CN113981000A - 一种COL4A3 p.P408H基因点突变小鼠模型及其构建方法、应用和试剂盒 - Google Patents
一种COL4A3 p.P408H基因点突变小鼠模型及其构建方法、应用和试剂盒 Download PDFInfo
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Abstract
本发明公开了一种COL4A3p.P408H基因点突变小鼠模型及其构建方法、应用和试剂盒。其包括以下步骤:(1)设计gRNA靶点序列;(2)将Cas9nickasemRNA、靶点序列以及Donor DNA混合物对受精卵进行注射;(3)在小鼠出生后进行基因鉴定,获得COL4A3p.P408H基因点突变小鼠模型,然后再利用高脂喂养联合腹腔注射链脲佐菌素构建糖尿病肾病小鼠模型。本发明利用CRISPR/Cas9技术剪切COL4A3基因的DNA,同时提供带有点突变的Donor同源模板,通过DNA的同源重组修复在特定外显子实现碱基替换,实现点突变,并以C57BL/6J小鼠为背景构建杂合型点突变小鼠模型。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种COL4A3 p.P408H基因点突变 小鼠模型及其构建方法、应用和试剂盒。
背景技术
随着人们生活方式与饮食结构的变化,糖尿病已经成为威胁全球健康的慢 性非传染性疾病之一,据2016年世界卫生组织发布的“全球糖尿病报告”统计, 截止2014年,糖尿病患病人数为4.22亿人,预计到2030年,全球将有4.39亿 糖尿病患者。糖尿病的激增,必将带来糖尿病并发症的大流行,糖尿病肾病 (Diabetic Kidney Disease,DKD)是糖尿病最常见的微血管并发症之一,是西方终 末期肾脏疾病(End-Stage Kidney Disease,ESKD)的首要原因;我国2011-2015 年的流行病学调查显示糖尿病合并慢性肾脏疾病(ChronicKidney Disease,CKD) 的患者达2430万,DKD已经超过肾小球肾炎,成为CKD的首要原因,ESKD 的第二位病因,DKD及ESKD所导致的肾脏替代治疗将给国家带来极其沉重的 卫生经济负担。
DKD发生及进展的病理生理机制复杂,它是遗传背景、环境因素及二者交 互作用的结果。环境因素包括饮食习惯、吸烟、血糖、血压、血脂等,严格管 控可控的危险因素降低了糖尿病患者心血管事件的发生,却未能有效地控制 DKD的发病率及其导致的ESKD。既往研究发现,在治疗原则相同的条件下, DKD临床表现有较强的异质性,呈现出不同的发展特点,部分患者肾功能可以 长期稳定,部分患者肾功能缓慢下降,而部分患者在血糖血压等控制达标的情 况下,肾功能仍然快速下降。目前,指示DKD进展的生物标记物仍以尿白蛋白和估算肾小球滤过率(estimated Glomerular Filtration Rate,eGFR)为主,但二者出 现明显改变时,已经错过最佳干预时机,寻找更有效的生物标记物早期识别和 分层管理DKD患者,并且早期、恰当、精准干预,合理配置医疗资源,也是有 效改善这部分患者预后的重要措施。
遗传信息是患者稳定携带且易于检测的,如果能找到与DKD发生发展相关 的遗传特征,可以成为早期识别和分层管理DKD人群的有效生物标记物。近年 来,DKD家族聚集现象及不同人种间临床表型的差异都提示遗传背景在DKD 发生发展中的重要作用。基于糖尿病家系的FIND(the Family Investigation of Nephropathy and Diabetes consortium)研究通过全基因连锁分析发现了一些可能 与DKD发生以及进展至ESKD相关的基因突变;同时,基于糖尿病人群的全基 因组关联分析也识别到多个与DKD肾脏表型相关的突变,然而,这些突变对 DKD的影响在不同队列难以重复,且尚未有研究在细胞或者动物水平验证这些突变在DKD模型中具体作用机制。
科学家们历来认为,探索DKD的遗传密码是DKD研究领域的“噩梦”。首 先,糖尿病本身的分型、遗传背景就极其复杂,DKD作为糖尿病的并发症,二 者“基因-基因”的交互作用更是难以分辨;其次,DKD患者往往合并肥胖、高血 压等其它代谢性疾病,这些因素如何影响或者修饰基因的表达未知使得“基因- 环境”的交互作用也需要纳入考虑;加之各研究对糖尿病合并慢性肾脏病与糖尿 病肾病的定义不统一,导致糖尿病伴其它肾病的患者纳入部分研究造成偏倚, 我们前期研究通过肾活检病理诊断发现37%的糖尿病伴CKD的患者其实是糖尿 病合并其它非糖尿病肾病,缺乏肾活检金标准诊断DKD可能对“基因型-肾脏临床表型”的相关性分析造成了偏倚;以上“基因-基因”的相互作用、“基因-环境” 的相互影响及“基因-表型”的相关偏倚使研究DKD的遗传易感性面临着巨大挑 战。
发明内容
针对现有技术中的上述不足,本发明提供一种COL4A3 p.P408H基因点突变 小鼠模型及其构建方法、应用和试剂盒,本发明采用CRISPR/Cas9基因编辑技 术构建全身COL4A3基因p.P408H杂合及纯合点突变的小鼠,以高脂饮食联合 链脲佐菌素的方法将野生型及突变型小鼠诱导为早期DKD模型,从肾脏形态学、 病理学、功能学、分子机制等层面观察该突变对早期DKD肾脏损伤的影响。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
利用CRISPR/Cas9技术剪切COL4A3基因的DNA,同时提供带有点突变的 Donor同源模板,通过DNA的同源重组修复在特定外显子实现碱基替换,实现 点突变,并以C57BL/6J小鼠为背景构建杂合型点突变小鼠模型。
一种COL4A3 p.P408H基因点突变小鼠模型的构建方法,包括以下步骤:
(1)点突变位置确定:由于人类第408位氨基酸为精氨酸(R),而小鼠为脯 氨酸(P),故本研究构建的全身点突变小鼠为COL4A3 p.P408H点突变,该点突 变位于21号外显子,故在21号外显子及两端内含子上分别设计gRNA靶点进 行基因组DNA的剪切;
(2)设计gRNA靶点:其靶点序列如SEQ ID NO.1、SEQ ID NO.2,或SEQ ID NO.3所示;
(3)将Cas9 nickase mRNA、靶点序列以及Donor DNA(SEQ ID NO.4) 混合物对受精卵进行注射;
(4)在小鼠出生后进行基因鉴定,获得COL4A3 p.P408H基因点突变小鼠 模型。
进一步地,点突变位于21号外显子,由脯氨酸突变为组氨酸。
进一步地,基因鉴定的过程为:抽提小鼠DNA,并采用引物COL4A3-F/R 进行PCR检测,以鉴定其是否突变完成。
进一步地,PCR反应体系为:0.5μL KODFX、5μL DNTPs、12.5μL 2x Buffer、2μLDNA、1μL COL4A3-F、1μL COL4A3-R,最后用H2O补足至25 μL。
进一步地,PCR反应程序为:94℃预变性2min;98℃变性10s;60℃退火 30s;68℃延伸30s,共35个循环;68℃延伸10min,最后16℃延伸2min。
一种COL4A3 p.P408H基因点突变杂合小鼠模型的构建方法,具体过程如 下:将上述构建得到的纯合点突变小鼠F0与野生型小鼠进行杂交培育得到F1 代,即为点突变杂合小鼠模型。
进一步地,野生型小鼠为野生型C57BK/6J。
上述方法构建的COL4A3 p.P408H基因点突变小鼠模型,COL4A3 p.P408H 基因点突变杂合/纯合型小鼠模型在糖尿病防治中的应用。
进一步地,糖尿病包括但不限于糖尿病肾病。
进一步地,对构建的小鼠模型采用高脂喂养联合腹腔注射链脲佐菌素,构 建得到糖尿病肾病模型。
一种构建COL4A3 p.P408H基因点突变小鼠模型的试剂盒,包括如SEQ ID NO.1、SEQ ID NO.2,或SEQ ID NO.3所示的gRNA靶点序列、Cas9 nickase mRNA,以及Donor DNA。
本发明的有益效果:
与野生型早期DKD小鼠相比,本发明构建的杂合及纯合型突变DKD小鼠 尿ACR增多、血清肌酐更高、肾脏病理改变更重、GBM出现厚薄不均、足突 融合等病理改变,且纯合型突变DKD小鼠上述肾脏表型较杂合型突变的DKD 小鼠严重,这一结果与临床现象一致。本发明首次证实COL4A3 p.P408H全身杂 合/纯合型点突变是加速DKD的进展的遗传背景,这有望成为识别DKD高风险 人群的重要新型稳定的生物标志物。
附图说明
图1为全身点突变小鼠模型的构建过程;
图2为gRNA靶点弧形检测结果;
图3为小鼠突变检测结果;其中,图3a为琼脂糖凝胶电泳检测结果,图中 M:BM2000Marker;NC:阴性对照;WT:野生型;图3b为测序结果;
图4为小鼠肾脏HE染色检测结果;
图5为小鼠肾脏MASSON染色检测结果;
图6为小鼠肾脏PAS染色检测结果;
图7为小鼠肾脏TUNEL染色检测结果;
图8为小鼠肾脏电镜检测;其中,图8a为损伤情况检测;图8b为厚度检 测;
图9为小鼠肾脏IV型胶原α3链表达量检测结果;
图10为小鼠肾脏内质网应激指标检测结果;
图11为小鼠肾脏细胞凋亡情况检测结果;
图12为小鼠肾脏组织纤维化检测结果;
图13为小鼠肾脏组织炎症相关指标检测结果。
具体实施方式
下面对本发明的具体实施方式进行描述,以便于本技术领域的技术人员理 解本发明,但应该清楚,本发明不限于具体实施方式的范围,对本技术领域的 普通技术人员来讲,只要各种变化在所附的权利要求限定和确定的本发明的精 神和范围内,这些变化是显而易见的,一切利用本发明构思的发明创造均在保 护之列。
实施例1构建全身点突变纯合/杂合小鼠模型
1、点突变位置确定:由于人类第408位氨基酸为精氨酸(R),而小鼠为脯氨 酸(P),故本研究构建的全身点突变小鼠为COL4A3 p.P408H点突变,该点突变 位于21号外显子,故在21号外显子及两端内含子上分别设计gRNA靶点进行 基因组DNA的剪切。
2、Cas9/gRNA靶点设计:共设计三个靶点备选,采用该公司靶点效率检测 盒(货号:VK007)进行活性分析,靶点序列如表1所示,靶点活性检测如图2所 示。
表1 gRNA靶点活性分析
由表1和图2的检测结果可知,构建的三个靶点序列活性均大于75%。
3、COL4A3 p.P408H全身点突变小鼠的构建:将Cas9nickase mRNA、Cas9 靶点gRNA与donor DNA共同显微注射至小鼠受精卵,此种方法在受精卵发育 早期就发生基因敲入,故小鼠嵌合率较高。
4、小鼠出生后两周抽提鼠尾基因组鉴定基因型,PCR检测是否获得发生基 因点突变的首建鼠F0。抽提小鼠DNA方法如下:
剪下小鼠尾巴尖部约4mm,放入无菌1.5mL EP管中做好标记,每管加入 60μL鼠尾裂解液(NaOH,PH=12),煮沸40min后加入180μLTris-HCl(PH=3), 震荡反应。提取3μL的DNA提取液,加入20μL的PCR反应体系中,引物具 体序列如表2所示,采用表3的PCR体系和反应条件进行检测,琼脂糖凝胶电 泳检测突变是否成功(图3)。
表2引物序列
表3 PCR反应体系和反应条件
如图3a中箭头所示,PCR样品条带大小约为365bp,该条带为扩增的目的 条带,根据图3b中通过峰形分析,可以看出CCTGGAAAAGACACT前的CCC 突变为CAT,即p.P408H。
5、点突变小鼠的繁育:将F0代首建鼠(雌性)与野生型C57BL/6J(雄性)进行 交配,按照上述方法鉴定小鼠基因型,获得目标小鼠。
实施例2 DKD模型的建立
1、SPF级小鼠饲养于恒定温度(21-25℃)、湿度(40-70%)环境中,日光灯模 拟规律昼夜交替,适应性饲养一周后,根据实验需求按随机分配的原则将小鼠 分为上述六组。各组小鼠饮水、饮食定期更换,小鼠血糖升高后每天更换饮水 及垫料保证鼠笼干燥清洁。
2、DKD组小鼠适应性喂养一周体重约16-20g时,更换为高脂饲料继续喂 养4周。注射STZ前应禁食12小时,不禁水,将配制好的STZ注射液按40mg/kg 的剂量腹腔注射,连续5天,注意应在30分钟内完成避免STZ失效。注射完成 7天后使用血糖仪测量小鼠尾静脉血糖水平,当随机血糖连续>16.7mmol/L时则 认为糖尿病模型造模成功,后每两周复测血糖及体重。
实施例3 DKD小鼠模型主要血液生化指标及尿ACR检测
收集标本前禁食12小时,不禁水,留取尿液及血液标本,尿液标本收集后 迅速离心(3000rpm,5分钟)取上清,检测ACR;根据小鼠体重腹腔注射水合氯 醛麻醉,采用摘取眼球取血法,收集血液标本后冰上静置2-3小时离心(3500rpm, 15分钟)取上清,检测血肌酐、尿素、甘油三酯、胆固醇等指标,以上样品送于 国家成都中药安全性评价中心和成都里来生物科技有限公司检测。其结果如下 (详见表4):
表4各组小鼠生化指标及尿液ACR
如表4数据所示:
1、血糖:各DKD组小鼠血糖高于非DKD组小鼠,与COL4A3+/++DKD组 相比,COL4A3+/Δ+DKD组和COL4A3Δ/Δ+DKD组血糖无差异。
2、血清肌酐:与COL4A3+/+组相比,COL4A3+/++DKD组血清肌酐无显著变 化;COL4A3+/Δ及COL4A3Δ/Δ组小鼠血清肌酐轻度增高,两组间比较无差异。与 COL4A3+/++DKD组相比,COL4A3+/Δ+DKD和COL4A3Δ/Δ+DKD组小鼠血清肌 酐显著上升,且COL4A3Δ/Δ+DKD组增幅大于COL4A3+/Δ+DKD组。
3、尿ACR:与COL4A3+/+组相比,COL4A3+/++DKD组小鼠尿ACR增加, 出现早期DKD肾脏表型;COL4A3+/Δ及COL4A3Δ/Δ组尿ACR增多,两组间比较 无差异。与COL4A3+/++DKD组相比,COL4A3+/Δ+DKD和COL4A3Δ/Δ+DKD组 小鼠尿ACR显著上升,两组间比较无差异。
根据上述检测结果可知:COL4A3+/Δ组及COL4A3Δ/Δ组血清肌酐和尿ACR 水平无差异;COL4A3+/Δ+DKD和COL4A3Δ/Δ+DKD组小鼠肾脏临床表型较 COL4A3+/++DKD组严重,主要表现为血清肌酐升高且尿ACR增多,其中 COL4A3Δ/Δ+DKD组血清肌酐高于COL4A3+/Δ+DKD组。
实施例4肾脏组织病理学检测
肾脏组织处理:小鼠麻醉后,开腹取出肾脏,剥去肾脏薄膜,将两个肾脏 分为四份分别置于4%多聚甲醛用于石蜡包埋、2.5%戊二醛固定用于电镜,余组 织分装置于冻存管中存于-150℃冰箱或液氮用于分子实验及备用。所有动物实验 均符合国家《动物实验管理条例》,且实验程序获得华西医院实验动物伦理委员 会批准。
4%多聚甲醛固定组织约24小时,自来水清洗固定液后采用浓度为70%、 80%、90%、100%的乙醇梯度脱水,再进行透明、浸蜡包埋为组织蜡块,并用 常规切片机自蜡块切取6-7μm的切片。实验前将石蜡切片放入60℃烘箱30分钟, 使蜡融化。
1、肾脏形态学改变检测,采用苏木素-伊红(Hematoxylin-Eosin,HE)染色进 行,其检测结果见图4(图4中+:wild type;Δ:c.1223G>A p.P408H;DKD: 糖尿病肾病,下同),具体过程如下:
1)脱蜡复水:将切片放入二甲苯I、II、III液中各浸泡5分钟,再依次将 其放入90%、80%、70%浓度的乙醇中各浸泡5分钟后双蒸水清洗2遍。
2)切片放入苏木素中染色10分钟后双蒸水清洗2遍。
3)分化:切片放入1%盐酸酒精液中5-10秒。
4)蓝化:清水冲洗数分钟镜下可见切片恢复蓝色。
5)复染:0.5%伊红染色5分钟,双蒸水清洗2-3遍。
6)脱水:依次将放切片放入70%、80%、90%、100%浓度的乙醇中各浸泡 5分钟。
7)透明:切片依次放入二甲苯I、II液中各浸泡5分钟。
8)封片:中性树胶封片。
如图4所示,与各非DKD组小鼠肾小球相比,各DKD组小鼠肾小球体积 增大、肾小球内细胞数增多,肾小管上皮细胞出现明显水肿空泡变性;与 COL4A3+/+组相比,COL4A3+/Δ和COL4A3Δ/Δ组小鼠肾小球大小和肾小管形态基 本正常;与COL4A3+/++DKD组相比,COL4A3+/Δ+DKD和COL4A3Δ/Δ+DKD组 小鼠肾小球内细胞数增多,肾小管上皮细胞水中空泡变性加重,其中COL4A3Δ/Δ +DKD组肾小球形态受损及肾小管空泡变性最严重。
2、肾组织纤维化程度检测,采用PAS(Periodic Acid-Schiff,PAS)染色进行, 检测结果如图5所示,具体过程如下:
1)脱蜡复水:同上述。
2)切片放入1%过碘酸氧化10分钟后双蒸水清洗2遍。
3)滴加Schiff染液,着色10分钟后蒸水清洗2遍。
4)放入苏木素中浸泡2分钟后蒸水清洗2遍。
5)分化:切片放入1%盐酸酒精液中5-10秒后清洗。
6)氨水返蓝清洗后镜下观察组织颜色。
7)脱水、透明、封片。
如图5所示,与COL4A3+/+组相比,COL4A3+/++DKD组小鼠肾脏纤维化轻 度增高,COL4A3+/Δ及COL4A3Δ/Δ组小鼠肾脏无明显纤维化表达;与COL4A3+/+ +DKD组相比,COL4A3+/Δ+DKD和COL4A3Δ/Δ+DKD组小鼠纤维化水平明显 升高,其中COL4A3Δ/Δ+DKD组小鼠肾小球及间质纤维化面积最大。
3、肾组织糖原沉积检测,采用Masson染色进行,检测结果如图6所示, 具体过程如下:
1)按上述步骤脱蜡复水、苏木素染色、分化、返蓝。
2)将切片置入Masson染料着色约10分钟,清洗2遍。
3)滴加1%磷钼酸溶液3-5分钟,倾斜玻片吸弃。
4)滴加1%苯胺蓝染色液5分钟,清洗2遍。
5)置入1%冰醋酸水溶液中数秒后清洗。
6)脱水、透明、封片。
如图6所示,与COL4A3+/+组相比,COL4A3+/++DKD组糖原沉积略有上升, COL4A3+/Δ及COL4A3Δ/Δ组小鼠出现肾脏糖原沉积增多,其中COL4A3Δ/Δ组小鼠 沉积更明显;与COL4A3+/++DKD组小鼠相比,COL4A3+/Δ+DKD和COL4A3Δ/Δ +DKD组小鼠肾脏系膜区糖原沉积明显增高,但两组间比较无显著差异。
4、肾组织细胞凋亡情况检测采用脱氧核糖核苜酸末端转移酶介导的缺口末 端标记法染色(TUNEL)进行,检测结果如图7所示,具体过程如下:
1)按上述步骤脱蜡复水,PBS清洗5分钟,重复两次。
2)滴加20μg/mL的蛋白酶K溶液,放置于37℃孵箱10分钟,PBS清洗5 分钟,重复两次。
3)滴加TUNEL反应混合液,避光放置入37℃孵箱中1小时,PBS清洗5 分钟,重复两次。
4)滴加DAPI染液,避光室温孵育5分钟,PBS清洗5分钟,重复两次。
5)封片后及时观察凋亡情况。
如图7所示,COL4A3+/+、COL4A3+/++DKD和COL4A3+/Δ组小鼠肾脏均未 观察到凋亡细胞;COL4A3Δ/Δ及COL4A3+/Δ+DKD组小鼠肾脏出现少量凋亡细胞, COL4A3Δ/Δ+DKD组小鼠肾脏则出现大量凋亡细胞。
5、透射电镜观察肾脏组织,其结果见图8,具体过程如下:
1)肾脏组织取材后放入3%戊二醛中固定过夜。
2)磷酸缓冲液漂洗10分钟,重复两次。
3)1%锇酸固定液固定2小时,磷酸缓冲液漂洗10分钟,重复两次。
4)丙酮梯度脱水后包埋、超薄切片。
5)室温下醋酸铀染色10-15分钟,枸橼酸铅染色1-2分钟。
6)透射电镜定位肾小球观察采集图片及分析。
图8a为)透射电镜观察各组小鼠肾脏超微结构,红色(图中未显)*指示足 突融合部位,箭头指示GBM增厚位置。图8b为GBM厚度测量统计分析, *P<0.05,**P<0.01****P<0.0001。
如图8所示,①COL4A3+/+组小鼠GBM厚薄均匀(129.3±1.50nm),足细胞足 突规律整齐;与COL4A3+/+组相比,COL4A3+/++DKD组小鼠GBM增厚 (129.3±1.50nm vs.212±9.65nm,P<0.0001),但未见明显足突融合; COL4A3+/Δ(128.7±3.58nm)、COL4A3Δ/Δ(123.1±2.10nm)组小鼠GBM厚薄未见明显 变化,但开始出现部分足突融合。
②分别与COL4A3+/Δ、COL4A3Δ/Δ组GBM厚度相比,COL4A3+/Δ +DKD(128.7±3.58nmvs.190.3±33.52nm,P<0.0001)、COL4A3Δ/Δ+DKD组 (123.1±2.10nm vs.212±9.65nm,P<0.0001)小鼠GBM都增厚。
③与COL4A3+/++DKD组GBM厚度(212±9.65nm)相比,COL4A3+/Δ+DKD 组小鼠GBM厚度无显著差异(212±9.65nm vs.190.3±33.52nm,P<0.01),但出现 厚薄不均,足突融合增多;COL4A3Δ/Δ+DKD组小鼠GBM厚度无显著差异(212±9.65nm vs.202.7±8.42,P>0.05),但GBM出现明显厚薄不均,“花篮状”改 变,足突融合增多。
④与COL4A3+/Δ+DKD组小鼠相比,COL4A3Δ/Δ+DKD组小鼠GBM厚度无 差异,但厚薄不均更加显著。
根据上述病理学检测可知,COL4A3+/Δ及COL4A3Δ/Δ组小鼠出现较轻微肾脏 病理变化,而COL4A3+/Δ+DKD、COL4A3Δ/Δ+DKD组肾脏病理形态、纤维化程 度、糖原沉积、细胞凋亡及超微结构(GBM厚度和足突融合)都较COL4A3+/+ +DKD组严重。
实施例4携带COL4A3 p.P408H点突变早期DKD小鼠肾脏IV型胶原α3链表 达
1、Western Blot蛋白表达检测
1)配制SDS-PAGE凝胶:双蒸水洗净和烘干玻璃板,安装玻璃板,注意 不能漏胶,根据所检测蛋白分子量选择适宜浓度的胶,按照说明书配制分离胶 混匀后注入玻璃板中,配制浓缩胶混匀注入后插上梳子,注意梳子和胶之间不 留有气泡,等待至少30分钟后取出待用。
2)蛋白上样及电泳:将胶板固定于电泳装置上,加满电泳液,观察是否漏 液,缓慢从两侧拔出梳子,加入3-5μL适宜的蛋白marker后按顺序加入样品, 如各组蛋白上样体积差距较大则加入1X Loading Buffer补齐,避免电泳不齐。 按照电极方向,正确安装电泳装置,接通电源,首先使用80V恒压使蛋白跑至 分离胶(约20分钟),调换电压至100-120V,当溴酚蓝染料到达分离胶底部时, 电泳完成。
3)转膜:将胶从电泳装置中取出,根据溴酚蓝提示去除多余凝胶,据凝胶 大小裁剪孔径为0.22μm的PVDF膜后完全浸润入甲醇中活化20-30秒待用;准 备转膜专用盆,倒入预冷的转膜液,将转膜夹子打开且黑色面置于盆底,按如 下顺序铺一层海绵垫,2张转膜液湿润过的转膜滤纸,电泳凝胶、激活的PVDF 膜、2张转膜液湿润过的转膜滤纸、一层海绵垫;注意每层之间应互相对齐,用 滚轮反复压走凝胶与PVDF膜之间的气泡;之后夹闭转膜夹,正确放置在转膜 槽中,加满转膜液,放置一块冰盒放置转膜温度过高,接通电源,恒流转膜。 一般以250mA,转膜1.5h-2h左右,且需将转膜槽置于含冰水混合物的盆中。
4)封闭:转膜结束后将PVDF膜取出,放置于混匀的封闭液中,根据目标 蛋白的分子量大小,将膜裁剪为相应条带,做好标记,室温摇床上封闭1h。
5)一抗的孵育:据抗体提供的说明书用TBST稀释抗体并混匀,转移入抗 体孵育盒中,将相应条带放入其中,注意避免使条带粘粘,影响孵育效果;将 抗体孵育盒放置入4℃冰箱的摇床中过夜。
6)二抗的孵育:取出一抗条带,用TBST洗膜5分钟,重复2次;按照说 明书比例用TBST稀释二抗,将对应的条带放入二抗孵育盒中,37℃摇床上孵 育1h;之后用TBST洗膜10分钟,重复两次。
7)条带曝光:按说明书配制ECL显影发光液,并注意避光;将条带表面的 水拭干后放置于曝光仪载物台上,均匀的将曝光液滴上条带,进行曝光。
8)每个指标重复三次,使用Image J对条带进行半定量分析。
首先确定各组小鼠IV型胶原α3链的表达,根据Western Blot检测结果可知: 与COL4A3+/+组小鼠相比,COL4A3+/Δ、COL4A3Δ/Δ组小鼠在正常或DKD状态下 IV型胶原α3链表达无明显差异(图9)。图9中柱状图为α3链蛋白较β-Tubulin 相对表达量统计。
实施例5携带COL4A3 p.P408H点突变早期DKD小鼠肾脏组织内质网应激相 关指标表达上调
提取各组小鼠肾脏总蛋白检测各组内质网应激指标,Western Blot检测(同 实施例4)显示:与COL4A3+/+组相比,COL4A3+/++DKD组ATF6和p-eIF2α表 达增高;COL4A3+/Δ组仅ATF6略有上调,COL4A3Δ/Δ组Bip、xBP1表达增高; 与COL4A3+/++DKD组相比,COL4A3+/Δ+DKD组Bip、ATF4、ATF6、xBP1及 p-eIF2α都显著增高,COL4A3Δ/Δ+DKD组Bip、ATF4、xBP1、p-PERK、及p-eIF2α 表达都显著上调,其中COL4A3Δ/Δ+DKD组p-eIF2α表达高于COL4A3+/Δ+DKD 组(图10)。
图10中,柱状图为蛋白较β-Tubulin相对表达量统计。*P<0.05vs.COL4A3+/+, **P<0.01vs.COL4A3+/+,***P<0.001vs.COL4A3+/+,****P<0.0001vs.COL4A3+/+; #P<0.05vs.COL4A3+/++DKD,##P<0.01vs.COL4A3+/++DKD,###P<0.001vs. COL4A3+/++DKD,$$P<0.01vs.COL4A3+/Δ+DKD。
以上结果表示:非DKD模型中,携带杂合型突变小鼠内质网应激指标无明 显增加,纯合型突变小鼠开始增加;而在早期DKD模型中,小鼠肾脏发生轻度 内质网应激,且携带杂合、纯合型突变会引起内质网应激指标明显上调。
实施例6携带COL4A3 p.P408H点突变早期DKD小鼠肾脏细胞凋亡指标上调
内质网持续应激后可导致凋亡,Western Blot检测(同实施例4)显示:与 COL4A3+/+组相比,COL4A3+/++DKD组出现Bax/Bcl2和CHOP表达上调,而 COL4A3+/Δ组、COL4A3Δ/Δ组肾脏组织的凋亡相关指标都无表达;与COL4A3+/+ +DKD相比,COL4A3+/Δ+DKD组Cleaved-caspase3和CHOP表达增强, COL4A3Δ/Δ+DKD组Bax/Bcl2、Cleaved-caspase3和CHOP都显著增加,其中, COL4A3Δ/Δ+DKD组Bax/Bcl2高于COL4A3+/Δ+DKD组(图11)。
图11中,柱状图为蛋白较β-Tubulin相对表达量统计。****P<0.0001vs. COL4A3+/+;#P<0.05vs.COL4A3+/++DKD,$P<0.05vs.COL4A3+/Δ+DKD。
以上结果说明,仅携带杂合及纯合型突变小鼠未引起肾脏凋亡蛋白表达增 加,而在早期DKD模型中即有细胞凋亡出现,且携带杂合、纯合型突变的DKD 小鼠肾脏细胞凋亡相关指标显著增高。
实施例7携带COL4A3 p.P408H点突变早期DKD小鼠肾脏组织纤维化相关指 标表达增加
Western Blot检测(同实施例4)结果显示:与COL4A3+/+组相比,COL4A3+/+ +DKD组纤维化指标α-SMA和TGFβ表达上调,COL4A3+/Δ组及COL4A3Δ/Δ组 表达水平无显著变化;与COL4A3+/++DKD组相比,COL4A3+/Δ+DKD组TGFβ 表达增加,COL4A3Δ/Δ+DKD组MMP9、TGFβ表达都显著增高,但α-SMA表 达无明显差异;COL4A3+/Δ+DKD组与COL4A3Δ/Δ+DKD组纤维化相关指标无显 著差异(图12)。图12中柱状图为蛋白较β-Tubulin相对表达量统计。*P<0.05vs.COL4A3+/+,**P<0.01vs.COL4A3+/+;#P<0.05vs.COL4A3+/++DKD,##P<0.01vs. COL4A3+/++DKD。
以上结果提示,携带杂合及纯合型突变的早期DKD小鼠肾脏的纤维化相关 指标表达明显增高。
实施例8携带COL4A3 p.P408H点突变早期DKD小鼠肾脏组织炎症相关指标 表达增加
Western Blot检测(同实施例4)显示:与COL4A3+/+相比,COL4A3+/++DKD 组炎症相关因子TNFR、TNFα、IL-6表达都上调,COL4A3+/Δ组及COL4A3Δ/Δ 组小鼠炎症指标无明显变化;与COL4A3+/++DKD组相比,COL4A3+/Δ+DKD及 COL4A3Δ/Δ+DKD组TNFR、TNFα、IL-6表达都显著增加,而两组间无明显差 别(图13)。图13中柱状图为蛋白较β-Tubulin相对表达量统计。*P<0.05vs. COL4A3+/+,**P<0.01vs.COL4A3+/+;#P<0.05vs.COL4A3+/++DKD,###P<0.001vs.COL4A3+/++DKD。
以上结果提示,DKD早期肾脏即表达炎症因子,携带杂合及纯合型突变的 早期DKD小鼠肾脏炎症相关指标的表达更高。
综上所述,与野生型早期DKD小鼠相比,杂合及纯合型突变DKD小鼠尿 ACR增多、血清肌酐更高、肾脏病理改变更重、GBM出现厚薄不均、足突融合 等病理改变,且纯合型突变DKD小鼠上述肾脏表型较杂合型突变的DKD小鼠 严重,这一结果与临床现象一致。本发明首次证实COL4A3 p.P408H全身杂合/ 纯合型点突变是加速DKD的进展的遗传背景,这有望成为识别DKD高风险人 群的重要新型稳定的生物标志物。
序列表
<110> 四川大学华西医院
<120> 一种COL4A3 p.P408H基因点突变小鼠模型及其构建方法、应用和试剂盒
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<170> SIPOSequenceListing 1.0
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aggagagaga ggaccccctg g 21
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cccacagtgt cttttccagg g 21
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<212> DNA
<213> 人工序列(Artificial Sequence)
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agggagtaaa ggagagagag g 21
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atagcaccca tgcaagaact tttcacaagt ccttgtatat aaagcttaca tcacagctca 60
gactcagtct tggctccttg taataactca cataagtcat cttgtgggac tgcttttaca 120
ctttgtctta atataataaa acataacaag gccaccgaga aagaaaggat atacatggtt 180
tggatttata tgtttttatg tagttttcaa ctcttgtaaa taagaattta tcattctatt 240
ttgaactgaa aaataaacat attcatcaga agaaaaatta aaaccttaca aatggctctt 300
tcttccaagg actgtcaagg cctggcctca gaggacccat tggatggcca ggcttgaaag 360
ggagtaaagg agagagagga ccccctggaa aagacactgt gggccctcct ggacccctgg 420
gatgtcctgg ctcaccaggt ccaccaggcc ctccaggacc tccaggatgt ccaggtaaag 480
atataaaatg ggctggttag ctttctgttt ccctgtgtta gcagcgcaat agaacttcca 540
atgaacaact acatccagct catctatact caggcatagg agcatgggcc tgtagtacag 600
tcctagaaaa tagaacataa aacaacaatg catctttgta agagctggcc agcctgtgtc 660
ttctcagttg tagataaact tctaaaggct catcttccac cagaaggatg gagga 715
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cacagggaaa cagaaagc 18
Claims (10)
1.一种COL4A3 p.P408H基因点突变小鼠模型的构建方法,其特征在于,包括以下步骤:
(1)设计gRNA靶点,其靶点序列如SEQ ID NO.1、SEQ ID NO.2,或SEQ ID NO.3所示;
(2)将Cas9 nickase mRNA、靶点序列以及Donor DNA混合物对受精卵进行注射;
(3)在小鼠出生后进行基因鉴定,获得COL4A3 p.P408H基因点突变小鼠模型。
2.根据权利要求1所述的构建方法,其特征在于,所述点突变位于21号外显子,由脯氨酸突变为组氨酸。
3.根据权利要求1所述的构建方法,其特征在于,所述基因鉴定的过程为:抽提小鼠DNA,并采用引物COL4A3-F/R进行PCR检测,以鉴定其是否突变完成。
4.根据权利要求3所述的构建方法,其特征在于,所述PCR反应体系为:0.5μL KODFX、5μL DNTPs、12.5μL 2x Buffer、2μL DNA、1μL COL4A3-F、1μL COL4A3-R,最后用H2O补足至25μL。
5.根据权利要求3所述的构建方法,其特征在于,所述PCR反应程序为:94℃预变性2min;98℃变性10s;60℃退火30s;68℃延伸30s,共35个循环;68℃延伸10min,最后16℃延伸2min。
6.一种COL4A3 p.P408H基因点突变杂合小鼠模型的构建方法,其特征在于,具体过程如下:将权利要求1或2构建得到的纯合点突变小鼠F0与野生型小鼠进行杂交培育得到F1代,即为点突变杂合小鼠模型。
7.根据权利要求3所述的构建方法,其特征在于,所述野生型小鼠为野生型C57BK/6J。
8.权利要求1~6任一项所述方法构建的COL4A3 p.P408H基因点突变小鼠模型,或权利要求6或7所述方法构建的COL4A3 p.P408H基因点突变杂合/纯合型小鼠模型在糖尿病防治中的应用。
9.根据权利要求8所述的应用,其特征在于,对构建的小鼠模型采用高脂喂养联合腹腔注射链脲佐菌素,构建得到糖尿病肾病模型。
10.一种构建COL4A3 p.P408H基因点突变小鼠模型的试剂盒,其特征在于,包括如SEQID NO.1、SEQ ID NO.2,或SEQ ID NO.3所示的gRNA靶点序列、Cas9 nickase mRNA,以及Donor DNA。
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