- | 1304303 | 巴马猪,冲胚注射 | 死亡(20天) |
16186 | ♀ | +/+ | 1304303 | 巴马猪,冲胚注射 | 死亡(14天) |
16186 | ♀ | +/- | 1304303 | 巴马猪,冲胚注射 | 存活 |
1643 | ♂ | -/- | 1235305 | 巴马猪,冲胚注射 | 存活 |
1655 | ♂ | -/- | 1309701 | 巴马猪,冲胚注射 | 死胎 |
1655 | ♀ | -/- | 1309701 | 巴马猪,冲胚注射 | 死胎 |
5.NPHS2基因敲除猪模型的鉴定及分析
5.1基因敲除小猪的基因型鉴定
敲除仔猪出生情况稳定后,一般是三天,进行耳朵打孔标记。采集耳组织,用DPBS将耳组织洗涤后,进行猪基因组DNA的提取。根据Cas9中靶基因的靶序列,进行PCR引物的设计,随后PCR测序,以明确该敲除猪为单等位基因还是双等位基因的突变猪。
对F0代基因鉴定
对出生F0进行基因鉴定,结果如下。“单下划线”表示gRNA序列,“倾斜字体+波浪下划线”表示EagI酶切位点,“双下划线”表示插入/错配的序列,“:”表示缺失的序列,右侧的“×数字”表示测到的次数。
Founder:
162901测序结果(单等位基因突变):
162902测序结果(单等位基因突变):
162903测序结果(双等位基因突变):
162904测序结果(双等位基因突变):
163002测序结果(双等位基因突变):30d死亡
163003测序结果(单等位基因突变):42d死亡
1618601(双等位基因打靶):20d死亡
1618603(单等位基因打靶):
164301(双等位基因打靶):
165501(双等位基因打靶):死胎
165502(双等位基因打靶):死胎
F1猪的产生及基因型鉴定
在F0代选取基因为单等位基因母猪:编号162901,双等位基因公猪:编号162904,进行交配,产生更为稳定的F1代。生产F1代3只,死亡1只。基因型如下:171901(单等位基因打靶):♀
171902(双等位基因打靶):♂
171903(双等位基因打靶):♀,37d死亡
5.2基因敲除成功F0代尿生化检测
将小型猪仰卧位固定于X型支架上,确保四肢被束缚,使用便携式超声探测仪于膀胱处查看,膀胱是否充盈,如有充盈,在超声探测指导下进行经皮膀胱穿刺留取洁净随机尿液标本,如未充盈,则挑取其他时间再次超声探测,直至取到洁净随机尿液标本为止。洁净尿管中4℃保存,24小时内送到生化科测定生化指标。
留取基因敲除成功的6只F0代猪的尿液,生化指标与正常对照组相比,尿蛋白肌酐比(P/C)明显升高(P<0.05)(见表10、图3)。
表10基因敲除成功F0代尿蛋白肌酐比检测结果
5.3F1代小型猪肾脏病理学检测
待敲除小猪出生后,实时监测,掌控健康状况,给予母乳喂养。状态较差的个体死亡后,尽量快速最迟不过24h内进行解剖,取肾组织,分成几部分:用于包埋石蜡切片的固定于4%多聚甲醛溶液;用于蛋白及RNA的提取的直接将组织分管-80℃冻存。健康的小猪继续母乳喂养,待观察结束时,采集标本时,按上述步骤进行。
使用PAS染色,观察F1代小型猪肾脏病理变化。PAS结果显示,与正常对照组相比,F1肾脏组织系膜细胞增多,系膜基质增多(见图4)。
5.4采用western blot方法检测podocin蛋白表达
采集敲除猪的肾脏组织,进行组织总蛋白质的提取,并使用western blot检测组织中podocin蛋白的表达情况。western blot具体操作步骤如下:
(1)肾组织总蛋白的提取:RIPA裂解液预冷,100mg组织块加500μl 1XRIPA裂解液进行组织匀浆,冰浴静置30min,期间颠倒混匀5次,4℃12000rpm,离心30min,析出的上层液体即为组织总蛋白,吸取留用。
(2)蛋白浓度测定及蛋白变性:置于37℃温箱孵育2h,检测样本650nm吸光度值。配制2×SDS上样缓冲液:2mL 20%SDS,2mL甘油,2mL 0.5M Tris(pH 6.8),1mL 1MDTT,0.5mL0.1%溴酚蓝,2.5mL ddH20,取100μg总蛋白,按体积加入2×SDS上样缓冲液,95℃水浴变性5min。变性后冰浴10min。
(3)电泳:配制5mL PAGE-SDS聚丙酰胺分离胶(6%~15%),缓慢倒入胶板,室温静置凝胶1小时,之后加入3mL 5%的积层胶,室温静置凝胶1小时。将标准蛋白Marker、各组样品蛋白分别加到配制完毕的凝胶中,使用1×Tris-甘氨酸电泳缓冲液电泳(积层胶电压控制到60V,分离胶电压控制到80-100V),观察溴酚蓝到达分离胶底部时,停止电泳。
(4)转膜:将硝酸纤维素膜与凝胶一起夹在用转移缓冲液浸泡过的滤纸中,从阴极到阳极依次为:3张滤纸-凝胶-硝酸纤维素膜-3张滤纸,置于半干电转膜仪中。控制在50mA/块恒流条件下,室温中转膜1至5小时(每1KD分子量对应1min)。
(5)丽春红S染色:将硝酸纤维素膜置于丽春红S液中进行染色2min(丽春红S液配制:丽春红0.5g,加冰乙酸1mL,溶解后加水定容至100mL)。待观察到出现蛋白条带后,寻找蛋白是否转移到硝酸纤维素膜。对蛋白marker的位置进行标记,使用ddH2O漂洗,反复3次,直至丽春红颜色褪去。
(6)膜的封闭与杂交:室温下使用1×封闭液封闭1小时,加入podocin抗体(1:1000比例稀释),4℃孵育24-48h后,用TBST将硝酸纤维素膜清洗3遍,每次清洗时间约5min(TBST配制:1500mM NaCl,100mM Tris,调pH值至7.6,并加1%的Tween20mL);分别加入过氧化物酶标记的山羊抗兔或鼠IgG抗体(1:1000比例稀释),孵育2小时于室温下;TBST反复洗膜后进行显影。设置β-Actin为内参蛋白,在凝胶图象分析软件中进行半定量分析。
本实施例的检测结果见图5A和图5B,结果显示,使用western blot对F1代猪肾脏中NPHS2基因编辑的足蛋白podocin进行检测,在模型组肾脏中podocin的表达明显低于正常对照组(P<0.05),表明NPHS2基因敲除成功,F1代更稳定。
5.5应用免疫荧光方法检测猪肾组织podocin蛋白表达
(1)肾脏组织切片(4μm)依次进行脱蜡;水化;二甲苯依次浸泡10min、15min;100%、95%乙醇分别浸泡10min;清水冲洗;
(2)0.01M PBS洗3×3min;
(3)组织抗原修复:室温下胰酶修复10min;
(4)分别滴30μL 3%过氧化酶溶液至切片上,室温下放置10min,用于阻断内源性过氧化物酶活性;
(5)PBS洗3×3min;
(6)将30μL封闭用山羊血清滴至切片上,室温封闭10min;
(7)弃去多余血清,在切片上滴加一抗50μL(1:50比例的podocin抗体),放于湿盒内,4℃条件下静置18h;
(8)PBS冲洗,5min×3次;
(9)将50μL兔二抗加至切片上,室温下静置孵育30min;
(10)PBS冲洗,5min×3次;
(11)将100μL过氧化物酶溶液滴至切片上,孵育30min,室温;再次进行PBS反复冲洗,3min×3次;
(12)将50μL新鲜配制的DAB溶液滴至组织上,置于显微镜下观察显色3min;
(13)自来水反复冲洗,室温下用苏木素复染1min;0.01M PBS冲洗返蓝;
(14)将切片依次置于:盐酸乙醇3秒、氨水3秒、85%乙醇30秒,95%乙醇1min、100%乙醇2min,二甲苯透明3min,中性树脂封片。
本实施例中,使用免疫荧光对F1代猪肾脏中NPHS2基因编辑的足蛋白podocin进行检测的结果参见图6,结果显示在模型组肾脏中Podocin表达分布在肾脏足细胞内,模型组与正常对照组相比,podocin蛋白表达量显著下降。
序列表
<110> 中国人民解放军总医院第一医学中心
<120> 一种用于敲除猪NPHS2基因的gRNA及其相关应用
<130> GAI20CN3540
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<170> PatentIn version 3.5
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cgcgcagaga gcccagcgga gccccgaccg ccggcagcca ccgtggtgga cgtggatgaa 240
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Claims (10)
1.一种gRNA,其核苷酸序列如SEQ ID No.2所示。
2.一种用于敲除NPHS2基因的打靶载体,其中,所述打靶载体含有权利要求1所述的gRNA。
3.根据权利要求2所述的打靶载体,其构建方法包括:合成5’端磷酸化寡核苷酸链的SEQ ID No.2所示的靶点序列;将靶点序列在初始载体骨架上进行克隆,得到打靶载体。
4.根据权利要求3所述的打靶载体,其中,所述初始载体包括pX330。
5.权利要求1所述的gRNA或权利要求2-4中任一项所述的打靶载体在制备用于敲除猪NPHS2基因的试剂盒中的应用。
6.一种制备猪NPHS2基因敲除的细胞的方法,包括:
将权利要求2-3中任一项所述的打靶载体与Cas9 mRNA转染猪的体细胞系,筛选获得NPHS2基因敲除的体细胞。
7.根据权利要求6所述的方法,其中,所述体细胞系包括成纤维细胞系。
8.一种制备NPHS2基因敲除的激素抵抗性肾病综合征猪模型的方法,该方法包括:
利用核移植技术,将去核卵母细胞与权利要求6或7中获得的NPHS2基因敲除的体细胞融合,构建重组卵母细胞;待重组卵母细胞发育成囊胚后,将囊胚移植到同期发情的母猪子宫内,制备基因敲除猪。
9.根据权利要求8所述的方法,其中,所述方法还包括将权利要求1所述的gRNA与Cas9mRNA注射到自然受精的受精卵中,将注射后的胚胎移植到同期发情的母猪子宫内,制备基因敲除猪。
10.一种用于敲除猪NPHS2基因的试剂盒,其中,所述试剂盒包括权利要求1所述的gRNA或权利要求2-4中任一项所述的打靶载体。
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