CN114058647B - 一种pig-a基因特异性敲除的小鼠模型及其构建方法和应用 - Google Patents
一种pig-a基因特异性敲除的小鼠模型及其构建方法和应用 Download PDFInfo
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Abstract
本发明创造提供了一种PIG‑A基因特异性敲除的小鼠模型及其构建方法和应用，使用Vav‑iCre构建了一个造血***PIG‑A基因特异性敲除的小鼠模型，通过对小鼠各项指标的检测证明该模型不仅存在稳定的GPI缺失同时还存在血管内溶血的疾病表型，是一个理想的PNH动物模型，可以用于PNH发病机制和治疗的相关研究中。
Description
技术领域
本发明创造属于动物实验模型及其制备技术领域，尤其是涉及一种PIG-A基因特异性敲除的小鼠模型及其构建方法和应用。
背景技术
阵发性睡眠性血红蛋白尿症(paroxysmal nocturnal hemoglobinuria，PNH)是一种后天获得性体细胞突变所致的造血干细胞膜缺陷引起的疾病。PNH发病的分子机制是造血干细胞X染色体上的磷脂酰肌醇聚糖-A(Pig-a)基因发生突变，导致糖基磷脂酰肌醇(GPI)合成障碍，使得细胞膜表面GPI锚连蛋白(GPI-AP)缺乏，主要的临床表现为溶血性贫血、血栓形成倾向和骨髓衰竭。
目前认为单纯PIG-A基因突变不足以导致PNH异常克隆获得增殖优势，导致PNH发病，既往文献报道的敲除Pig-a的动物模型进一步验证了这个结论。文献报道全基因组Pig-a基因敲除小鼠(Conventional Knockout，KO)大部分都胎死宫内，存活小鼠的GPI-AP缺失的血细胞比例很低(约10-30％)，且GPI-AP随出生后时间的延长进一步减少。此后，使用Cre/loxP技术构建出的条件性Pig-a基因敲除小鼠(Conditional Knockout，CKO)，获得了具有更高嵌合率、更高GPI-AP缺失比例(约40-90％)的小鼠，CKO小鼠GPI-AP缺失的血细胞比例在出生后基本保持稳定或轻度下降。Tae-Hoon Shin等使用CRISPR/Cas9技术敲除了猕猴造血干细胞的PIG-A基因，再将编辑过的造血干细胞回输至猕猴体内从而获得PNH猕猴模型，PNH猕猴模型体内各系血细胞PNH异常克隆比例在出生后逐渐下降，后维持在一定水平。但无论是在小鼠模型还是猕猴模型中，并没有观察到PNH比例的增加和贫血、血红蛋白尿、血栓等典型临床表现，无法真正用于PNH相关的研究中。
发明内容
有鉴于此，本发明创造旨在克服现有技术中的缺陷，提出一种PIG-A基因特异性敲除的PNH小鼠模型及其构建方法和应用。
为达到上述目的，本发明创造的技术方案是这样实现的：
一种Pig-a基因特异性敲除的小鼠模型的构建方法，包括如下步骤：
(1)通过基因打靶技术构建Pig-a基因特异性敲除的打靶载体；
(2)验证打靶载体后进行质粒提取及线性化后转染至小鼠ES细胞(胚胎干细胞)；
(3)使用新霉素或嘌呤霉素进行阳性ES细胞的筛选；
(4)将阳性ES细胞注射入小鼠的囊胚中，将注射后的囊胚移植到假孕母鼠体内，生产出小鼠，即为F0代小鼠，提取F0代小鼠尾部DNA，并进行基因型鉴定；
(5)将F0代Flox纯合子小鼠与Vav-iCre工具小鼠交配获得的F1代小鼠，通过基因型鉴定，筛选出的F1代小鼠中的CKO纯合子小鼠和/或CKO杂合子小鼠即为PIG-A基因特异性敲除的小鼠模型。
优选的，步骤(1)的具体步骤为：在Pig-a基因外显子3和外显子5两侧***同向LoxP位点，构建打靶载体。
优选的，所述步骤(3)中使用的G418遗传霉素的浓度为：200μg/ml，嘌呤霉素的浓度为0.8μg/mL。
优选的，小鼠的基因型鉴定采用PCR鉴定，所使用的引物序列为：
Pia-a：
正向引物：5’-GACTTCTGAACAAAATGAAGGCAGT-3’(SEQ ID NO:1)；
反向引物：5’-GTGCACAGCTGATTAGAAATCTAGG-3’(SEQ ID NO:2)；
Vav-iCre：
正向引物：5’-GGTGTTGTAGTTGTCCCCACT-3’(SEQ ID NO:3)；
反向引物：5’-CAGGTTTTGGTGCACAGTCA-3’(SEQ ID NO:4)。
本发明还提供一种由上述构建方法得到的Pig-a基因特异性敲除的小鼠模型。
本发明还提供一种上述构建方法得到的Pig-a基因特异性敲除的小鼠模型用于作为阵发性睡眠性血红蛋白尿症(PNH)研究的模型。
本发明还提供一种上述构建方法得到的CKO纯合子小鼠组成的PIG-A基因特异性敲除的小鼠模型在评价药物在治疗PNH效果中的应用。
本发明还提供一种上述构建方法得到的CKO杂合子小鼠组成的PIG-A基因特异性敲除的小鼠模型在PNH异常克隆增殖优势相关的研究中的应用。
本发明还提供一种上述构建方法得到的Pig-a基因特异性敲除的小鼠模型在筛选预防或者治疗阵发性睡眠性血红蛋白尿症药物中的应用
本发明还提供一种上述构建方法得到的Pig-a基因特异性敲除的小鼠模型在制备预防或者治疗阵发性睡眠性血红蛋白尿症药物中的应用。
相对于现有技术，本发明创造具有以下优势：
本发明使用Vav-iCre构建了一个造血***PIG-A基因特异性敲除的小鼠模型，通过对小鼠各项指标的检测证明该模型不仅存在稳定的GPI缺失同时还存在血管内溶血的疾病表型，是一个潜在的理想的PNH动物模型，可以用于PNH发病机制和治疗的相关研究中。
附图说明
图1为Pig-a基因特异性敲除的打靶方案的示意图；
图2中图2A为PCR鉴定小鼠基因型，同一样品重复两次(外框)，每只小鼠(内框)左孔鉴定loxp，右孔鉴定Cre；图2B为qRT-PCR检测小鼠骨髓细胞中Pig-a的mRNA表达水平；图2C为WB检测小鼠骨髓细胞中Pig-a的蛋白表达水平(*p<0.05,**p<0.01,***p<0.001，****p<0.0001)；
图3为小鼠外周血各系血细胞锚蛋白和锚连蛋白检测结果(图3A为锚连蛋白CD24/CD48表达，图3B为锚蛋白FLAER表达)；
图4为CKO小鼠外周血各系血细胞锚蛋白和锚连蛋白随出生后时间延长的变化(△代表CKO纯合子，■代表CKO杂合子)；
图5为四组小鼠外周血细胞计数、溶血相关指标和补体水平检测结果(图5A为四组小鼠的白细胞计数、红细胞计数、血红蛋白浓度和血小板计数；图5B为四组小鼠的血清中LDH、TBIL、IBIL C5b-9水平对比；图5C为四组小鼠血浆游离血红蛋白(FHb)浓度对比；图5D为四组小鼠血清中C3a、C5a、C5b-9水平对比)，*p<0.05,**p<0.01,***p<0.001，****p<0.0001；
图6为四组小鼠骨髓和脾脏病理(HE染色，10×40)(图6A为四组小鼠骨髓病例，图6B为四组小鼠脾脏病理)。
具体实施方式
除有定义外，以下实施例中所用的技术术语具有与本发明创造所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂，如无特殊说明，均为常规生化试剂；所述实验方法，如无特殊说明，均为常规方法。
下面结合实施例来详细说明本发明创造。
1、Pig-a基因特异性敲除的小鼠模型的构建方法
1.1Pig-a基因特异性敲除载体构建
在Pig-a基因外显子3和外显子5两侧***同向LoxP，构建打靶载体并验证后，进行质粒提取及线性化，打靶方案见图1。需要说明的是线性化处理技术为本领域的常用技术，具体试验过程在本实施例中不进行赘述。
1.2电转ES细胞及阳性克隆筛选鉴定
将线性化的打靶载体质粒电转至C57BL/6N小鼠的ES细胞中，使打靶载体中的外源的基因序列整合到内源基因组中进行表达，表达后加入新霉素挑选出来抗药克隆，采用PCR、核型分析和Southern Blot鉴定得到阳性克隆ES细胞。将得到的药物抗性ES克隆进行培养。需要说明的是电转技术、PCR、核型分析和Southern Blot鉴定技术为本领域的常用技术，具体试验过程在本实施例中不进行赘述。
1.3F0代Flox纯合子小鼠繁育
将培养得到的阳性ES细胞进行显微注射，将注射后的囊胚移植到假孕母鼠体内，生产得到F0代小鼠，F0代小鼠出生后1周进行编号，出生后3周剪取待测小鼠1cm左右鼠尾应用鼠尾鉴定试剂盒(购自美国Bimake公司)提取基因组DNA，PCR鉴定Pig-a基因型(引物序列见表1)。
Flox纯合子雌鼠基因型为Pig-a[flox/flox])、Flox纯合子雄鼠基因型为Pig-a[flox/Y]。
1.4繁育和基因型鉴定
将Flox纯合子小鼠与Vav-iCre小鼠交配获得F1代小鼠，出生后1周对F1小鼠进行编号，出生后3周剪取待测F1小鼠1cm左右鼠尾应用鼠尾鉴定试剂盒提取基因组DNA，使用PCR鉴定Pig-a和Vav-iCre基因型(引物序列见表1)。
基因型Piga[flox/flox,Vav-icre]为雌性CKO纯合子，Piga[flox/Y,Vav-icre]为雄性CKO纯合子，Piga[flox/+,Vav-icre]为雌性CKO杂合子。
应当说明，对F0-F1代小鼠进行基因型鉴定，其采用的基因型鉴定为生物学检测中一种常用技术，其具体的实现过程将不进行赘述。当然，为适应本申请的基因型鉴定，其基因型鉴定过程中使用的PCR引物信息如表1所示。
表1基因引物序列
PCR反应体系为：小鼠基因组1μl,正向引物(10mM)1μl，反向引物(10mM)1μl，10XBuffer 2.5μl，ddH2O 16.5μl，Mg2+(25mM)2μl,dNTPs(10mM)0.5μl,Taq(5U/μl)0.25μl。
PCR反应程序为：94℃5min，94℃15s，58℃15s，72℃1min，72℃5min，共35个循环。
2.验证实验
将所有小鼠均在在独立通气笼盒(IVC)***(SPF级)中进行饲养，本发明后续实验各组均选取鼠龄相近(相差不超过1周)的CKO纯合子小鼠、CKO杂合子小鼠、Flox鼠和C57BL/6N小鼠四组进行分析。
实验数据应用统计学软件GraphPad Prism8进行分析。本实施例中的计量资料以均值±标准差(MEAN±SD)表示。使用Shapiro-Wilk检验对多组数据进行正态性检验，p＞0.05为符合正态。符合正态分布且方差齐的多组数据比较使用卡方检验，组间比较使用Holm-Sidak’s多重t检验；方差不齐或不符合正态分布的多组数据比较使用Kruskal-Wallis检验，组间比较使用Dunn’s多重t检验定义。定义p＜0.05为存在统计学差异。
2.1qRT-PCR
处死小鼠后，分离出双侧股骨，PBS冲洗骨髓腔获得骨髓细胞悬液，过滤后加入1ml的红细胞裂解液，充分混匀后静置8min，1500r/min离心5分钟后弃上清，PBS清洗一次弃上清，沉淀即为小鼠骨髓细胞。
PCR结果可见正常C57BL/6N的小鼠和Vav-iCre鼠能扩增出216bp大小的Pig-a片段，Flox纯合子和CKO纯合子能扩增出256bp大小的突变型Pig-a片段，Flox杂合子和CKO杂合子可以扩增出一条216pb大小的正常片段和一条256bp大小的异常片段；正常C57BL/6N小鼠和Flox小鼠不表达Vav-iCre，扩增后没有条带，Vav-iCre鼠、CKO小鼠能扩增出390bp大小的目的片段(图2A)。
qRT-PCR结果显示正常C57BL/6N、Flox鼠、CKO杂合子和CKO纯合子骨髓细胞的Pig-a mRNA相对表达量为1.017±0.1850、0.8082±0.1392、0.00098±0.00038和0.000047±0.0002654，四组比较有统计学差异(p＜0.0001)。CKO杂合子小鼠和CKO纯合子小鼠骨髓细胞Pig-a mRNA相对表达量明显低于正常C57BL/6N小鼠(p＜0.0001，p＜0.0001)和Flox鼠(p＜0.0001，p＜0.0001)，Flox鼠骨髓细胞Pig-a的mRNA相对表达量低于正常C57BL/6N小鼠(p＝0.0005)(图2B)。
鉴定基因型后将Flox鼠、CKO杂合子和CKO杂合子分笼饲养，定期监测小鼠的体重、生存和尿色，CKO小鼠与Flox鼠、正常C57BL/6N小鼠相比均无明显差异。
2.2Western Blot蛋白质免疫印迹试验
细胞计数后取约107个小鼠骨髓细胞，加入RIPA裂解液、PMSF、磷酸化酶抑制剂混合液(100：1：1)100μl，充分混匀后冰上裂解30min，每5min弹匀一次。将裂解后的标本12000r/min 4℃离心15min，吸取上清液按照体积3：1比例加入4×蛋白上样缓冲液，混匀后100℃加热变性10min，所得即为小鼠骨髓细胞总蛋白。使用Tris-MOPS-SDS电泳液、4-20％蛋白预制胶，分别在每孔加入上述蛋白样品5μl和蛋白预染Marker 5μl，110V电压至蛋白跑至凝胶底端时终止电泳。使用转膜仪A，将电泳凝胶中蛋白转至PVDF膜上。将PVDF膜置于5％封闭液中，室温摇床封闭1小时后按照Marker位置及目的蛋白分子量裁剪PVDF膜，分离Pig-a和Gapdh区域条带，分别置于Pig-a一抗和Gapdh一抗(用5％封闭液按1：1000稀释)中，4℃摇床孵育过夜。回收一抗，TBST摇床上清洗5min，重复三次后，将条带放入二抗中(1：2000稀释)室温摇床孵育1小时。回收二抗，加入TBST摇床上清洗5min，重复三次。避光将ECL显影液A和B以1:1的比例混合成为曝光工作液(现用现配)，并均匀覆盖在PVDF膜上，曝光仪选择化学发光模式、自动曝光、高分辨率模式曝光并储存图像。
结果发现：与正常C57BL/6N小鼠相比，Flox鼠的Pig-a蛋白表达水平无变化，CKO杂合子的Pig-a蛋白表达明显减低，CKO纯合子几乎没有Pig-a蛋白表达(图2C)。
2.3FCM流式细胞术检测
分别于各组小鼠5w、8w、3m、4m、6m、8m、10m和12m使用流式细胞术检测外周血细胞表达锚蛋白和锚连蛋白的表达。固定小鼠后从小鼠内眦静脉丛取外周血(150-200)μl至1.5ml EDT抗凝的EP管内。每只小鼠设1管同型对照管和四个试验管，试验管中分别使用BV421-TER119、CD45R-PE单抗、CD3-APC、Gr1-PreCp Cy5.5单抗标记红细胞、B淋巴细胞、T淋巴细胞和粒细胞，检测不同血细胞表面锚蛋白(FLAER)和锚连蛋白(CD24/CD48)表达水平。使用BECKMAN COULTER流式细胞仪上机检测；应用CytExpert分析软件，每管收集20000个细胞。
结果发现：Flox鼠和正常C57BL/6N小鼠各系外周血细胞锚蛋白和锚连蛋白表达正常；CKO纯合子小鼠各系外周血细胞锚蛋白和锚连蛋白表达几乎完全缺失；CKO杂合子小鼠外周血各系血细胞锚蛋白和锚连蛋白缺失比例在不同细胞种类略有差异，RBC、T细胞缺失比例最高，其次是粒细胞，B细胞缺失比例约20-30％(图3)。随着出生后时间的延长，CKO纯合子外周血各系血细胞的锚蛋白和锚连蛋白缺失比例稳定；CKO杂合子外周血各系血细胞的缺失比例逐渐下降，至出生后3月左右达到稳定并维持终身(图4)。
2.4小鼠血常规、血生化检测
取外周血约50μl至EDTA抗凝的EP管内，全自动血常规分析仪行血常规化验；取外周血约600μl至无抗凝剂的EP管内，室温静置20min，3000r/min离心20分钟后，收集上清即为血清，取100μl血清使用全自动血生化分析仪行LDH、TBIL、IBIL化验。使用试剂盒检测小鼠血浆游离血红蛋白含量。
对正常C57BL/6N鼠、Flox鼠、CKO杂合子小鼠和CKO纯合子小鼠的进行外周血细胞计数、溶血相关指标和补体水平检测，结果见表2。
表2小鼠各项化验数据结果
*四组比较，有统计学差异(p＜0.05)
四组小鼠的白细胞计数、红细胞计数、血红蛋白浓度和血小板计数均有显著差异(p＜0.0001，p＜0.0001，p＜0.0001，p＝0.0008)(图5A)。CKO纯合子小鼠的白细胞计数、红细胞计数、血红蛋白浓度和血小板计数均明显低于正常C57BL/6N鼠(p＜0.0001，p＜0.0001，p＜0.0001，p＝0.0011)和Flox鼠(p＜0.0001，p＜0.0001，p＝0.0652，p＝0.0062)。CKO杂合子的白细胞计数、红细胞计数均明显低于正常C57BL/6N鼠(p＜0.0001，p＜0.0001)和Flox鼠(p＜0.0001，p＜0.0001)；CKO杂合子的血红蛋白浓度低于正常C57BL/6N鼠(p＝0.0002)。CKO纯合子和CKO杂合子、正常C57BL/6N鼠和Flox鼠的上述指标比较均无统计学差异。
四组小鼠的血清LDH、TBIL、IBIL均有显著差异(p＝0.0001，p＜0.0001，p＝0.0002)(图5B)。CKO纯合子小鼠血清的LDH、TBIL、IBIL均明显高于正常C57BL/6N鼠(p＝0.0002，p＝0.0001，p＝0.0004)和Flox鼠(p＝0.0003，p＝0.0001，p＝0.0003)。CKO杂合子小鼠血清的TBIL水平明显高于正常C57BL/6N鼠(p＝0.0120)和Flox鼠(p＝0.0098)。CKO纯合子小鼠血清LDH和IBIL水平高于CKO杂合子(p＝0.0140，p＝0.0041)。正常C57BL/6N鼠和Flox小鼠的上述指标比较无统计学差异。
肉眼观察小鼠血浆颜色可以发现，CKO杂合子、CKO纯合子的血浆比正常C57BL/6N鼠和Flox鼠颜色偏红，CKO纯合子较CKO杂合子更红，四组小鼠血浆游离血红蛋白(FHb)浓度比较具有统计学差异(p＝0.0052)(图5C)。CKO纯合子FHb水平高于正常C57BL/6N小鼠和Flox鼠(p＝0.0181，p＝0.0363)，CKO杂合子FHb水平高于正常C57BL/6N小鼠(p＝0.0381)，CKO纯合子和CKO杂合子、正常C57BL/6N鼠和Flox鼠比较均无统计学差异。
2.5ELISA检测
使用小鼠TCC C5b-9、C3a、C5a ELISA试剂盒(SBJ-M0335、SBJ-H1735、SBJ-H1734，SenBeiJia Biological Technology Co.,Ltd.)检测小鼠血清补体C3a、C5a、C5b-9水平。在酶标包被板上待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl，用封板膜封板后置37℃温育30min，使用清洗缓冲液清洗板子3次。每孔加入酶标试剂50μl，用封板膜封板后置37℃温育30min，再次洗涤5次。每孔加入显色剂A 50μl，再加入显色剂B 50μl，轻轻震荡混匀，37℃避光显色15min，之后每孔加入终止液50μl终止反应，此时孔中液体由蓝色转为黄色。
检测结果见图5D，虽然CKO小鼠血清C3a、C5a高于正常C57BL/6N鼠和Flox鼠，但四组比较没有统计学差异(p＝0.1703，p＝0.1138)。小鼠血清C5b-9水平四组比较具有显著统计学差异(p＝0.0027)，其中CKO杂合子和CKO纯合子的血清C5b-9水平高于正常C57BL/6N小鼠(p＝0.0297，p＝0.0018)，余各组间比较无统计学差异。
2.6病理检测
分别取20w CKO杂合子、CKO纯合子、Flox鼠和正常C57BL/6N小鼠，处死小鼠后取出一侧股骨和脾脏，分别置于预先装有4％多聚甲醛的1.5ml EP管中。石蜡包埋后切片(厚度3～5μm)，将蜡片附贴于载玻片上，置于烤箱中烘烤(80℃，30～60min)。HE染色后使用显微镜镜下观察组织结构。
由图6可见CKO小鼠的脾脏增大伴有结节。取4组小鼠的骨髓和脾脏标本切片并行HE染色。4组小鼠骨髓粒红比例大致正常，正常C57BL/6N小鼠和Flox小鼠骨髓造血组织容量约90％VOL，CKO杂合子和CKO纯合子小鼠骨髓造血组织容量约为95％VOL(图6A)。4组小鼠脾脏结构大致正常，正常C57BL/6N小鼠和Flox小鼠脾脏内含铁血黄素颗粒细胞可见，CKO杂合子小鼠脾脏内含铁血黄素颗粒细胞易见，CKO纯合子小鼠脾脏内含铁血黄素颗粒细胞可见小簇分布，成块聚集(图6B)。
综上所述，对构建的小鼠进行PNH异常克隆比例的检测发现CKO纯合子小鼠各系血细胞锚蛋白和锚连蛋白完全缺失，在仅有异常克隆细胞的情况下，能始终保持完全敲除的状态；CKO杂合子小鼠各系血细胞锚蛋白和锚连蛋白缺失比例在不同细胞、不同小鼠略有差异，杂合子小鼠体内同时存在正常和异常克隆，随着出生时间的推移异常克隆比例逐渐降低，出生后3月左右异常克隆比例基本稳定，这一结果也再次证实了单纯敲除PIG-A基因的异常克隆不具有增殖优势。
疾病相关的化验提示CKO小鼠尤其是CKO纯合子存在轻度溶血，Flox鼠和正常C57BL/6N均无明显异常。CKO小鼠的白细胞、血小板、血红蛋白均减低，骨髓活检提示骨髓造血组织容积增加，这提示血细胞的减低与骨髓造血功能无关，与髓外血细胞的破坏有关。CKO小鼠的溶血相关指标，包括LDH、TBIL、IBIL均明显升高，提示小鼠存在溶血。血浆游离血红蛋白升高、血清C5b-9水平升高和脾脏的含铁血黄素颗粒细胞增多均证明CKO小鼠存在血管内溶血。
通过上述实验结果可知，本发明成功构建了一个不仅存在GPI缺失同时还存在轻度溶血的造血***PIG-A基因特异性敲除的PNH小鼠模型：CKO纯合子小鼠体内仅有异常克隆且终生稳定；CKO杂合子虽然溶血表型较纯合子轻，但其体内同时存在异常克隆和正常造血，出生3个月后PNH异常克隆比例可以达到稳定且维持终身。两者的不同特点使纯合子小鼠适用于新药治疗效果的研究，杂合子小鼠则更适用于PNH异常克隆增殖优势相关的研究。
以上所述仅为本发明创造的较佳实施例而已，并不用以限制本发明创造，凡在本发明创造的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明创造的保护范围之内。
序列表
<110> 天津医科大学总医院
<120> 一种PIG-A基因特异性敲除的小鼠模型及其构建方法和应用
<141> 2021-11-18
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence
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<210> 2
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<212> DNA
<213> Artificial Sequence
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gtgcacagct gattagaaat ctagg 25
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<211> 21
<212> DNA
<213> Artificial Sequence
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<212> DNA
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caggttttgg tgcacagtca 20
Claims (3)
1.一种CKO纯合子小鼠组成的PIG-A基因特异性敲除的小鼠模型的应用，其特征在于：所述小鼠模型在以下应用中的一种或多种；
评价药物在治疗阵发性睡眠性血红蛋白尿症效果中的应用；
筛选预防或者治疗阵发性睡眠性血红蛋白尿症药物中的应用；
制备预防或者治疗阵发性睡眠性血红蛋白尿症药物中的应用；
所述PIG-A基因特异性敲除的小鼠模型的构建方法包括如下步骤：
（1）通过基因打靶技术构建Pig-a基因特异性敲除的打靶载体；
（2）验证打靶载体后进行质粒提取及线性化后转染至供体小鼠ES细胞；
（3）使用G418遗传霉素或嘌呤霉素进行阳性ES细胞的筛选， G418遗传霉素的浓度为200μg /ml，嘌呤霉素的浓度为0.8μg/mL；
（4）将阳性ES细胞注射入供体小鼠的囊胚中，将注射后的囊胚移植到假孕母鼠体内，生产出小鼠，即为F0代小鼠，提取F0代小鼠尾部DNA，并进行基因型鉴定；
小鼠的基因型鉴定采用PCR鉴定，所使用的引物序列为：
Pia-a：
正向引物：5’-GACTTCTGAACAAAATGAAGGCAGT-3’；
反向引物：5’-GTGCACAGCTGATTAGAAATCTAGG-3’；
Vav-iCre：
正向引物：5’-GGTGTTGTAGTTGTCCCCACT-3’；
反向引物：5’-CAGGTTTTGGTGCACAGTCA-3’；
（5）将F0代Flox纯合子小鼠与Vav-iCre工具小鼠交配获得的F1代小鼠，通过基因型鉴定，筛选出的F1代小鼠中的CKO纯合子小鼠和/或CKO杂合子小鼠即为PIG-A基因特异性敲除的小鼠模型。
2.根据权利要求1所述的应用，其特征在于：步骤（1）的具体步骤为：在Pig-a基因外显子3和外显子5两侧***同向LoxP位点，构建打靶载体。
3.根据权利要求1所述的应用，其特征在于：供体小鼠为C57BL/6N小鼠。
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| CN102958535A (zh) * | 2009-11-05 | 2013-03-06 | 亚力史剑桥公司 | 阵发性夜间血红蛋白尿、溶血性贫血和涉及血管内和血管外溶血的疾病状态的治疗 |
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| CN102958535A (zh) * | 2009-11-05 | 2013-03-06 | 亚力史剑桥公司 | 阵发性夜间血红蛋白尿、溶血性贫血和涉及血管内和血管外溶血的疾病状态的治疗 |
| CN109197781A (zh) * | 2018-09-14 | 2019-01-15 | 徐州医科大学 | Aurka-cko1-n条件性基因敲除小鼠模型的构建方法 |
| CN112029765A (zh) * | 2020-07-30 | 2020-12-04 | 南京医科大学附属逸夫医院 | 一种Metrnl基因条件性敲除小鼠模型制作方法 |
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