CN112608940B - 一种先天性白内障疾病动物模型构建方法及应用 - Google Patents
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Abstract
本发明涉及一种先天性白内障疾病动物模型的构建方法,先针对小鼠GJA8基因的第二个外显子区域设计gRNA靶标位点序列,扩增同源臂和cKO区域,分别连接到载体上;将构建好的质粒电转ES细胞向囊胚中注射阳性克隆,并转移入小鼠体内,出生小鼠经发育成熟后,得到首建者小鼠;再与野生型小鼠交配,得基因突变的杂合子小鼠F1代;最后将基因型相同的杂合子小鼠F1代交配,得基因突变纯合子小鼠F2代。本发明提供的先天性白内障疾病动物模型的构建方法,可得到表现出先天性白内障的典型特征如表现出晶状体变性混浊、晶状体上皮凋亡等特征,该动物模型可用于先天性白内障疾病的研究和筛选治疗先天性白内障疾病的药物中,其应用前景广阔。
Description
技术领域
本发明属于生物学基因编辑技术领域,涉及一种先天性白内障疾病动物模型构建方法及应用。
背景技术
先天性白内障是指在出生时或出生后不久出现的晶体混浊,其发生率为0.6-6/10000人口,是造成儿童失明及弱视的重要原因,占儿童致盲性眼病第二位。基于我国庞大人口技术,先天性白内障患者可达百万,给家庭及社会带来沉重负担。虽然先天性白内障手术技法不断改进,且成功率不断提高。但儿童眼部解剖学、生理学特点及术后炎症反应较成人复杂,其弱视、后发障等术后并发症易发生,因此患者视力恢复较难达到期望效果。更为困难的是,目前对该病病因和病理机制仍缺乏深入系统研究,大量先天性白内障致病位点及基因致病机制尚不清楚,给预防、诊断及针对性治疗干预带来障碍。
先天性白内障防治措施乏力的根本原因在于该病在早期胚胎及初生期的病因与病理机制缺乏深入研究。造成这种问题的原因主要有以下几个方面:1)因医学伦理范畴及该病的随机性,人类先天性白内障胚胎无法获取。2)临床表现的异质性强。该病临床表型差异较大,严重者在出生时即开始显现发病表型,并很快失明,而轻微患者一直到青年时才显现发病表型。3)致病基因多、遗传模式复杂。目前已知与先天性遗传性白内障确切相关基因已超过40个,包括以下几大类:晶状体蛋白基因(CRYAA、CRYAB、CRYBA1\A3、CRYBB1、CRYBB2、CRYGC、CRYGD)、缝隙连接蛋白基因(GJA8、GJA3)、水通道蛋白基因(MIP)、晶状体内在膜蛋白(LIM2)、细胞骨架蛋白基因(BFSF2/CP49)、转录调控因子基因(PITX3、PAX6、MAF、HSF4)、氨基葡萄糖N-乙酰转移2(GCNT2)等。上述基因的单核苷酸突变是产生遗传性白内障主要原因。临床工作中可通过基因疾病诊断与遗传咨询在数量繁多基因突变中确定致病突变。4)致病分子机制复杂。已发现先天性白内障致病基因参与晶状体及相关细胞生化过程非常复杂,如细胞骨架、离子通道、RNA剪接、内质网压力等,这使得其致病分子机制研究增加难度,也给治疗干预带来诸多障碍。目前尚缺乏晶状体变性疾病动物模型,给进一步深入系统研究该疾病带来不便。
发明内容
有鉴于此,本发明的目的在于提供一种先天性白内障疾病动物模型的构建方法,利用该构建方法可以得到先天性白内障疾病动物模型,可用于研究者深入系统研究先天性白内障疾病,以及用于先天性白内障疾病的药物筛选。
为达到上述目的,本发明提供如下技术方案:
1.一种先天性白内障疾病动物模型的构建方法,包括以下步骤:
1)针对小鼠GJA8(C.134G>T)基因的第二个外显子区域设计gRNA靶标位点序列,序列如SEQ ID NO.1所示;扩增同源臂和cKO区域,分别连接到载体上,最后质粒构建如图9所示;将构建好的质粒电转ES细胞,通过鉴定后再向囊胚中注射阳性克隆,并转移入小鼠体内,出生小鼠即为F0,经发育成熟后,得到发生基因突变的首建者小鼠;
2)将带有突变的首建者小鼠与野生型小鼠交配,得到基因突变的杂合子小鼠F1代;
3)将基因型相同的杂合子小鼠(+/-)F1代相互交配,得到GJA8(C.134G>T)基因突变纯合子小鼠(-/-)F2代。
进一步,构建方法还包括步骤4):将步骤3)的F2代小鼠与Cre工具小鼠进行杂交,从而获得F1’代GJA8(C.134G>T)mut/mut;WT/KO杂合子小鼠,将GJA8(C.134G>T)mut/mut;WT/KO杂合子小鼠进行交配,获得F2’代GJA8KO/KO纯合子非突变小鼠。
进一步,步骤1)中同源臂和cKO区域包括第二外显子5’端A片段,B片段,B1片段和3’端D片段。
进一步,A片段大小为3123bp,克隆A片段的引物序列如SEQ ID NO.5和SEQ IDNO.6所示;B片段大小为704bp,克隆B片段的引物序列如SEQ ID NO.7和SEQ ID NO.8所示,B1片段大小为1234bp,克隆B1片段的引物序列如SEQ ID NO.9和SEQ ID NO.10所示;D片段大小为2136bp,克隆D片段的引物序列如SEQ ID NO.11和SEQ ID NO.12所示。
进一步,片段分2次连接到载体上,先用NruI/AscI双酶切质粒,将A片段连接至载体;再用PmlI/NotI双酶切质粒,将B片段、B1片段和C片段融合后连接至载体。
进一步,还包括对首建者小鼠CRISPR靶点位置附近片段进行测序。
进一步,测序反应引物如SEQ ID NO.3和SEQ ID NO.4所示。
进一步,得到基因突变的杂合子小鼠F1代后进行DNA测序,确认目标基因发生GJA8(C.134G>T)突变从而目标蛋白被失活。
本发明的另一目的在于提供一种由上述构建方法得到的先天性白内障疾病动物模型。
本发明的另一目的在于提供先天性白内障疾病动物模型在筛选治疗先天性白内障疾病药物中的应用。
本发明的另一目的在于提供一种用于构建先天性白内障疾病动物模型的核酸分子。
本发明的有益效果在于:目前转基因小鼠多采用打靶或者cas9方法进行操作,得到单一点突变或者基因功能缺失小鼠,功能较单一,费用和繁殖周期过长。在发明中,采用点突变与基因缺失共嵌套的模式进行载体设计及遗传小鼠构建。首先,在构建质粒过程中,在原有的2号外显子中引入c.134G>T点突变,同时,在点突变序列后设计插入loxP位点。紧接2号外显子后面加上polyA位点。原有DTA元件后及Neo筛选基因盒的两端加入SDA位点以便于与fleep工具鼠进行后期繁殖,对两侧SDA位点进行剪切。该小鼠采用CRISPR-Cas9技术进行嵌套小鼠的载体插入。得到的小鼠结果为:fleep工具小鼠配繁情况下为GJA8(c.134G>T)点突变小鼠,而与Cre工具小鼠交配后可于F1代获得GJA8(c.134G>T)点突变与CX50缺失小鼠,再进行二次繁育,使Cre工具小鼠与GJA8(c.134G>T)点突变与CX50缺失小鼠交配获得CX50条件性缺失小鼠。该小鼠既可以研究点突变造成的CX50功能紊乱也可以研究GJA8基因缺失后造成的CX50功能缺失效应。在研究中,既可以应用Cre工具小鼠进行基因缺失的研究也可以采用前房注射AAV或LV病毒进行Cre外源性引入,从而诱导晶状体局部组织区域的GJA8基因缺失,从而可以进行精细的CX50功能研究。本发明提供的先天性白内障疾病动物模型的构建方法,可得到表现出先天性白内障的典型特征如表现出晶状体变性混浊、晶状体上皮凋亡等特征,该动物模型可用于先天性白内障疾病的研究和筛选治疗先天性白内障疾病的药物中,其应用前景广阔。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为gRNA序列靶向GJA8(C.134G>T)基因的结构示意图;
图2为设计载体的酶切位点图谱;
图3为测序得到的移码突变结果图;
图4为本发明实施例提供的GJA8(C.134G>T)点突变敲除小鼠基因型鉴定图;
图5为野生型小鼠和小鼠模型在4个月时,小鼠的晶状体对比图;
图6为本发明实施例提供的CRISPR靶向序列设计示意图。
图7-图9为本发明质粒构建各步骤示意图。
图10为测序突变点示意图。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法,通常按照常规条件或按照制造厂商所建议的条件。
目前转基因小鼠多采用打靶或者cas9方法进行操作,得到单一点突变或者基因功能缺失小鼠。在本发明中,我们采用点突变与基因缺失共嵌套的模式进行载体设计及遗传小鼠构建。首先,在构建质粒过程中,在原有的2号外显子中引入c.134G>T点突变,同时,在点突变序列后设计插入loxP位点。紧接2号外显子后面加上polyA位点。原有DTA元件后及Neo筛选基因盒的两端加入SDA位点以便于与fleep工具鼠进行繁殖,对两侧SDA位点进行剪切。该小鼠采用CRISPR-Cas9技术进行嵌套小鼠的载体插入。得到的小鼠结果为:fleep工具小鼠配繁情况下为Cx50(W45L)点突变小鼠,而与Cre工具小鼠交配后可于F1代获得Cx50(W45L)点突变与CX50缺失小鼠,再进行二次繁育,使cre工具小鼠与Cx50(W45L)点突变及CX50缺失小鼠交配获得CX50条件性缺失小鼠。
实施例1
图6为本发明实施例提供的CRISPR靶向序列设计示意图,如图6所示,本实施例采用CRISPR-Cas9技术点突变GJA8(c.134G>T)基因构建先天性白内障疾病动物模型,具体步骤如下:
1、设计靶向点突变GJA8(c.134G>T)基因的gRNA序列,获得F0代GJA8(c.134G>T)点突变小鼠
1)针对小鼠GJA8(C.134G>T)基因的第二个外显子区域设计gRNA靶标位点序列,如下:
5’-CCTGTATCACATGCGGCTCTCGG-3’(SEQ ID NO.1);体外合成RNA,与Cas9核酸内切酶一起显微注射到小鼠受精卵中,DTA和Neo cassette(SDA-Neo-SDA)是质粒骨架自带,将同源臂和cKO区域带入骨架即可,如图7所示;用于连接转化的片段的引物如下面表格所示,该项目会扩增4个片段,大小分别是3123bp/704bp/1234bp/2136bp,后三个片段在连接前会进行体外融合,片段扩展具体引物见下面表1。BACs(Bacterial artificial chromosomes)
表1
分两次连接到载体骨架,第一步是5arm酶切位点为(NruI/AscI),第二步是CKO+3arm酶切位点是(PmlI/NotI)。用于载体筛选的引物表2:
表2
用NruI/AscI双酶切骨架质粒,再与从BAC上扩增下来的5arm片段连接,如图8所示;
2)PmlI/NotI双酶切步骤1的质粒,再与从BAC上扩增下来的片段(cKO1,cKO2-FLAG,loxP-3arm三片段融合:扩增cKO1和cKO2的引物带有点突变位点)连接,如图9所示;
3)再将质粒电转C57BL/6ES细胞,通过鉴定后再向囊胚中注射阳性克隆,并转移入小鼠体内,出生小鼠即为F0,F0小鼠向F1传代过程中自删元件将发挥作用,采用fleep工具小鼠对Neo元件两端SDA进行基因重组,从而在基因组层面将Neo被去除,从而在F1时将得到下面的模型,经发育成熟后,得到发生基因突变的首建者小鼠(founder)。
gRNA序列靶向GJA8(C.134G>T)基因的结构示意图如图1所示,图2为设计载体的酶切位点图谱。
实施例2
利用小鼠的基因组DNA,对本CRISPR靶点位置附近片段进行测序,确定首建者小鼠带有影响阅读框的移码突变。
具体如下:
(1)提取扩增用样品
①剪步骤1得到的首建者小鼠小鼠尾梢少许组织样本,置于干净的1.5ml离心管中;
②在离心管中加入100μl裂解液(40mM NaOH,0.2mM EDTA溶液),并在金属浴100℃加热1h;
③取出离心管,冷却至室温后,加入100μl的中和液(40mM Tris-HCl,pH5.5),10000g离心2min后,取上清用于小鼠基因型鉴定。
(2)小鼠基因型鉴定
①PCR扩增:按照如下体系配置PCR反应体系。
②纯化:
扩增出的PCR片段需要使用FastAP(已加入外切酶、反应buffer和ddH2O)纯化以去除引物及其他干扰片段,具体的纯化体系为:
PCR片段4.5μL
FastAP 1.5μL。
反应条件为:
步骤1. 37℃30min
步骤2. 80℃15min
步骤3. 12℃for保存。
③测序
经过上述步骤的纯化片段随后用于测序反应。具体的测序反应体系如表3所示,反应条件如表4所示:
表3测序反应体系
引物组
F1:5’-GTGGCCAATTAGATTTTGCCTTCT-3’SEQ ID NO.3
R1:5’-GATGGAGTGGAGACGAAGATGAT-3’SEQ ID NO.4
表4反应条件
测序反应结束后,每孔中加入50μL的70%酒精,然后12,000×g离心30min使DNA扩增片段沉淀于96孔板底,随后打开盖子,缓缓倒掉上清液体,然后倒扣96孔板,1,000×g离心1min,敞口避光放置约30min以使酒精彻底挥发干净,然后加入10μL ddH2O以重新溶解DNA片段,最后上机测序。
测序得到的移码突变结果见图3,相对于序列SEQ ID NO.21,GJA8(c.134G>T),Mut:NM_008123.3,c.134G/T。可见,第134个碱基共1个碱基被替换了(TGG->TTG)。测序结果如SEQ ID NO.2所示,突变位点图示见图10。
图4为本发明实施例提供的GJA8(C.134G>T)点突变敲除小鼠基因型鉴定图;图中WT代表野生型,Hom代表基因敲除纯合基因型,Het代表基因敲除杂合基因型。野生型扩增片段为198bp(WT),敲除纯合基因型扩增片段为262bp(hom),敲除杂合基因型(Het)扩增片段为198bp+262bp。
实施例3
(1)获得F1代GJA8(C.134G>T)基因敲除小鼠
将带有突变的首建者小鼠(founder)与野生型小鼠交配,得到基因突变的杂合子小鼠(WT/mut)F1代,并进行DNA测序,确认目标基因发生了图3的移码突变从而目标蛋白被失活,实现GJA8(C.134G>T)基因敲除。
(2)获得F2代GJA8(C.134G>T)基因突变小鼠
将基因型相同的突变杂合子小鼠(WT/mut)相互交配,得到GJA8(C.134G>T)基因突变纯合子小鼠(mut/mut)F2代。
(3)获得F1’代GJA8基因敲除小鼠
将上述F2代GJA8(C.134G>T)mut/mut小鼠与Cre工具小鼠进行杂交,从而获得F1’代GJA8(C.134G>T)mut/mut;WT/KO杂合子小鼠,将GJA8(C.134G>T)mut/mut;WT/KO杂合子小鼠进行交配,获得F2’代GJA8KO/KO纯合子非突变小鼠。
因此,利用本发明设计的gRNA序列,能够引导Cas9快速、高效、特异性敲除小鼠的GJA8(C.134G>T)基因,利用本发明方法可以构建GJA8(C.134G>T)基因突变GJA8(C.134G>T)+/mut小鼠模型(杂合突变型)和GJA8(C.134G>T)mut/mut小鼠模型(纯合突变型),以及GJA8WT/KO杂合子和GJA8KO/KO纯合子四种小鼠,均可用于后续的功能性研究。
实施例4试验例
以下通过试验例具体说明由实施例1-3得到GJA8(C.134G>T)基因敲除小鼠表现出的晶状体变性疾病特征。
敲除型小鼠模型在4个月时,纯合GJA8(C.134G>T)突变小鼠的晶状体混浊,如图5所示。CX50是重要的缝隙连接蛋白,对晶状体透明维持极其关键,因此,可知GJA8基因在晶状体变性疾病中发挥重要作用,图5中左边图为前照法照片,右边为后照法照片。
结果发现在4个月时,KO小鼠的晶状体厚度明显变薄:晶状体表现为空泡样改变,而杂合子与野生型无明显改变。
可见,利用本发明方法构建的GJA8(C.134G>T)基因敲除小鼠模型,其具有典型的晶状体变性疾病特征,可以用于晶状体变性疾病研究。
综上,本发明实施例提供的通过CRISPR-Cas9技术特异性靶向敲除GJA8(C.134G>T)基因的核苷酸序列,能够引导Cas9快速、高效、特异性敲除小鼠的GJA8(C.134G>T)基因,得到先天性白内障疾病小鼠模型,为GJA8(C.134G>T)基因相关疾病的研究提供了可靠的动物模型,其具有广阔的应用。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 中国人民解放军陆军特色医学中心
<120> 一种先天性白内障疾病动物模型构建方法及应用
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
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cctgtatcac atgcggctct cgg 23
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<211> 15342
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 60
ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 120
ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata 180
cgactcacta tagggcgaat tggagctcca ccgcccgggc tggttctttc cgcctcagaa 240
gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg 300
ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca 360
atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc 420
acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag 480
cgagctctag gatctgcatt ccaccactgc tcccattcat cagttccata ggttggaatc 540
taaaatacac aaacaattag aatcagtagt ttaacacatt atacacttaa aaattttata 600
tttaccttag agctttaaat ctctgtaggt agtttgtcca attatgtcac accacagaag 660
taaggttcct tcacaaagag atcgcctgac acgatttcct gcacaggctt gagccatata 720
ctcatacatc gcatcttggc cacgttttcc acgggtttca aaattaatct caagttctac 780
gcttaacgct ttcgcctgtt cccagttatt aatatattca acgctagaac tcccctcagc 840
gaagggaagg ctgagcacta cacgcgaagc accatcaccg aaccttttga taaactcttc 900
cgttccgact tgctccatca acggttcagt gagacttaaa cctaactctt tcttaatagt 960
ttcggcatta tccactttta gtgcgagaac cttcgtcagt cctggatacg tcactttgac 1020
cacgcctcca gcttttccag agagcgggtt ttcattatct acagagtatc ccgcagcgtc 1080
gtatttattg tcggtactat aaaacccttt ccaatcatcg tcataatttc cttgtgtacc 1140
agattttggc ttttgtatac ctttttgaat ggaatctaca taaccaggtt tagtcccgtg 1200
gtacgaagaa aagttttcca tcacaaaaga tttagaagaa tcaacaacat catcaggatc 1260
catggcacgc gcttctacaa ggcgctggcc gaagaggtgc gggagtttca cgccaccaag 1320
atctgcggca cgctgttgac gctgttaagc gggtcgctgc agggtcgctc ggtgttcgag 1380
gccacacgcg tcaccttaat atgcgaagtg gacctcggac cgcgccgccc cgactgcatc 1440
tgcgtgttcg aattcgccaa tgacaagacg ctgggcgggg tttgctcgac attgggtgga 1500
aacattccag gcctgggtgg agaggctttt tgcttcctct tgcaaaacca cactgctcga 1560
cattgggtgg aaacattcca ggcctgggtg gagaggcttt ttgcttcctc ttgaaaacca 1620
cactgctcga tttgttagca gcctcgaatc aacccgggcg atcctaggcg atgagatcta 1680
gctgtcgcga tgtgccacca agcctgtgac ctgaggataa taccagaaac cccatggtag 1740
aagagaactc tcctctgtct cctgccccac acctcctcat actgctctct tgttgagaac 1800
ttcttttcct tttccaatat caactatcct gaacattcct aattttgtta gaaattttgt 1860
ccagcacatg ttcctataaa ttatgaatct actgagatta attactgggc tattatacta 1920
gccaacttac tggattgtta tctttacact gtacagactt ttttttctag attgtggctg 1980
tgaaacatca ggatttttgt taggcctgag agcacttgtt atatgtaagc acatgcctgg 2040
atattataac tgactcaatg gggacacttt tctgacatgc gtgcttctct gtcgagagtt 2100
cgctactgtg ataatcaagc aggttgaggt gaatgacctc aagcaaatcc cgcatgtaac 2160
atgtaaatgt gtttctcttt tgtatatttc cctggtacaa ttcttttaat atctaaacac 2220
gttcattagt gttttattaa aacattttta aactatctgg aaaaaaaaaa accctcactc 2280
ccgaaagttc tctgacctct acacctgcac acacatacta aatacatcta gaacatattt 2340
taataaccta acacttcccc agcttctctc gatcctctct tcttgacagg agacgtggcc 2400
tgtttcactg ctgtgctaat agaatgattt ataccaattt acagttgccc gtaacttggc 2460
acaaaacttt tagtaggcat aagaaaaaaa ttcaaagccc ccaaacaagc ctaatgtgtc 2520
aggataggtt acacagggaa tgctttgcac tggctctcat ttagaaatga ctgacgtggc 2580
tgtcagtaag gccctgggcc atgactgtgt atcaggacac tgtgcgttaa gagtcactgg 2640
tctacacatt tatacgcagg atgcttccca aaacaagtac catctgcccc aagtgtggga 2700
agggctggtc tgccccagat gtgggaaggg ctggttcgcc tttgtttttt gattcatgat 2760
agccagttgg gctcatctta ccttttcctt ttctttcctt tttccctcct agtttgcata 2820
tcctcgcaga ttctctttct ttttttttta ttacgtattt tcttcaatta catttccaat 2880
gctatcccaa aagtcccccg taccctaccc cccacttcct tacccaccca ttcccgtacc 2940
ctacccccca cttccttacc cacccattcc catttttttg gccctggcgt tcccctgtac 3000
tggggcatat aaagtttgtg tgtccaatgg gcctctcttt ccagtgatgg ccgactaggc 3060
catcttttga tacatatgca gctagagtca agagctccag ggtactggtt agttcatatt 3120
gttgttctac ctatagggtt gcagatccct ttagctcctt gggtactttc tctagctcct 3180
ccattgggag tcctgtgatc catccaatag ctgactgtga gcatccactt ctgtgtttgc 3240
taggccccgg catagtctca caagagacag ctgttgcata cactagcaag attttgctga 3300
aaggacgcag attctctttc ttagtctaaa ttttataaac tggcttactt ttatgtgttc 3360
ttcctctcac tgctgctatg gaggaagaat cccgagggtc cccgaatttt ggtgataact 3420
gtctctcctc aggagctgag aggttcagat cttaaagcca ttgctcttgg ccctgtccct 3480
ggtgatgggt ggaggtccca cactaaggtg tggctttgat ctgcattgtg ctggctcggg 3540
gaggagaaag ccaaacgtga gcccagaatg tcctattctt agagagccag attatggaca 3600
ctgtaataat aagctatcaa gttagaatgc actaaagcta gagttgaact atttgataag 3660
aaatgggcct ccctggatct gtcttgtgtg tggggttaat gcttggattg tccacccaat 3720
tgtgatagtt cctgcttact ttttcttttt tttttttaag atttatttat tatatgtaag 3780
tacactgtag ctgtcttcag acgcaccaga agagggcatc cgatctcatt acagatggtt 3840
atgagccgcc atgtggttcc tgggatttga actcaggacc tccagaagag cagtcagtgc 3900
tcttaacctc tgagccatct ctccagcccc ctcctgctta tttctggatg gtgtccagaa 3960
gaaacccata gaagagctct ccaacatgag ccattgcctt gccctaaggg caggcatgag 4020
agggttccag agctccagag atagtcagct ccgagagaca actgacctaa actgatacgg 4080
gagggttgct tttatcatag ttctgaatga gtggtccact ttgtatcccc caatcgactc 4140
agtcctcttc tcgtcagccc ctgctatgct agaggcatta tatagtttaa tagtattttc 4200
ccctacagaa caaattccta gccttattta aacagccacc tgtaacaaac cctgcagcct 4260
attcattccg tgaaacactg aacgtgtcct agcgggcttc ctgtaatgca atcccctcct 4320
aaagccctcg ctctgctctt gggatttaaa agggtaataa taaaaatata ctgccctcaa 4380
tttgtaaaac tattaagagg ggtttccagc tctcaacccc aggaaaagaa agcataaatt 4440
aggactgcag aagtcagaga aacgtgtagc ccacatcaca ggcagtattt cttaatggtt 4500
tgagaatcca acctatggga tcgggtacag aactgctagc tagcatttag ctacctgatc 4560
ttagaccatc tgtttagcct caaaagcccc tgttttctca cacattgaaa gcaagctgga 4620
aacaaacaaa cacatctgtg tgtgcgggaa ttggctatgc agctgtaact acaataggca 4680
ggtccctacc atcagatagc taaggtctaa caggactaag ataaacgaaa tgatgcaaca 4740
tcaaagggct gtccataatg gcgcgccata acttcgtata gcatacatta tacgaagtta 4800
tgtttaaaca accgcggcac ttaagtagtt aactttaaat aatgccaatt atttaaagtt 4860
aataggcgat cgcaccatat gaatctcgag gttatgtacc tgactgatga agttcctata 4920
ctttctagag aataggaact tcgaagggtt ccgcaagctc tagtcgagcc ccagctggtt 4980
ctttccgcct cagaagccat agagcccacc gcatccccag catgcctgct attgtcttcc 5040
caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc 5100
agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc 5160
agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca 5220
gtcgaggctg atcagcgagc tctagagaat tgatcccctc agaagaactc gtcaagaagg 5280
cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg 5340
tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga 5400
tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc 5460
accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcatc gccgtcgggc 5520
atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc 5580
agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt 5640
ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca 5700
tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc 5760
ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct 5820
gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca 5880
ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc 5940
cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc 6000
ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat ggccgatccc 6060
atggtttagt tcctcacctt gtcgtattat actatgccga tatactatgc cgatgattaa 6120
ttgtcaacag gctgcaggtc gaaaggcccg gagatgagga agaggagaac agcgcggcag 6180
acgtgcgctt ttgaagcgtg cagaatgccg ggcctccgga ggaccttcgg gcgcccgccc 6240
cgcccctgag cccgcccctg agcccgcccc cggacccacc ccttcccagc ctctgagccc 6300
agaaagcgaa ggagcaaagc tgctattggc cgctgcccca aaggcctacc cgcttccatt 6360
gctcagcggt gctgtccatc tgcacgagac tagtgagacg tgctacttcc atttgtcacg 6420
tcctgcacga cgcgagctgc ggggcggggg ggaacttcct gactagggga ggagtagaag 6480
gtggcgcgaa ggggccacca aagaacggag ccggttggcg cctaccggtg gatgtggaat 6540
gtgtgcgagg ccagaggcca cttgtgtagc gccaagtgcc cagcggggct gctaaagcgc 6600
atgctccaga ctgccttggg aaaagcgcct cccctacccg gtagaatttc gacgacctgc 6660
agccaaagcg ctagagccag acatgataag atacattgat gagtttggac aaaccacaac 6720
tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt 6780
aaccattata agctgcaata aacaagttta gctatccatc agtcgagaat tggctcccac 6840
ggacactccg ccccttctga gataccgcat cagtgtttca ggcttcttcc atgtcccttc 6900
ctgcataatc tcgaccatag acacctgttt ctcggccatg tctattgcgg cacccaccct 6960
ggcggaatgg ccggtccagg tcctatacct ccctttattg ggggtggcgt cccgtttgtt 7020
cagcagcacc caggcatcac tgaaaatctt ctccatggca ggggcggtca ggggcgtggt 7080
agtaatgcgg gctttgtttg accggtgtat aggtgggaag agcacagcgt ctggatgttc 7140
tctaagccca ctgacgtcca gccagtcatt gagcactgct gtggttcgcc gggagagcac 7200
tttgtcaagg cccgcagctg tggtgattgt cttagtgtga ctgatgtgca gggtcactgt 7260
gtctcctgtc tgatccaagt ccccaaccct gatgcgtgat atttcagaca ttctcatgag 7320
ggtgttatag gctacgaaca ggaaagccct gttgcgcagg tcgaccagcc gctcgctcct 7380
gctgagcagc acatccagca gcttcaggtc gtcccagcgc agggggattg cctgtcctgt 7440
cctctcaccc ttttcggttg cggcttctct tctaattctt ctcatggcaa gactcactga 7500
cttgtcgtcg gacaagggtg gcagtccgca gtgggacaga agcatattaa gcatagcata 7560
gtgcttgtcg atggtagtgg aggccaggtc tgcatcgtgc aactgaagaa agtattcgcg 7620
tgccatttca ggactaattg gaaaccatgc aagctgccga gcatggcacc atctggccca 7680
gctatgaaag accaacctca aatccctcaa agtgttagga gcgtacgccc cctggtcatt 7740
cataaacctc atgaagtttt cagcggcctc ctgatactct ttgccgatgt ttcgcaggaa 7800
tccaccagat gaaccactaa taatcagctc agaaaccttc ctcttcttct taggcatggc 7860
cgcaggaaag cagagccctg aagctcccat caccggccaa taagagccaa gcctgcagtg 7920
tgacctcata gagcaatgtg ccagccagcc tgaccccaag ggccctcagg cttgggcaca 7980
ctgtctctag gaccctgaga gaaagacata cccatttctg cttagggccc tgaggatgag 8040
cccaggggtg gcttggcact gaagcaaagg acactggggc tcagctggca gcaaagtgac 8100
caggatgctg aggctttgac ccagaagcca gaggccagag gccaggactt ctcttggtcc 8160
cagtccaccc tcactcagag ctttaccaat gccctctgga tagttgtcgg gtaacggtgg 8220
acgccactga ttctctggcc agcctaggac ttcgccattc cgctgattct gctcttccag 8280
ccactggctg accggttgga agtactccag cagtgccttg gcatccaggg catctgagcc 8340
taccaggtcc ttcagtacct cctgccaggg cctggagcag ccagcctgca acacctgcct 8400
gccaagcaga gtgaccactg tgggcacagg ggacacaggg tggggcccac aacagcacca 8460
ttgtccactt gtccctcact agtaaaagaa ctctagggtt gcggggggtg ggggaggtct 8520
ctgtgaggct ggtaagggat atttgcctgg cccatggagc tagcttggct ggacgtaaac 8580
tcctcttcag acctgaagtt cctatacttt ctagagaata ggaacttcgg aattcgatat 8640
caagctaagc ttgattaact ttaaataatg ccaattattt aaagttagta gcgtcgcacg 8700
tggggtgtcc agtaaatgct cattctcttc caattctttt caagtatgtg atcattactt 8760
agcctgaaac agcccaaagt agggcagcta tctacaattt cattcacata ggaatgttgc 8820
tggaagccag gtacccatag tatatggggt tgccttcagc attaggattc atgaatcttc 8880
catcaacgta tggtcttgat ttaaattcat gttaccgtag ctttctattt caggtccaca 8940
gtgaatctca ataaattcta gttttacagc taccgattca cggatccttt caaacaacat 9000
gtgaggtgaa tgaggacaga tgttgttagt aaccctactt tatagattag tattaaacac 9060
actttaagag ccctttcatc tcttagcact agtcggcaca gatgaggcac ttgatagaag 9120
ctgttggata ctatgattgt tccatcagtt ccaaaaggaa agtcactcca agagctagga 9180
aagagatcat ctcagagttg cactgtggcc aattagattt tgccttctgc ttccttggta 9240
gtgagcaatg ggcgactgga gtttcctggg aaacatcttg gaagaggtga atgagcactc 9300
cactgtcatc ggcagagtct ggctcacagt gctcttcatc ttccgcatcc tcatcctcgg 9360
gacagcagcg gagtttgtgt tgggcgatga gcaatctgat tttgtatgca acacccagca 9420
gccaggctgt gagaatgtct gctacgatga ggcctttccc atctcacaca tccgcctctg 9480
ggtgctgcag atcatcttcg tctccactcc atcgctgatg tacgtggggc acgcggtaca 9540
ccacgttcgc atggaggaga agcgaaagga ccgtgaagct gaggagctct gtcagcagtc 9600
gcgcagcaac gggggtgaga gggtaccaat cgccccagac caggccagca tccggaagag 9660
cagcagcagt agcaaaggca ccaagaagtt ccggctggag ggcacactgc taaggaccta 9720
tgtctgccac atcatcttca agaccctctt tgaggtgggc ttcatcgtgg gccattactt 9780
cctgtatggt ttccgcatcc tgcccctcta tcgctgcagc cggtggccct gccccaatgt 9840
ggtagactgc tttgtatccc ggcctactga gaagaccatc ttcatcctct tcatgttatc 9900
agtcgctttt gtgtcactct tcctcaacat catggagatg agccacctgg gcatgaaagg 9960
aatccggtct gccttcaaga ggcctgtaga gcaaccactg ggggagattg ctgagaagtc 10020
cctccactcc attgcagttt cctccatcca gaaagccaag ggctaccagc ttctagaaga 10080
agagaagatc gtatcacact atttcccttt gacagaggtt ggaatggtgg agaccagccc 10140
tctttcggcc aagcctttta gtcagtttga ggagaagatc ggcacaggac ccctggcaga 10200
tatgtcacgg agttaccaag aaaccctgcc ttcttatgct caggtggggg tccaggaagt 10260
ggagcgggaa gagccgccta tagaagaggc tgtggaaccg gaagtgggag agaagaagca 10320
agaagcagag aaggtggccc cagaagggca ggagacagtt gcagtgccag acagggagag 10380
agtagagacc cctggagtgg ggaaggagga tgagaaagaa gagctgcaag ctgaaaaggt 10440
aaccaagcaa gggctgtctg ctgagaaggc accctcactc tgtccggagc tgacaaccga 10500
tgacaatcgg cccttgagca ggctgagtaa agccagcagc agggccaggt cagatgatct 10560
caccatagac tacaaagacg atgacgacaa gtgaataact tcgtatagca tacattatac 10620
gaagttatgt gcaccctggt gcagaagaaa gatatcccac ctccagccaa tacagaccaa 10680
gataaaccca gctggtgcca atatactttg agtcttatgt gtcccttggt ccacccccac 10740
ccctggtttg atagatgctg ttctagaaca atgcacgctg tacaactagg ggactgcctc 10800
tcccacctgt atgcgctgtc aaggtaaacc cgctgtccta gtgacgttcc tatgcctgcc 10860
ctggtcttct atcccaactg ctgttaggaa ggtggggatg ggggctttgc tttcctcctc 10920
cattctccat ggaaacccat caggtcagaa tcatgattac ctcataacct tttcctaatc 10980
ttctctcgtg ctcactcact cagtcagtga gattctgtac cccagcacgt ccccagcaga 11040
cgccacctac cctacatgag agatctcaaa ctcaaaagcc tataggaccc aggccagtga 11100
tttaaatagc aaatgtttcc atgctgtaat atgacagaga gaggtagggt catgaaagat 11160
ggagagcggg ataacagccc gccccttcca ggcagtggtt tccttgatgc ctgggcatct 11220
ggtttgtttg tttgtttgtt tgtttgtttt tcttggttgt tgttttatga gaaacaaaaa 11280
tataattgga attttaacaa gttaatttta aatacaatat gcggaccaaa tgcaatgtat 11340
ccaaatttaa cctgtgtggt agtaagttgc aatatcagtt ttttacatta atgagcccag 11400
atttgctaag aaattggtct cacttttttt ttaatggtct tggcctattt tttgtggttt 11460
cccaaccgca tcttatcact ttgaaatgtc ttaagtcatc agtccagaga tgcccagagt 11520
tcttgctcac ctttctgttg ggtcactgtg ccccagggtt taaagcaatc gagcctttgt 11580
atcagaaaga attcagccca gagcacagtg agagaacaag tgagctatca gacctctccc 11640
tgtgaaatga aaagttttta ggctttcaag ccaattgcag ttttcttccc tgaagcaaca 11700
aaaatccttt tttaaaattc tcttcttcca acatcagtca acctaaaatc tgcatctcct 11760
ttcaccaagg ctgggagctg gacaagttcc aaactggaag atatctgccc ataggcctct 11820
cctgagggag gcagctctca agcattgctc agacagaatg agcaccaaaa agctgccagc 11880
agaaatgtga caggggggaa acatgtctta ctctattacc tacaaactgg acgctcagtc 11940
agtacccagt ccaagctgta tccctaaaag agatgcagag actagagggg cctctcatag 12000
gcctcatccc ctataagcct tctacaaacc tcactgagcc catcagggga aaaaaatatc 12060
aagtatacgt atatatttca cacagggaac aatttcactc aatcaatcga tttttcagaa 12120
cagatgatat atagatagat aaatgatagt tggatagata gatcagtaga tagagcaatg 12180
atagatagat agatagatag atagatagat agatagatag atagatagat atagatagat 12240
atagatagac agatagacaa acatgttaga tgatatgaat aaaatcaggc gatggtgacc 12300
ttgatatcta actatctgat tttaaaaaaa aagaaggcag aaattcatgg caagaggctt 12360
cctgatcagc ccaggatagc catgcatgca gcccaacaca aaatcataaa agtacagtct 12420
tagtttgtgt aactattgct atgatgaaac accatgatca aaaagcaagc tagggaggaa 12480
agggtttatt tggcttatgt atccacactg tcatctatca ctgaaggaaa cccagacagc 12540
aatttaaaca gggcaggaac caggaggcag gagctgatgc agaagccaca gagggggaac 12600
tgcttattgt catgcttgct cctcatggct tcgcacagct tgcttgctta tagaccccag 12660
gcctaccacc tacaatgggc tggcggccgc gtaccagctt ttgttccctt tagtgagggt 12720
taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 12780
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 12840
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 12900
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 12960
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 13020
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 13080
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 13140
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 13200
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 13260
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 13320
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 13380
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 13440
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 13500
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 13560
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 13620
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 13680
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 13740
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 13800
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 13860
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 13920
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 13980
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 14040
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 14100
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 14160
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 14220
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 14280
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 14340
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 14400
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 14460
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 14520
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 14580
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 14640
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 14700
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 14760
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 14820
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 14880
cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg 14940
ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct 15000
tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc 15060
ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg 15120
atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 15180
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg 15240
tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc 15300
tgatttaaca aaaatttaac gcgaatttta acaaaatatt aa 15342
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gtggccaatt agattttgcc ttct 24
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gatggagtgg agacgaagat gat 23
<210> 5
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
taggcgatga gatctagctg tcgcgatgtg ccaccaagcc tgtgacc 47
<210> 6
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgtatgctat acgaagttat ggcgcgccat tatggacagc cctttgatgt tgc 53
<210> 7
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atttaaagtt agtagcgtcg cacgtggggt gtccagtaaa tgctcattct c 51
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
acacaaactc cgctgctgtc cc 22
<210> 9
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gacagcagcg gagtttgtgt tgggcgatga gcaatctgat ttt 43
<210> 10
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcacttgtcg tcatcgtctt tgtagtctat ggtgagatca tctgacctgg cc 52
<210> 11
<211> 85
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aagacgatga cgacaagtga ataacttcgt atagcataca ttatacgaag ttatgtgcac 60
cctggtgcag aagaaagata tccca 85
<210> 12
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aagggaacaa aagctggtac gcggccgcca gcccattgta ggtggtaggc c 51
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cgactgcatc tgcgtgttcg 20
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gttacatgcg ggatttgctt gagg 24
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cctgtaatgc aatcccctcc taaa 24
<210> 16
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
catcgccttc tatcgccttc t 21
<210> 17
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cagcaccatt gtccacttgt cc 22
<210> 18
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tgggaaaggc ctcatcgtag cag 23
<210> 19
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aatcgagcct ttgtatcaga a 21
<210> 20
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gttgtgtgga attgtgagcg g 21
<210> 21
<211> 1323
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atgggcgact ggagtttcct gggaaacatc ttggaagagg tgaatgagca ctccactgtc 60
atcggcagag tctggctcac agtgctcttc atcttccgca tcctcatcct cgggacagca 120
gcggagtttg tgtggggcga tgagcaatct gattttgtat gcaacaccca gcagccaggc 180
tgtgagaatg tctgctacga tgaggccttt cccatctcac acatccgcct ctgggtgctg 240
cagatcatct tcgtctccac tccatcgctg atgtacgtgg ggcacgcggt acaccacgtt 300
cgcatggagg agaagcgaaa ggaccgtgaa gctgaggagc tctgtcagca gtcgcgcagc 360
aacgggggtg agagggtacc aatcgcccca gaccaggcca gcatccggaa gagcagcagc 420
agtagcaaag gcaccaagaa gttccggctg gagggcacac tgctaaggac ctatgtctgc 480
cacatcatct tcaagaccct ctttgaggtg ggcttcatcg tgggccatta cttcctgtat 540
ggtttccgca tcctgcccct ctatcgctgc agccggtggc cctgccccaa tgtggtagac 600
tgctttgtat cccggcctac tgagaagacc atcttcatcc tcttcatgtt atcagtcgct 660
tttgtgtcac tcttcctcaa catcatggag atgagccacc tgggcatgaa aggaatccgg 720
tctgccttca agaggcctgt agagcaacca ctgggggaga ttgctgagaa gtccctccac 780
tccattgcag tttcctccat ccagaaagcc aagggctacc agcttctaga agaagagaag 840
atcgtatcac actatttccc tttgacagag gttggaatgg tggagaccag ccctctttcg 900
gccaagcctt ttagtcagtt tgaggagaag atcggcacag gacccctggc agatatgtca 960
cggagttacc aagaaaccct gccttcttat gctcaggtgg gggtccagga agtggagcgg 1020
gaagagccgc ctatagaaga ggctgtggaa ccggaagtgg gagagaagaa gcaagaagca 1080
gagaaggtgg ccccagaagg gcaggagaca gttgcagtgc cagacaggga gagagtagag 1140
acccctggag tggggaagga ggatgagaaa gaagagctgc aagctgaaaa ggtaaccaag 1200
caagggctgt ctgctgagaa ggcaccctca ctctgtccgg agctgacaac cgatgacaat 1260
cggcccttga gcaggctgag taaagccagc agcagggcca ggtcagatga tctcaccata 1320
tga 1323
Claims (6)
1.一种先天性白内障疾病动物模型的构建方法,其特征在于,包括以下步骤:
1)针对小鼠GJA8基因的第二个外显子区域设计gRNA靶标位点序列,序列如SEQ IDNO.1所示,体外合成RNA,与Cas9核酸内切酶一起显微注射到小鼠受精卵中;扩增同源臂和cKO区域,分别连接到载体上,最后质粒构建如图9所示;将构建好的质粒电转ES细胞,通过鉴定后再向囊胚中注射阳性克隆,并转移入小鼠体内,出生小鼠即为F0,经发育成熟后,得到发生基因突变C.134G>T的首建者小鼠;
2)将带有突变的首建者小鼠与野生型小鼠交配,得到基因突变的杂合子小鼠F1代;
3)将基因型相同的杂合子小鼠WT/mut-F1代相互交配,得到GJA8-C.134G>T基因突变纯合子小鼠mut/mut-F2代。
2.根据权利要求1所述的一种先天性白内障疾病动物模型的构建方法,其特征在于,构建方法还包括步骤4):将步骤3)的F2代小鼠与Cre工具小鼠进行杂交,从而获得F1’代GJA8-C.134G>Tmut/mut;WT/KO杂合子小鼠,将GJA8-C.134G>Tmut/mut;WT/KO杂合子小鼠进行交配,获得F2’代GJA8KO/KO纯合子非突变小鼠。
3.根据权利要求1所述的一种先天性白内障疾病动物模型的构建方法,其特征在于,步骤1)中同源臂和cKO区域包括第二外显子5’端A片段,B片段,B1片段和3’端D片段;其中A片段大小为3123bp,克隆A片段的引物序列如SEQ ID NO.5和SEQ ID NO.6所示;B片段大小为704bp,克隆B片段的引物序列如SEQ ID NO.7和SEQ ID NO.8所示,B1片段大小为1234bp,克隆B1片段的引物序列如SEQ ID NO.9和SEQ ID NO.10所示;D片段大小为2136bp,克隆D片段的引物序列如SEQ ID NO.11和SEQ ID NO.12所示。
4.根据权利要求3所述的一种先天性白内障疾病动物模型的构建方法,其特征在于,片段分2次连接到载体上,先用NruI/AscI双酶切质粒,将A片段连接至载体;再用PmlI/NotI双酶切质粒,将B片段、B1片段和C片段融合后连接至载体。
5.根据权利要求1所述的一种先天性白内障疾病动物模型的构建方法,其特征在于,得到基因突变的杂合子小鼠F1代后进行DNA测序,确认目标基因发生GJA8-C.134G>T突变从而目标蛋白被失活。
6.由权利要求1-5任一项所述的先天性白内障疾病动物模型的构建方法构建所得的模型小鼠在筛选治疗先天性白内障疾病药物中的应用。
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Non-Patent Citations (4)
Title |
---|
Identification of a Novel GJA8(Cx50) Point Mutation Causes Human Dominant Congenital Cataracts;Xiang-Lian Ge等;《Scientific Reports》;20140218;第4卷;全文 * |
Mus musculus gap junction protein, alpha 8 (Gja8), mRNA, Accession No. NM_008123.2;Xin,L.等;《Genebank数据库》;20120708;第1页"ORIGIN"和第2页比对结果 * |
PREDICTED: Mus musculus hexokinase domain containing 1 (Hkdc1), transcript, Accession No. XR_004936154.1;/;《Genebank数据库》;20200921;第1页"ORIGIN"和第2、3页比对结果 * |
利用DNA同源重组工程和CRISPR/Cas9构建SCN9A人源化小鼠;段秋月;《中国优秀硕士学位论文全文数据库》;20190915(第09期);全文 * |
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