CN113549638A - 一种构建x连锁低血磷性佝偻病的小鼠模型的方法 - Google Patents
一种构建x连锁低血磷性佝偻病的小鼠模型的方法 Download PDFInfo
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Abstract
本发明公开了一种构建X连锁低血磷性佝偻病的小鼠模型的方法，本发明通过对XLHR患者及其家系进行基因筛查，在PHEX基因的第4号外显子上发现一个突变位点(c.C426G)，随之通过CRISPR/Cas9技术对小鼠PHEX基因第4号外显子相应位点进行基因编辑引入突变位点，可以获得具有X连锁低血磷性佝偻病患者的临床表型(如低血磷、高尿磷、四肢及尾巴短小、骨骼矿化异常等)的小鼠。
Description
技术领域
本发明涉及一种构建X连锁低血磷性佝偻病的小鼠模型的方法，属于基因编辑技术领域。
背景技术
低磷性佝偻病是儿童常见的代谢性骨病，发病率约为1:25,000，由于遗传性或获得性的 原因致使肾小管对磷酸盐的重吸收障碍，大量磷从尿中丢失，导致血磷降低和骨矿化障碍所 致的一组骨骼疾病，典型表现为：佝偻病的骨骼异常、低磷血症、高碱性磷酸酶血症，该病 致畸、致残率高，给社会和家庭带来沉重负担。
遗传性低磷性佝偻病包括常染色体显性遗传(ADHR,OMIM#193100)、常染色体隐性遗 传(ARHR,OMIM#241520、OMIM#613312)、X连锁遗传(XLHR,OMIM#307800、 OMIM#300554)、伴高钙尿症的遗传性低磷性佝偻病(HHRH,OMIM#241530)等。其中X连 锁低磷性佝偻病最为常见，在活产婴儿中发病率为1:20,000，占家族性低磷性佝偻病的80％ 以上，由PHEX(phosphate regulating endopeptidase homolog X-linked)基因异常所致， 50％-70％的XLHR患者中可检测到PHEX基因突变。另外，成纤维生长因子23(FGF23)突 变可引起ADHR，ARHR的致病基因包括：牙基质蛋白1(DMP1)、核苷酸外焦磷酸酶/磷 酸二脂酶1(ENPPI)、序列相似家族20(FAM20C)，而HHRH的致病基因是溶质转运家 族34成员3(SLC34A3)，这些基因异常所致的低磷性佝偻病比较罕见。
染色体内肽酶同源性磷酸调节基因(PHEX基因)位于染色体Xp22.11，包括22个外显 子，大小220kb，主要在软骨、成骨细胞和成牙本质表达。PHEX蛋白由749个氨基酸构成，属于脑啡肽酶(M13)锌指-金属内肽酶家族，是一种II型锌依赖性跨膜糖蛋白，包含三个结构域：含20个氨基酸的N末端胞内结构域、含25个氨基酸的单次跨膜结构域和一个含 704个氨基酸的胞外结构域。PHEX基因突变可引起磷代谢异常。至今为止，已发现481种 PHEX基因突变(The Human Gene Mutation Database，http://www.hgmd.cf.ac.uk/ac/index.php)，其中错义/无义突变约38％、小片段缺失突变约21％、拼接位点突变约占18％、小片段插 入突变约12％、大片段缺失突变约9％、其他约占2％。PHEX基因突变可导致PHEX蛋 白无法折叠、功能缺陷，从而影响其细胞内转运及内肽酶活性，最终导致骨矿化异常及低血 磷。然而PHEX蛋白磷代谢调节时作用的底物仍不清楚，PHEX在XLHR中发挥的作用仍未 完全阐明。目前，对于该病的发病机制和病因仍有待进一步探索，从本质上揭示XLH的病因 和发病机制对于XLH的治疗至关重要。
发明内容
本发明克服了上述现有技术的不足，提供一种构建X连锁低血磷性佝偻病的小鼠模型的 方法，本发明通过对XLHR患者及其家系进行基因筛查，在PHEX基因的第4号外显子上发 现一个突变位点(c.C426G)，随之通过CRISPR/Cas9技术对小鼠PHEX基因第4号外显子相 应位点进行基因编辑引入突变位点，可以获得具有X连锁低血磷性佝偻病患者的临床表型(如 低血磷、高尿磷、四肢及尾巴短小、骨骼矿化异常等)的小鼠。
一种小鼠PHEX基因SNP位点，所述SNP位点为小鼠PHEX基因第4号外显子426bp 处，变异类型为C/G。
进一步的，上述SNP位点在构建X连锁低血磷性佝偻病的小鼠模型中的应用。
一种利用上述SNP位点构建X连锁低血磷性佝偻病的小鼠模型的方法，包括如下步骤：
(1)以PD274载体为骨架构建插入上述小鼠PHEX基因SNP位点的SgRNA表达载体；
(2)利用步骤(1)的SgRNA表达载体在体外转录获取SgRNA；
(3)将Cas9mRNA和步骤(2)获得的SgRNA混合后加入到小鼠受精卵中；体外培养1-2小时后将存活的受精卵移植入假孕母鼠的输卵管；
(4)获得阳性转基因小鼠模型，检测到PHEX c.426G，即为X连锁低血磷性佝偻病的小鼠模型。
进一步的，上述Cas9mRNA和步骤(2)获得的SgRNA混合后的溶液中，SgRNA终浓 度为30ng/uL，Cas9 mRNA终浓度为200ng/uL。
利用上述方法获得X连锁低血磷性佝偻病的小鼠模型，该模型中的小鼠自发产生如下的 X连锁低血磷性佝偻病表型特征：低血磷、高尿磷、四肢及尾巴短小、骨骼矿化异常。
上述X连锁低血磷性佝偻病的小鼠模型在筛选预防或治疗X连锁低血磷性佝偻病药物中 的应用。
有益效果：
(1)通过对近期收治的一个XLHR患者及其家系进行基因筛查，发现了一个新的突变 位点(c.C426G)，随之通过CRISPR/Cas9技术对该位点进行基因编辑引入突变位点，进而影 响PHEX蛋白正常表达，对应的142位氨基酸C变成W，从而模拟X连锁低血磷性佝偻病 患者的临床表型，如低血磷、高尿磷、四肢及尾巴短小、骨骼矿化异常等。
(2)本发明首次通过Crispr/Cas9基因编辑技术对小鼠PHEX基因进行定点突变，首次 人工基因编辑构建PHEX点突变小鼠模型。利用本发明，一方面可应用于XLHR发病机制研究，进一步阐明PHEX调磷机制。另一方面，可应用于XLHR治疗研究，为未来治疗XLHR 患者提供新的药物治疗靶点。
基于Crispr/Cas9技术构建的小鼠模型相比既往小鼠模型能够更加稳定地遗传，并有希望 作为XLHR分子机制研究的首选模型。
具体实施方式
为了使本技术领域人员更好地理解本申请中的技术方案，下面结合实施例对本发明作进 一步说明，本发明是以PD274载体为骨架构建插入有小鼠PHEX基因编辑靶位点的SgRNA 表达载体。体外转录获取SgRNA，纯化回收后按照一定摩尔比例与Cas9mRNA混合，显微 注射小鼠受精卵，并将注射后的小鼠受精卵移植代孕，以获得PHEX定点突变小鼠。以下结 合实施例对本发明做详细的阐释。需要指出的是，本实施例仅用于解释本发明，而非对本发 明的权利要求做出限制或限定。
实施例1、T1和T2位点插入有小鼠PHEX基因编辑靶位点的SgRNA表达载体的构建
(1)主要试剂与材料来源
pDR274载体购自Addgene。单链oligo由深圳华大基因有限公司合成。BsaI限制性内切 酶购自Fermentas。T4 DNA连接酶购自Fermentas。DH5α感受态细胞购自北京天根生物科 技有限公司。重组载体按照北京天根生物科技有限公司提供的小提中量超纯质粒抽提试剂盒 说明书进行。DNA割胶回收试剂盒购自Qiagen公司。测序委托深圳华大基因有限公司完成。
(2)操作步骤
一、限制性内切酶BsaI酶切质粒PDR274载体，反应体系如表1：
表1限制性内切酶BsaI酶切质粒PDR274载体反应体系
| 成分 | 初始浓度 | 用量 | 终浓度 |
| FD buffer | 10× | 1μL | 1× |
| Bas I | 10units/μL | 1μL | 1unit/μL |
| pDR274质粒 | 100ng/μL | 2μL | 20ng/μL |
| ddH2O | 6μL | ||
| 总体积 | 10μL |
在冰上将以上成分依次加入，充分混匀。放在37℃水浴锅中酶切过夜。按照Qiagen公 司DNA回收试剂盒的步骤回收上述酶切产物。
二、单链oigo退火形成双链DNA，反应体系如表2：
表2单链oigo退火形成双链DNA反应体系
混合均匀后瞬时离心，置于PCR仪中95℃孵育3min，然后自然冷却20min。
三、T4 DNAligase连接反应，反应体系如表3：
表3 T4 DNA ligase连接反应反应体系
| 线性化pDR274载体 | 2μL |
| 双链DNA | 1.75μL |
| T4 DNAligase | 1μL |
| 10XT4 DNALigase buffer | 1μL |
| ddH2O | 4.25μL |
| 总体积 | 10μL |
在冰上将以上成分依次加入，充分混匀。放在37℃水浴锅中连接过夜结束后置于冰上， 用于转化。
四、转化大肠杆菌D5a，转化体系如表4：
表4转化大肠杆菌D5a
| 成分 | 用量 |
| 连接产物 | 10μL |
| DH5α感受态细胞 | 100μL |
| 总体积 | 110μL |
将以上成分混匀，冰浴30min；42℃热激90s；加入不含有抗生素的液体LB培养基500μL， 置于37℃摇床以200rpm/min的转速复苏45min；在台式离心机上以8000rpm/min的转速离心， 将菌体沉淀下来，然后去除450μlLB培养基；将剩余的100μL样品涂布在卡那霉素LB平板 上，置于37℃温箱过夜培养16h挑取细菌单克隆送深圳华大测序。测序正确的单克隆大摇后 提取质粒，-20℃保存。
实施例2、SgRNA体外转录
(1)主要试剂与材料来源
PCR引物对由华大基因有限公司合成。DNA割胶回收试剂盒购自Qiagen公司。MEGAshortscript kit(AM1354)SgRNA体外转录试剂盒购于Ambion公司。Taq酶和10×PCRBuffer 购自大连宝生物工程有限公司。
(2)操作步骤
一、体外转录gRNA模板准备
以SgRNA表达质粒为模板做PCR，SgRNA的序列为：TTTCTCCAGCATGTCAATGA (SEQ IDNO.1)，反应体系如表5：
表5以SgRNA表达质粒为模板做PCR反应体系
| 组分 | 用量 |
| 10×buffer | 2μL |
| dNTP | 1.6μL |
| 上游引物(10uM) | 1μL |
| 下游引物(10uM) | 1μL |
| rTaq酶 | 0.2μL |
| Cas9gRNA表达质粒 | 1μL |
| DEPC H2O | Up to 20μL |
PCR程序为：95℃3min，(94℃30s+58℃30s+72℃30s)×35cycles，72℃5min，16℃10min， 1.5％琼脂糖凝胶电泳检测PCR结果。PCR产物120bp左右，切胶回收，用DEPC水洗脱DNA 后测浓度，作为后续体外转录的DNA模板。
二、体外转录SgRNA：
以获得的PCR产物为模板进行体外转录，转录体系如表6：
表6体外转录
| 组分 | 用量 |
| T710×Reaction buffer | 2μL |
| T7(A++C++U) | 8μL |
| 模板DNA | 6μL |
| T7 Enzyme Mix | 2μL |
| 无酶水 | 2μL |
以上混合后，37℃反应4h(推荐使用37℃恒温培养箱处理，尽量不要使用水浴)，反应 结束后，加入2u1 TURBO DNase，37℃处理30min，电泳检测后80℃保存。
三、SgRNA纯化:
加入80uL DEPC水和20μL 3mol/L醋酸钠，混匀后加入等体积的1:1苯酚/异戊醇混合物， 充分混匀后12000～15000rpm4℃离心l5min，转移上清至新的EP管中；加入等体积氯仿，充 分混匀后12000～15000rpm4℃离心15min，转移上清至新的EP管中；加入等体积异丙醇，-80℃ 静置30min后，12000～15000rpm4℃离心30min，此时可见乳白色RNA沉淀；吸弃上清，加 入500μ70％冰乙醇洗涤沉淀，12000～15000rpm4℃离心10min，吸弃上清；室温自然晾干，待 乙醇完全挥发后加入适量DEPC水溶解RNA沉淀，测定OD值及浓度，-80℃保存备用。
四、SgRNA与Cas9 mRNA混合：
将SgRNA与Cas9 mRNA按照1:5摩尔比例混合，使其终浓度为SgRNA30ng/uL、Cas9mRNA200ng/uL。
实施例3、小鼠受精卵显微注射
(1)主要试剂与材料来源
试验动物为购于湖北省医学科学院实验动物中心【生产许可证SCXK(鄂)2003205】的 SPF级昆明小鼠。孕马血清促性腺激素(PMSG)和人绒毛膜促性腺激素(HCG)购于宁波 市三生药业有限公司，透明质酸酶购于上海生物制品厂。M2、M16细胞培养液为自配。
(2)操作步骤
供体鼠与受体鼠的超排：选取6周龄、30g以上的母鼠，腹腔注射5～10IU PMSG，注射 后46～48h，再注射5～10IU HCG进行超排。注射HCG的当天，供体鼠与种公鼠合笼，受体鼠与结扎公鼠合笼，第二天上午检査阴栓，选取有阴栓的母鼠分别为供体鼠和受体鼠。
二、受精卵的获取及培养：
将供体鼠脱颈处死，在无菌操作下迅速取岀输卵管置于200uM培养液中，在实体显微镜 下刺破输卵管膨大部或用钟表镊子轻轻压出包含受精卵的卵丘细胞团，转入含30ug/mL玻璃 酸酶的M2液中消化，移岀已游离的受精卵于新鲜的M2中，用M2洗涤3次，然后将受精卵转入M16培养液中，采用套皿法培养，置于37℃、5％C02、饱和湿度培养箱中培养，选出 雌雄原核清晰的受精卵待注射。
三、受精卵显微注射：
将已配制好的Cas9 mRNA和SgRNA混合液在电镜下吸入注射针内，选取雌雄原核清晰 的受精卵，将注射针快速刺入胞质中，注射2pL基因，迅速退出注射针。
实施例4、胚胎样品单细胞PCR检测
一、设计引物，序列如表7：
表7
设计好的引物交由深圳华大基因有限公司合成。
二、样品的消化与模板的制备
将体外培养48h的小鼠胚胎收集到灭菌PCR管中，残余的培养液应尽量少，加入5μLNP40 细胞裂解液(0.45％NP40、0.6％蛋白酶K瞬时离心后在PCR仪中37℃孵育1h，55℃孵育lh； 取出瞬时离心，再将其放入PCR仪中95℃孵育10min；完成上述步骤后吸取上清，转移至新 的灭菌PCR管中作为模板保存。
三、双重巢式PCR检测：
巢式第一轮PCR检测：20μLraq反应体系：在灭菌PCR管中加入0.2μL rTag酶，2uL10× Pcr Buffer，1.6μL dNTP，4μL Template，上、下游引物(第1对引物)各lμL，补加ddH20至20μL，轻弹混匀后瞬时离心。将混好的反应体系放入PCR仪内进行反应，反应程序为： 94℃
预变性5min；(⑨4℃变性30s、52℃退火30s、72℃延伸lmin)×35cycles；72℃总延伸 7min。
巢式第二轮PCR检测20μL rTag反应体系：在灭菌PCR管中加入0.2μL rTag酶，2uL10×PCR Buffer，1.6uL dNTP,lμL第一轮P(R反应产物，上、下游引物(第2对引物) 各lμL，补加ddH2O至20μL，轻弹混匀后瞬时离心。将混好的反应体系放入PCR仪内进行 反应，反应程序为：94℃预变性5min；(94℃变性30s、52℃退火30s、72℃延伸30s)×35cycles； 72℃总延伸7min。完成上述反应后取5μL产物进行2％琼脂糖凝胶电泳。
实施例5、小鼠胚胎移植、分娩及小鼠模型产生的X连锁低血磷性佝偻病表型特征
一、将当天的同步发情的受体母鼠，后腿部肌肉注射速眠新(10ng/kg)麻醉。采用头左 尾右的方式放置，剪去被毛，在最后一根肋骨与脊髓旁lcm处剪开一横切口，用镊子夹住脂 肪垫将卵巢、输卵管和子宫角部分拉岀，在实体显微镜下避开血管撕开腹膜(有时以防岀血， 可先滴些肾上腺素)，找到输卵管伞口，勿动。筛选岀轮廓清晰，且透明带与卵之间有卵周隙 的注射过的受精卵(不少于20枚)吸入移卵管中，从伞口吹入，直到从输卵管中看到气泡为 止，将子宫、输卵管和卵巢放回腹腔，抹上青霉素、链霉素粉，缝合，用碘酊消毒，做好耳 号标记，放置笼中，苏醒灵按1.5mg/kg量后腿部肌肉注射解麻醉，待其苏醒。
二、待母鼠苏醒后转入动物房中饲养，待其分娩，若足日不能顺产，则注射催产素助。 仔鼠产后剪其尾巴编号，进行PCR检测，C426G基因型表示构建成功。
统计结果表明，如表8删除效率统计表所示共显微注射受精卵195枚，生崽51只，获得 阳性F0代小鼠4只。本发明提供的小鼠PHEX基因编辑位点，为对该基因进行精确编辑提供了有效的靶标，为探明PHEX基因的生物学功能和信号通路提供了可靠的手段和素材。
表8删除效率统计表
构建的小鼠模型具有X连锁低血磷性佝偻病患者的临床表型(如低血磷、高尿磷、四肢 及尾巴短小、骨骼矿化异常等)。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本 技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化 或等效替换均落入本发明的保护范围内。
SEQUENCE LISTING
<110> 山东第一医科大学附属省立医院（山东省立医院）
<120> 一种小鼠PHEX基因SNP位点及其应用
<130> 2021
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> 序列
<400> 1
tttctccagc atgtcaatga 20
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<212> DNA
<213> 人工序列
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caagcagttt ggaatctctc ctg 23
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<211> 27
<212> DNA
<213> 人工序列
<400> 3
gtactcagac actcatgtcc atgtacc 27
Claims (6)
1.一种小鼠PHEX基因SNP位点，其特征在于，所述SNP位点为小鼠PHEX基因第4号外显子426bp处，变异类型为C/G。
2.如权利要求1所述的SNP位点在构建X连锁低血磷性佝偻病的小鼠模型中的应用。
3.一种利用上述SNP位点构建X连锁低血磷性佝偻病的小鼠模型的方法，其特征在于，包括如下步骤：
(1)以PD274载体为骨架构建插入如权利要求1所述的小鼠PHEX基因SNP位点的SgRNA表达载体；
(2)利用步骤(1)的SgRNA表达载体在体外转录获取SgRNA；
(3)将Cas9mRNA和步骤(2)获得的SgRNA混合后加入到小鼠受精卵中；体外培养1-2小时后将存活的受精卵移植入假孕母鼠的输卵管；
(4)获得阳性转基因小鼠模型，检测到PHEX c.426G，即为X连锁低血磷性佝偻病的小鼠模型。
4.如权利要求3所述的方法，其特征在于，所述的Cas9mRNA和步骤(2)获得的SgRNA混合后的溶液中，SgRNA终浓度为30ng/uL，Cas9 mRNA终浓度为200ng/uL。
5.如权利要求3所述的方法，其特征在于，所述的X连锁低血磷性佝偻病的小鼠模型自发产生如下的X连锁低血磷性佝偻病表型特征：低血磷、高尿磷、四肢及尾巴短小、骨骼矿化异常。
6.利用如权利要求3所述的方法获得的X连锁低血磷性佝偻病的小鼠模型在筛选预防或治疗X连锁低血磷性佝偻病药物中的应用。
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| CN117025747A (zh) * | 2023-05-16 | 2023-11-10 | 山东大学齐鲁医院 | 检测样本中slc34a3基因变异的试剂在制备筛查低血磷性佝偻病/骨软化症患者的产品的应用 |
| CN117025747B (zh) * | 2023-05-16 | 2024-04-09 | 山东大学齐鲁医院 | 检测样本中slc34a3基因变异的试剂在制备筛查低血磷性佝偻病/骨软化症患者的产品的应用 |
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