CN110384074A - 一种Kir7.1基因失活的小鼠模型及其构建方法 - Google Patents
一种Kir7.1基因失活的小鼠模型及其构建方法 Download PDFInfo
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供了一种Kir7.1基因失活的小鼠模型，所述小鼠模型的Kir7.1基因失活是通过将Kir7.1基因第13位的亮氨酸突变为脯氨酸实现Kir7.1基因失活。另外还提供了一种Kir7.1基因失活的小鼠模型的构建方法，包括以下步骤：用N‑乙基‑N‑亚硝基脲腹腔注射雄性小鼠，将其与雌性小鼠交配产生G1代小鼠；G1代雄性小鼠与雌性小鼠交配产生G2代雌性；将G2代雌性小鼠与G1代雄性小鼠交配得到的G3代幼鼠进行气管检测和全外显子测序分析来筛选和鉴定Kir7.1基因失活小鼠。本方法产生的Kir7.1基因失活小鼠模型，机制较明了，症型较明确，模型稳定可重复。可用于筛选治疗气道软骨缺陷的药物，评价治疗气道软骨缺陷的方法。
Description
技术领域
本发明涉及基因工程技术领域，尤其涉及Kir7.1基因失活的小鼠模型及其构建方法。
背景技术
气管由内胚层起源的上皮和中胚层起源的软骨，结缔组织以及平滑肌组成。平滑肌提供气管收缩所需要的弹性，软骨赋予气管刚性从而防止气道塌陷。在人体内，气管形成缺陷可引起先天性气管塌陷这类疾病，新生儿中的发病率1/3000，临床表现为气道软骨缺陷并可能导致呼吸窘迫和死亡。
钾离子通道是特异性允许钾离子通过质膜，并阻碍包括钠离子在内的其它离子通透的一类通道蛋白，是生物界中分布最广泛的离子通道，分为钙活化型钾离子通道，内向性整流型钾离子通道，串联孔域型钾离子通道和电位控制型钾离子通道。近年来的研究表明，钾离子通道在器官发育中发挥着重要功能。Kir7.1是内向性整流型钾离子通道中的一员，人类Kir7.1基因的突变会导致snowflake玻璃体视网膜变性和Leber先天性黑朦。相对于其它组织和器官开展的工作，Kir7.1在呼吸系统和消化系统中的发育和生理功能的研究很有限。
到目前为止，在人体中鉴定出的Kir7.1基因的突变包括错义突变R162W，这与雪花玻璃体视网膜病变有连锁关系。无义突变Arg166X和错译突变Leu241Pro与Leber先天性黑蒙(LCA)相连锁。至今为止，尚未鉴定出小鼠Kir7.1基因的突变。同时，有研究证明Kir7.1基因突变不是简单的点突变，利用CRISPR-Cas9系统定点产生的突变小鼠，不能得到Kir7.1基因错义突变的表型。
发明内容
本发明的目的在于提供一种Kir7.1基因失活的小鼠模型。
本发明的另一目的在于提供一种Kir7.1基因失活的小鼠模型的构建方法。
本发明的又一目的在于提供一种Kir7.1基因失活的小鼠模型的应用。
由于小鼠生长周期短，易于饲养管理，容易获得已经定性的近交交配鼠和遗传突变体，作为实验动物较为经济实惠，是基因组结构、个体发育过程、组织细胞结构等与人类相近的模式生物体系。同时，小鼠是目前研究哺乳动物气管发育的良好模型，气管的结构和功能与人类似；小鼠中成熟的细胞谱系示踪，基因敲除，人工化学诱变等技术能够快速有效的确定特定细胞和基因的体内功能。
本发明通过使Kir7.1基因失活来验证该基因在调控气管软骨、平滑肌和上皮细胞的形成过程中所起的作用。本发明人通过将小鼠的Kir7.1基因通过错义突变，使其第13位的亮氨酸变为脯氨酸实现Kir7.1基因失活，Kir7.1基因错义突变编码的氨基酸序列如SEQID NO：1所示，编码该氨基酸序列的核苷酸序列如SEQ ID NO：2所示。Kir7.1基因失活的小鼠气管平滑肌具有气管平滑肌细胞骨架组装障碍、平滑肌细胞收缩力降低、气管上皮细胞分化缺陷、食管形成缺陷的表型。
上述Kir7.1基因失活小鼠模型的构建方法，包括以下步骤：用N-乙基-N-亚硝基脲腹腔注射雄性小鼠；5～10周后，将其与雌性小鼠交配产生G1代小鼠；G1代雄性小鼠与雌性小鼠交配产生G2代雌性；将G2代雌性小鼠与G1代雄性小鼠交配得到的G3代幼鼠进行气管检测和全外显子测序分析来筛选和鉴定Kir7.1基因失活小鼠。
上述方法中优选地，所述小鼠为C57BL/6J小鼠。
上述方法中优选地，所述N-乙基-N-亚硝基脲的浓度为100mg/kg；腹腔连续注射3次。
上述方法中优选地，气管检测包括：检测小鼠的气管、软骨、平滑肌细胞、细胞骨架以及上皮细胞的异常情况。
本发明人通过气管检测发现Kir7.1失活小鼠气管平滑肌细胞表现出空间排列和细胞极性的异常，以及气管平滑肌表现出细胞骨架组装缺陷；还观察到Kir7.1的失活能强烈抑制刚出生小鼠气管的形成，包括气管延长的障碍和软骨形成的缺陷；并且显示Kir7.1基因失活小鼠的气管上皮纤毛细胞分化的标记性蛋白Acetylatedα-Tubulin的量显著下降。
本发明的Kir7.1失活小鼠模型可用于筛选治疗气道软骨缺陷的药物的用途。
本发明的有益效果：本方法产生的Kir7.1基因失活小鼠模型，机制较明了，症型较明确，模型稳定可重复。在失活的Kir7.1基因调控下，具有气管塌陷、气管短小的缺陷，可用于筛选治疗气道软骨缺陷的药物，评价治疗气道软骨缺陷的方法。
附图说明
图1为Kir7.1基因敲除的小鼠表现出气管形成的缺陷示意图(图1a和1b显示阿尔新蓝染色检测野生型和基因敲除小鼠气管软骨的形成及定量分析结果，表明基因敲除小鼠表现出气管的缩短和软骨环的断裂。图1c和1d显示α-SMA抗体染色检测野生型和基因敲除小鼠的平滑肌的排列及定量分析结果，表明基因敲除小鼠表现出气管平滑肌排列的缺陷和面积的减小。图1e和1f显示阿尔新蓝染色(Alcain blue)检测野生型和突变体小鼠气管软骨的形成及定量分析结果，表明突变体小鼠表现出气管的缩短和软骨环的断裂。图1g和1h显示α-SMA抗体染色检测野生型和基因突变小鼠的平滑肌的排列及定量分析结果，表明突变体小鼠表现出气管平滑肌排列的缺陷和面积的减小。图1i显示荧光实时定量检测Kir7.1mRNA在小鼠气管中的表达量的结果，表明Kir7.1mRNA在小鼠胚胎期各阶段的气管中稳定表达。图1j显示KIR7.1抗体染色检测KIR7.1蛋白在野生型和突变体小鼠气管中的表达结果，表明KIR7.1蛋白表达于小鼠气管平滑肌并且突变蛋白表达稳定)。
图2为Kir7.1基因失活的小鼠气管平滑肌表现出细胞骨架组装的障碍和收缩力的降低示意图(图2a显示刚出生的野生型和突变体小鼠的气管收缩力、Kir7.1的抑制剂VU590处理的野生型和突变体小鼠的气管收缩力以及洗除VU590后野生型和突变体的气管收缩力，结果显示突变体小鼠的气管收缩力与VU590处理的野生型小鼠的气管收缩力明显降低，并且VU590的抑制效力呈现可逆性。图2b和2c显示α-SMA抗体染色和Phalloidin染色检测刚出生的野生型和突变体小鼠气管平滑肌的细胞骨架和定量分析，结果显示突变体小鼠气管平滑肌的细胞骨架组装障碍)。
图3为Kir7.1基因失活的小鼠表现出气管上皮细胞分化的缺陷示意图(图3a，3b，3c和3d显示刚出生的野生型和突变体小鼠的气管冰冻切片的acetylated alpha-tubulin，CC10，KRT5和DAPI染色及定量分析，结果显示突变体小鼠气管上皮纤毛细胞数量减少)。
图4为Kir7.1基因失活的小鼠表现出食管形成的缺陷示意图(图4a和4b显示刚出生的野生型和突变体小鼠食道示意图及定量分析结果，表明突变体小鼠表现出食道延伸的缺陷。图4c显示KIR7.1，α-SMA和CDH1抗体染色检测小鼠胚胎期第12.5和13.5天KIR7.1在气道中的表达，结果显示KIR7.1表达在小鼠食道的平滑肌细胞中。图4d，4e和4f显示α-SMA抗体染色检测小鼠胚胎期第11.5至13.5天食道的平滑肌细胞的形状及定量分析，结果显示从小鼠胚胎期第11.5至13.5天食道的平滑肌细胞的核展弦比升高。图4g，4h和4i显示α-SMA抗体整体染色检测野生型和突变体小鼠胚胎期第14.5天的食道平滑肌和定量分析，结果显示突变体小鼠表现出食道平滑肌细胞排列异常。图4j和4k显示α-SMA和GM130抗体染色检测野生型和突变体小鼠胚胎期第14.5天的食道平滑肌细胞的极性和定量分析，结果显示突变体小鼠表现出食道平滑肌细胞极性的异常。图4l显示Phalloidin染色检测野生型和突变体小鼠胚胎期第14.5天的食道平滑肌细胞的骨架，结果显示突变体小鼠表现出食道平滑肌细胞骨架组装的障碍)。
图5为细胞外钾离子浓度的升高引起小鼠气道平滑肌细胞排列和细胞骨架组装的障碍示意图(图5a和5b显示DiBAC4(3)荧光检测氯化钾处理后小鼠气管平滑肌膜电势的变化和定量分析，结果显示氯化钾处理后小鼠气管平滑肌的膜电势呈现去极化。图5c和5d显示α-SMA抗体染色检测氯化钾处理后小鼠气管平滑肌和定量分析，结果显示氯化钾处理后小鼠表现出气管平滑肌面积缩小。图5e，5f，5g和5h显示α-SMA抗体和Phalloidin染色检测氯化钾处理后小鼠气管平滑肌及定量分析，结果显示氯化钾处理后小鼠表现出平滑肌细胞排列和形状的异常，以及细胞骨架组装的障碍。图5i，5j和5k显示α-SMA抗体染色检测氯化钾处理，氯化钾和ouabain联合处理后小鼠气管平滑肌和定量分析，结果显示ouabain可部分的挽救氯化钾处理引起的小鼠气管平滑肌的缺陷)。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例及其附图对本发明做进一步的详细描述。
实施例1构建Kir7.1基因失活突变小鼠模型
1、诱变：N-乙基-N-亚硝基脲诱导小鼠基因突变具体步骤如下：
用100mg/kg剂量的N-乙基-N-亚硝基脲腹腔连续注射C57BL/6J的雄性小鼠3次。10周后，将注射过N-乙基-N-亚硝基脲的小鼠与C57BL/6J的雌性小鼠交配产生G1代小鼠。G1代雄性小鼠与C57BL/6J雌性小鼠交配产生G2代雌性。将G2代雌性小鼠与其G1代雄性小鼠交配得到G3代的幼鼠进行气管和肺的分析来筛选突变体。
3、鉴定：全外显子测序分析具体步骤如下：
使用Agilent SureSelect Mouse All Exonkit V1捕获野生型和突变的小鼠外显子，并使用IlluminaHiSeq 2000测序。将序列读数与C57BL/6J小鼠基因组(mm10)做比对，并使用CLCBioGenomicWorkbench和GATK软件进行分析，找出突变体中基因的突变位点。
实施例2验证Kir7.1基因失活突变小鼠的表型
使用阿尔新蓝染色、Phalloidin染色、抗体染色以及荧光实时定量PCR的方法鉴定Kir7.1在气管发育过程中的表达模式和突变体小鼠的表型(实验中的抗体来源及稀释倍数：α-SMA(1:1000,Sigma-Aldrich,C6198)；anti-CDH1(1:500,Santa Cruz,sc-59778)；anti-GM130(1:50,R&D systems,AF8199)；anti-KIR7.1(1:50))；Alexa Fluor488Phalloidin(1:50,ThermoFisher Scientific，A12379)。
1、阿尔新蓝染色具体步骤如下：
分别将野生型、Kir7.1基因敲除以及Kir7.1基因失活小鼠的解剖的气管在95％乙醇中固定12小时，然后用溶解在80％乙醇和20％乙酸中的0.03％阿尔新蓝染色过夜，气管在2％KOH中清洗除去背景染色。
使用阿尔新蓝染色(Alcain blue)的方法检测小鼠气管发育和形成的异常。本发明观察到Kir7.1的失活能强烈抑制刚出生小鼠气管的形成，包括气管延长的障碍和软骨形成的缺陷。同时这样的表型在Kir7.1基因敲除的小鼠(Kir7.1-/-)里也被观察到。证明本发明所观察到的气管形成缺陷型的表性是特异性的由Kir7.1基因的失活造成的。请参阅图1，其显示Kir7.1基因的错译突变小鼠和Kir7.1基因敲除的小鼠均表现出气管延长的障碍和软骨形成的缺陷。
2、α-SMA抗体染色具体步骤如下：
分别将野生型、Kir7.1基因敲除的小鼠以及Kir7.1基因失活的小鼠的气管或食道组织在4％多聚甲醛中于4℃固定过夜，在10％蔗糖和30％蔗糖溶液中于4℃孵育24小时，OCT包埋，并做10μm厚度的冰冻切片。将切片在4％多聚甲醛中于4℃固定10分钟，然后在通透溶液(0.3％Triton X-100/PBS)中在室温下孵育15分钟，在封闭溶液(5％FBS/PBS/3％BSA)中孵育1小时，在一抗中于4℃孵育过夜，洗涤，在二抗体中于室温下孵育2小时，洗涤，在DAPI溶液中于室温下孵育10分钟，洗涤，然后封片用于成像。
α-SMA是平滑肌细胞的标记性蛋白。利用α-SMA抗体免疫染色的方法结合激光共聚焦显微镜成像分析，本发明检测了平滑肌细胞的空间排列，利用顺式高尔基体基质蛋白GM130抗体免疫染色的方法，检测了平滑肌细胞中高尔基体的位置，以此比较野生型和Kir7.1突变体中平滑肌细胞的极性。本发明观察到Kir7.1的失活导致了平滑肌细胞空间排列和细胞极性表现出随机化的趋势。这些结果表明Kir7.1介导的平滑肌异常是通过改变平滑肌细胞的空间排列和极性来实现。请参阅图2和图4，其显示Kir7.1基因突变的小鼠的平滑肌细胞表现出空间排列和细胞极性的异常。
3、整体组织抗体染色具体步骤如下：
分别将野生型、Kir7.1基因敲除以及Kir7.1基因失活小鼠的气管或食道在4％多聚甲醛中于4℃固定过夜，在通透溶液(0.3％Triton X-100/PBS)中在室温下孵育过夜，在封闭溶液(5％FBS/PBS/3％BSA)中孵育4小时，在一抗中于4℃孵育过夜，洗涤，在二抗体中于4℃孵育过夜，洗涤，样品保存于50％的甘油中用于成像。
利用抗体免疫染色的方法结合激光共聚焦显微镜成像分析，检测气管上皮细胞分化的标记性蛋白Acetylatedα-Tubulin，CC10和KRT5的表达，比较野生型和Kir7.1突变体中上皮细胞的分化状态。本发明发现Kir7.1基因突变引起了Acetylatedα-Tubulin标记的纤毛细胞数量的减少，而club细胞的标记蛋白CC10和基底细胞的标记蛋白KRT5表达正常，从而证明了Kir7.1基因特异性的介导小鼠气管纤毛细胞的分化。请参阅图3，其显示Kir7.1基因突变小鼠的气管上皮纤毛细胞分化的标记性蛋白Acetylatedα-Tubulin的量显著下降。
4、Phalloidin染色具体步骤如下：
分别将野生型，Kir7.1基因失活小鼠或氯化钾处理的气管或食道在4％多聚甲醛中于4℃固定过夜，在通透溶液(0.3％Triton X-100/PBS)中在室温下孵育过夜，在封闭溶液(5％FBS/PBS/3％BSA)中孵育4小时，在Alexa Fluo488Phalloidin溶液中于4℃孵育过夜，洗涤，样品保存于50％的甘油中用于成像。
针对细胞骨架，利用Phalloidin染色检测F-肌动蛋白在Kir7.1突变体小鼠气道和食道平滑肌中的量。本发明发现Kir7.1的失活能引起气管平滑肌细胞中F-肌动蛋白的量降低。同时升高平滑肌细胞外的钾离子浓度可引起小鼠气道平滑肌面积降低，排列异常，细胞形状改变以及细胞骨架紊乱，其表型类似于Kir7.1突变体，另外利用可以降低细胞内钾离子浓度的化合物ouabain处理，可以部分挽救氯化钾处理引起的平滑肌缺陷。请参阅图2和图5，其显示Kir7.1突变体小鼠的气管平滑肌表现出细胞骨架组装的缺陷，氯化钾处理导致类似于Kir7.1突变体的表型，而ouabain处理部分挽救了氯化钾处理引起的表型。
5、荧光实时定量PCR具体步骤如下：
使用miRNeasy Mini Kit(Qiagen)提取气管的总RNA，利用Maxima First StrandcDNA Synthesis Kit(ThermoFisher Scientific)合成cDNA，使用Eco实时PCR系统(Illumina)和Maxima SYBR Green/Fluorescein qPCR Master Mix试剂(ThermoFisherScientific)进行实时定量PCR的检测。反应程序如下：步骤一，55℃，2分钟；步骤二，95℃，10分钟；步骤三95℃，10秒；步骤四，60℃，30秒；从步骤三到步骤四循环40次；步骤五，95℃，15秒；步骤六，55℃，15秒，步骤七，95℃，15秒。
荧光实时定量检测出Kir7.1mRNA在野生型小鼠气管中的表达量。结果显示Kir7.1mRNA在小鼠胚胎期各阶段的气管中稳定表达，请参阅图1i。
6、乙酰胆碱诱导气管收缩的具体步骤：
将分离到的气管制作成2mm厚的气管环并保持在Krebs溶液(119mM NaCl，4.7mMKCl，2.5mM CaCl2，1.17mM MgSO4，20mM NaHCO3，1.18mM KH2PO4，0.027mM EDTA，11mM葡萄糖)中。将气管环安装在线状肌电图系统(610-M，Danish Myo Technology)上，并对于每个气管环施加2mN的静息张力。通过加入乙酰胆碱来诱导气管收缩，同时记录气管的收缩力。
为了检测Kir7.1基因对气管生理上的影响，本发明检测了气管在乙酰胆碱诱导条件下的收缩。实验中，气管装在金属肌动扫描仪上，在通入氧气37℃的条件下，记录气管的收缩力。本发明观察到，乙酰胆碱诱导条件下，Kir7.1突变小鼠的气管收缩力很大程度的降低。利用Kir7.1的抑制剂VU590处理后，野生小鼠气管同样表现出收缩力的降低。洗除VU590后，小鼠气管的收缩力基本恢复到未加药前水平，表明VU590对气管收缩的抑制呈现可逆性。请参阅图2，其显示在乙酰胆碱诱导下，Kir7.1突变体小鼠气管和VU590处理的野生型气管的收缩均受到削弱，VU590洗除后，野生型小鼠气管的收缩基本恢复。
综上所述，本发明构建的Kir7.1基因失活小鼠具有气管缩短和软骨环断裂，气管平滑肌排列缺陷和面积减小，气管平滑肌表现出细胞骨架组装障碍和收缩力降低，气管上皮细胞分化缺陷以及其食管形成缺陷的表型。通过上述表型本发明的小鼠动物模型可用于筛选治疗具有上述气管和食道缺陷的药物。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
SEQUENCE LISTING
<110> 广州医科大学
<120> 一种Kir7.1基因失活的小鼠模型及其构建方法
<130> 7.8
<160> 2
<170> PatentIn version 3.3
<210> 1
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taa 1083
Claims (8)
1.一种Kir7.1基因失活的小鼠模型，其特征在于，所述小鼠模型Kir7.1基因的第13位的亮氨酸突变为脯氨酸，使Kir7.1基因失活。
2.如权利要求1所述的Kir7.1基因失活的小鼠模型，其特征在于，所述失活Kir7.1基因编码如SEQ ID NO：1所示的氨基酸序列。
3.如权利要求2所述的Kir7.1基因失活的小鼠模型，其特征在于，所述Kir7.1基因的核苷酸序列如SEQ ID NO：2所示。
4.一种Kir7.1基因失活的小鼠模型的构建方法，其特征在于，包括以下步骤：用N-乙基-N-亚硝基脲腹腔注射雄性小鼠；5～10周后，将其与雌性小鼠交配产生G1代小鼠；G1代雄性小鼠与雌性小鼠交配产生G2代雌性；将G2代雌性小鼠与G1代雄性小鼠交配得到的G3代幼鼠进行气管检测和全外显子测序分析来筛选和鉴定Kir7.1基因失活小鼠。
5.如权利要求4所述的小鼠模型的构建方法，其特征在于，所述小鼠为C57BL/6J小鼠。
6.如权利要求4所述的小鼠模型的构建方法，其特征在于，所述N-乙基-N-亚硝基脲的浓度为100mg/kg；腹腔连续注射3次。
7.如权利要求4所述的小鼠模型的构建方法，其特征在于，气管检测包括：检测小鼠的气管、软骨、平滑肌细胞、细胞骨架以及上皮细胞的异常情况。
8.如权利要求1-3任一项所述的Kir7.1基因失活的小鼠模型在筛选治疗气道软骨缺陷的药物中的用途。
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| CN114630579A (zh) * | 2019-11-05 | 2022-06-14 | 弗尔斯蒂血液研究所基金会股份有限公司 | 胎儿/新生儿同种免疫性血小板减少症的鼠模型 |
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