CN112779286B - 一种Alport综合征小鼠模型的构建方法及其应用 - Google Patents
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Abstract
本发明属于动物模型的制备方法领域,具体为一种基于CRISPR/Cas9技术的Col4a5基因Del‑ATGG突变动物模型的构建。本发明通过CRISPR/Cas9基因剪辑技术,敲除小鼠Col4a5基因上的特定位点,使得其基因失活,诱导产生Alport综合征。本发明还提供了评价该Alport综合征小鼠模型的方法。该遗传病模型将助力于Alport综合征的发病机制和治疗靶点等领域的研究。
Description
技术领域
本发明属于利用基因修饰技术制作基因突变动物模型技术领域,具体涉及一种基于CRISPR/Cas9技术的Col4a5基因Del-ATGG突变动物模型的构建方法。
背景技术
CRISPR/Cas(Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated)***是原核生物中一种抵抗外源基因侵入的获得性免疫***,在细菌和古生菌中CRISPR***整合侵入宿主的噬菌体或者质粒DNA片段到CRISPR位点,然后通过相应的CRISPR RNAs(cr RNAs)引导Cas核酸内切酶对外源DNA序列进行切割,从而抵抗病毒或者噬菌体的入侵。CRISPR/Cas***分为3种类型(Type I、Type II和Type III),其中TypeII***因其简单、高效以及功能多样化等优点,自2013在哺乳动物细胞中成功实现基因编辑后,现已被广泛用于多种模式生物的基因组修饰技术应用中。II型***可以通过crRNA的引导利用单个Cas9核酸酶对DNA靶位点进行精确充分地切割。CRISPR/Cas9***相关载体构建简单快速,便于操作,实验周期较短,且广泛适用于多个物种。在基因编辑的过程中,CRISPR/Cas9***需要一段特殊的引导RNA,即sgRNA(single guide RNA),sgRNA的序列选择限制性较小,只需要基因组序列中出现PAM(NGG)区即可。在sgRNA的引导下,Cas9蛋白可以对基因组实现定点切割,引起DNA双链断裂,继而引发细胞的自主修复,修复的过程可以是随机的片段缺失或***,也可以利用特定的模板修复,从而实现对基因组的永久性修饰。
Alport综合征是一种遗传性基底膜疾病,由于肾小球疾病、可变感音神经性听力损失和各种眼部异常而导致进行性肾功能衰竭。Alport综合征是一种遗传异质性疾病,其所有形式都是由编码IV型胶原的基因突变引起的,而IV型胶原是基底膜的主要结构成分,Ⅳ型胶原有α1~α6共6种α链,分别由COL4A1~6基因编码,根据组织分布的不同,这6种α链螺旋组成三种不同的三聚体:α1α1α2、α3α4α5、α5α5α6。在胚胎形成时期,α1α1α2三聚体广泛分布于各组织基底膜中;随着组织分化,肾小球基底膜、耳蜗、眼、睾丸、肺等组织中的α1α1α2前体逐渐被α3α4α5三聚体替代,而Bowman囊、皮肤、食管、平滑肌等组织的α1α1α2(Ⅳ)三聚体则被α5α5α6(Ⅳ)替代。研究认为,α1α1α2(Ⅳ)三聚体易被内切蛋白酶水解,由α3α4α5(Ⅳ)三聚体组成的Ⅳ型胶原交联结构更加稳定、抗蛋白水解能力更强。因此,α3α4α5(Ⅳ)三聚体对维持肾小球基底膜结构完整和功能稳定是必不可少的。在肾小球基底膜上,只有足细胞能合成并分泌α3α4α5(Ⅳ)三聚体,而α1α1α2(Ⅳ)三聚体可由足细胞、内皮细胞及系膜细胞合成,因此,足细胞是Alport综合征发病机制的关键细胞。当编码α3~α5链的基因发生突变时,足细胞不能合成成熟的α3α4α5三聚体,导致肾小球基底膜结构稳定性降低并且出现分层。而基底膜和足细胞直接存在相互作用,异常的肾小球基底膜可能通过影响两者间的整合素复合体导致足细胞损伤,损伤的足细胞传递触发周围完整足细胞产生二次损伤,足细胞的这种损伤传递机制形成了一个恶性循环,加速了足细胞融合和肾小球硬化,肾小球滤过屏障受损出现蛋白。
大约85%的患者为X染色体连锁的显性遗传的Alport综合征(X-linked dominantinheritance Alport Syndrome,XLAS),是由COL4A5基因突变导致。几乎所有的Alport综合征的患者都会有程度不一的血尿,多表现为镜下血尿;部分AS患者会出现蛋白尿,且随着疾病的进展逐渐加重,甚至达到肾病综合征的水平。通常XLAS的男性或常染色体隐性遗传Alport综合征(autosomal recessive Alport syndrome,ARAS)的患者常在20岁进展为终末期肾病,而XLAS女性或常染色体显性遗传Alport综合征(Autosomal Dominant AlportSyndrome,ADAS)患者多在60岁后出现肾功能衰竭。
国外目前已经建立2种不同的XLAS小鼠模型,2004年,Michelle N.Rheault针对小鼠Col4a5基因建立G5X突变的Alport综合征突变小鼠模型;2019年,日本研究者KentarouHashiami利用CRISPR-associated技术建立Col4a R471X突变的Alport综合征突变小鼠模型。但由于突变位置不同,小鼠病情的严重程度也不一致。
目前,还没有COL4A5基因中间区域发生突变的动物模型,因此利用CRISPR/CAS9技术构建COL4A5 Del-ATGG小鼠模型,对研究该基因在Alport综合征疾病发生发展过程中的机制将会有很大的帮助作用。
发明内容
本发明的目的是提供一种基于CRISPR/Cas9技术的Col4a5基因Del-ATGG突变动物模型的构建方法。
为实现以上目的,本发明采用了以下技术方案:
一种基于CRISPR/CAS9技术的COL4A5基因Del-ATGG突变动物模型的构建方法,通过以下步骤实现:
(1)在小鼠Col4a5基因17号外显子致病突变位点附近设计合适的sgRNA;其中sgRNA的核苷酸序列为:ACCTTTTCACCATCTCTTCC;
(2)利用体外转录技术获得sgRNA;
(3)将合成后的sgRNA以及Cas9 mRNA共注射转至鼠受精***中,移植入受体母鼠生产Del-ATGG基因小鼠模型;
在本发明所述的小鼠模型构建方法中,还包括通过引物鉴定小鼠基因型的方法,所述引物的序列如SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示。
(6)进一步对Del-ATGG小鼠进行血液/尿液生化指标***指标评价及病理组织学检查学检查。所述血液/尿液生化指标***指标包括血液白蛋白,血液肌酐,血液尿素氮及尿液ACR(Albumin/Urine Creatinine Ratio,尿微量白蛋白和肌酐的比值);所述病理组织学检查包括HE染色和PAS染色,肾脏电子显微镜检查,IV型胶原α5链染色。
本发明在血液/尿液生化指标***指标评价及病理组织学检查学检查中成功验证了Alport综合征的临床表型及病理特征,将助力Alport综合征的发病机制和治疗靶点、新药开发等领域的研究。
附图说明
图1为Col4a5基因Del-ATGG突变小鼠构建策略图;
图2为F0代缺失阳性小鼠PCR产物与PCR产物测序图谱结果;
图3为野生型和Col4a5-Del ATGG小鼠肾脏病理图;
图4为野生型和Col4a5-Del ATGG小鼠肾脏电镜结果图;
图5为野生型和Col4a5-Del ATGG小鼠IV型胶原α5链染色图;
图6为野生型和Col4a5-Del ATGG小鼠血液/尿液生物化学指标。
具体实施方式
下面结合附图对本发明做进一步的详细说明,通过参考下述的一些具体实施例可进一步理解本发明,这些实施例仅用于说明本发明,其无意于对本发明的范围做出任何限制。可以对本发明做出多种改动和变化而不脱离本发明的实质,因此,这些改动和变化同样在本申请要求保护的范围内。
实施例1 Col4a5-Del ATGG小鼠的构建
1.确定敲除基因的基本信息
(1)目的基因名称(MGI号):Col4a5(MGI:88456)
(2)目的基因在Ensembl数据库中的编号:ENSMUSG00000031274
(3)方案对应转录本(Ensembl号):Col4a5-202(ENSMUST00000112931.7)
(4)突变位点:Deletion ATGG(980-983)of Mouse Col4a5
2.构建Col4a5-Del ATGG小鼠策略,如图1所示。
3.体外转录sgRNA,同时构建同源重组载体(Donor vector)。将Cas9、gRNA、Donorvector同时注射到小鼠的受精卵中。经受精卵显微注射,胚胎移植,获得F0代小鼠。
实施例2对小鼠进行基因型鉴定。
(1)所用的引物如SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示;
(2)测序引物为SEQ ID No.2;
(3)PCR反应体系:
反应成分 | 体积(ul) |
2xTaq Master Mix,Dye Plus,(Vazyme P112-03) | 12.5 |
双蒸水 | 9.5 |
测序引物(10pmol/μl) | 1 |
下游引物(10pmol/μl) | 1 |
模板(≈100ng/μl) | 1 |
(4)PCR反应条件:
实施例3 DNA电泳鉴定
将PCR产物加入1.5%的琼脂糖凝胶内,120V 30min,观察结果如图2,获得阳性反应确认Col4a5基因Exon17删除ATGG(980-983)。
实施例4 Col4a5-Del ATGG小鼠的病理组织学检查
采用石蜡切片及HE染色和PAS染色方法观察及小鼠肾脏组织形态变化。
石蜡切片步骤如下:
(1)固定:取待检组织切成小块,10%中性***溶液内固定24小时
(2)洗涤与脱水:用流水冲洗,乙醇浓度梯度顺序脱水(50%→70%→80%→90%→95%→100%,100%三次)。
(3)透明:二甲苯作为透明剂,用1/2乙醇+1/2二甲苯→二甲苯一次→二甲苯二次,每步约为1小时。
(4)浸蜡与包埋:低熔点石蜡I、II、III中放置各25min(温度设置为62摄氏度);高熔点石蜡I缸25min(温度设置为64摄氏度);组织处理程序结束后,取出标本,置于包埋机的蜡槽中,进行包埋(温度设置为62摄氏度);
(5)切片:在切片机上切下4μm切片,漂在干净的水面上,在50-57摄氏度温水中展开,再将组织切片捞起;在70摄氏度烤箱中烘烤15-20min;从烤箱中取出切片,室温稍冷却;
(6)染色:二甲苯I、II、III中放置各7min,100%乙醇III、95%乙醇III、80%乙醇、蒸馏水各1min;。苏木精染液中15分钟,水洗,0.5%盐酸酒精10s;2%碳酸锂水溶液1min;流水冲洗10min,80%乙醇、95%乙醇III,80%乙醇,100%乙醇IIIIII、二甲苯I、II各1min。镜检控制,直至细胞核及核内染色质清晰为止,约10秒;
(7)封片:擦去切片周围多余的二甲苯,切勿干涸,迅速滴加适量中性树胶,再加盖玻片封固。
显微镜下观察HE染色和PAS染色切片,结果如图3,与野生型雄性小鼠相比,Del-ATGG雄性小鼠可见节段毛细血管硬化,系膜轻度增宽,局灶性毛细血管襻内皮细胞增多;PAS染色可见囊外明显纤维化。
实施例5 Col4a5-Del ATGG小鼠肾脏透射电镜检查
(1)前固定:快速切取大小为0.5~1mm3的样品块,一分钟内把组织浸入4%戊二醛中固定4小时以上,4℃保存;
(2)漂洗I:1M磷酸缓冲液15min*3次;
(3)后固定:1%四氧化锇2小时后,4℃冰箱保存;
(4)漂洗II:1M磷酸缓冲液15min*3次;
(5)脱水:室温下梯度丙酮脱水:30%丙酮→50%丙酮→70%丙酮→80%丙酮→90%丙酮,每步均需15分钟;最后100%丙酮30min*3次;
(6)浸透:包埋剂室温浸透3小时使包埋剂逐步渗入组织细胞内。100%丙酮:包埋液=1:1在37℃温箱中孵育60min。纯包埋液在37℃温箱中孵育5小时;
(7)包埋:用包埋剂将样品包埋在包埋板中;
(8)聚合:将包埋版放入烤箱中:37℃24小时;60℃48小时;
(9)超薄切片:用超薄切片机将样品切成厚度50nm的超薄切片;
(10)电子染色:超薄切片先柠檬酸铅染色10min,去二氧化碳的双氧水清洗3次,再用醋酸铀染色30min,Milli-Q水清洗3次,待超薄切片干燥后即可上透射电镜观察。
电镜结果如图4:在28周野生型雄性小鼠肾脏电镜可见:基底膜(BM)清晰,无明显断裂,大部分厚薄均匀,个别区域可见增厚;Del-ATGG雄性半合子小鼠肾脏电镜可见:基底膜大面积厚薄不均,局部严重增厚(黑箭头);足突(FP)大面积融合或消失(白箭头),足突间隙变宽。
实施例6 Col4a5-Del ATGG小鼠肾脏免疫胶原染色
如图5,可以见到在野生型小鼠肾组织肾小球毛细血管襻以及包曼囊壁染色均为阳性;Del-ATGG雄性半合子小鼠肾组织IV型胶原α5链:肾小球毛细血管襻以及包曼囊壁染色均为阴性。
实施例7 Col4a5-Del ATGG小鼠血液/尿液生物化学检查
每4周将Col4a5-Del ATGG小鼠放置于24小时生理笼,收集小鼠24小时尿液;休息5天后,再对小鼠进行眼眶取血。采集后的尿液3000rpm离心20min后,在全自动生化分析仪上进行分析。采集后的血清待凝固后4000rpm离心20min后,在全自动生化分析仪上进行分析。数据与野生型小鼠数据如图6,可以看出,Col4a5-Del ATGG小鼠肾功能明显下降,能判定其患有Alport综合征。
SEQUENCE LISTING
<110> 中国人民解放军东部战区总医院
<120> 一种Alport综合征小鼠模型的构建方法及其应用
<130> 2020.11.30
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 54
<212> DNA
<213> Mus musculus
<400> 1
ggtttgcctg gtgatcctgg ttaccctgga gaaccaggaa gagatggtga aaag 54
<210> 2
<211> 26
<212> DNA
<213> Mus musculus
<400> 2
ccttcttctg aatgttcata tccagg 26
<210> 3
<211> 23
<212> DNA
<213> Mus musculus
<400> 3
accctggaga accaggaaga gtg 23
<210> 4
<211> 26
<212> DNA
<213> Mus musculus
<400> 4
cactggaata gagatcaaac ctcctc 26
Claims (7)
1.一种Alport综合征小鼠模型的构建方法,其特征是利用CRISPR/Cas9技术在小鼠Col4a5基因的17号外显子上删除980-983位点的4个碱基ATGG;
1)设计表达针对鼠源Col4a5基因17号外显子的sgRNA质粒,所述鼠源Col4a5基因17号外显子序列如SEQ ID NO:1所示,sgRNA序列名称为:
Gps00000073-Col4a5-S1,sgRNA序列(5’-3’)为:ACCTTTTCACCATCTCTTCC,PAM为:TGG;
2)利用体外转录技术获得相应sgRNA;
3)将sgRNA及Cas9 mRNA或Cas9蛋白转至鼠受精***中,并将受精卵移植入受体母鼠生产小鼠。
2.根据权利要求1所述的一种Alport综合征小鼠模型的构建方法,其特征在于:还包括利用引物鉴定小鼠基因型的步骤。
3.根据权利要求2所述的一种Alport综合征小鼠模型的构建方法,其特征在于:鉴定小鼠基因型使用的引物序列如SEQ ID NO:2和SEQ ID NO:4或SEQ ID NO:3和SEQ ID NO:4所示。
4.根据权利要求1所述的一种Alport综合征小鼠模型的构建方法,其特征在于:还包括对小鼠模型进行血液、尿液生化指标***指标评价或病理组织学检查评价。
5.根据权利要求4所述的一种Alport综合征小鼠模型的构建方法,其特征在于:所述血液、尿液生化指标***指标包括血液白蛋白、血液肌酐、血液尿素氮或尿液ACR(Albumin/Urine Creatinine Ratio,尿微量白蛋白和肌酐的比值)。
6.根据权利要求4所述的一种Alport综合征小鼠模型的构建方法,其特征在于:所述病理组织学检查包括HE染色和PAS染色、肾脏电子显微镜检查或IV型胶原α5链染色。
7.根据权利要求1所述的方法获得的Col4a5基因Del-ATGG小鼠作为Alport综合征研究的模型鼠的用途。
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