CN112063654B - 一种点突变血小板无力症小鼠模型的构建方法 - Google Patents
一种点突变血小板无力症小鼠模型的构建方法 Download PDFInfo
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Abstract
本发明提供一种点突变血小板无力症小鼠模型的构建方法，包括如下步骤：(1)载体设计和构建、体外转录：(1‑1)根据基因信息及实验要求，设计gRNA和Donor Oligo的序列；(1‑2)合成Donor Oligo，构建gRNA载体；(1‑3)将gRNA载体和Cas9载体进行体外转录；(2)显微注射及F0代鼠的鉴定；(3)F1代鼠的繁殖和鉴定：将步骤(2)中性成熟的阳性F0鼠分别与野生型鼠配繁一代，最终提供至少3只经PCR和测序验证的F1代点突变GT小鼠模型；(4)冷冻保种。本发明首次用CRISPR/Cas9技术精准编辑构建点突变血小板无力症小鼠模型，该模型精准编辑小鼠的基因，比以往通过抗体注射诱导或者基因敲除技术构建的GT小鼠模型更为精准。为深入研究GT的发病机制及探索新的治疗方法提供了很好的模型。
Description
技术领域
本发明属于医学技术领域，具体涉及一种点突变血小板无力症小鼠模型的构建方法。
背景技术
血小板无力症(Glanzmann’s thrombasthenia,GT)是一种单基因遗传病。17号染色体的ITGA2B或ITGB3基因缺陷是其致病原因。当ITGA2B或ITGB3发生突变，血小板膜表面糖蛋白αIIbβ3则出现质或量的异常，使血小板聚集功能异常，引起出血。目前，通过抗体注射诱导或者基因敲除技术构建的GT小鼠模型，不能精准编辑小鼠的基因。
发明内容
本发明要解决的技术问题是提供一种点突变血小板无力症小鼠模型的构建方法，能够精准编辑小鼠的基因，为深入研究GT的发病机制及探索新的治疗方法提供了很好的小鼠模型。
为解决上述技术问题，本发明的实施例提供一种点突变血小板无力症小鼠模型的构建方法，包括如下步骤：
(1)载体设计和构建、体外转录
(1-1)根据基因信息及实验要求，设计gRNA和Donor Oligo的序列；
(1-2)合成Donor Oligo，构建gRNA载体；
(1-3)将gRNA载体和Cas9载体进行体外转录；
(2)显微注射及F0代鼠的鉴定
(2-1)将步骤(1)得到的gRNA和Cas9 mRNA与Donor Oligo共注射到受精卵；
(2-2)将显微注射后的受精卵送回到代孕鼠输卵管中；
(2-3)鼠出生后进行PCR基因分型和序列分析，获得阳性F0鼠；
(3)F1代鼠的繁殖和鉴定
将步骤(2)中性成熟的阳性F0鼠分别与野生型鼠配繁一代，最终提供至少3只经PCR和测序验证的F1代点突变GT小鼠模型；
(4)冷冻保种。
其中，步骤(1-1)中的基因信息如下：
(1-1-1)小鼠的ITGA2B基因gene位于小鼠的11号染色体上(GenBank accessionnumber:NM_010575.2；Ensembl:ENSMUSG00000034664)；
(1-1-2)步骤(1-1-1)的基因共具有30个外显子，起始密码子ATG位于1号外显子，终止密码子TGA位于30号外显子，Q887位于26号外显子；
(1-1-3)选择外显子26为靶向位点。
其中，步骤(1-2)的具体步骤为：
(1-2-1)设计靶向载体和供者寡核甘酸的gRNA，左右侧翼分别结合130bp或120bp的同源序列；
(1-2-2)供者寡核苷酸的Q887X(CAG to TAG)突变位点通过同源配对引进26号外显子；同时引进一个无义突变(CAG to CAA or CTG to TTA)来防止同源配对后gRNA结合并重新切割序列。
其中，步骤(1-1)中，gRNA靶向序列如下：
gRNA1：ATGCCTGTCTGCGCTCACGCTGG；
gRNA2：GCCCTGGCTTGGGCCCCTGCAGG。
其中，步骤(1-1)中，Donor Oligo序列如下：
供者寡核苷酸序列1：
GGTGGACTGGAAACTATCCACGCCCAGCCCTTCTTCCATTCGCCCCGTCCATCACCAACGTGAGCGCAGATAG；
其中，CAA为沉默突变，TAG为突变序列；
GCATTCCTGCAGGGGCCCAAGCCAGGGCAGCAGGACCCAGTTCTGGTGGTGAGAAGGCTC；
供者寡核苷酸序列2：
AAACTATCCACGCCCAGCCCTTCTTCCATTCGCCCCGTCCATCACCAGCGTGAGCGCAGATAGGCATTCTTAC；
其中，TAG为突变序列，TTA为沉默突变。
本发明的上述技术方案的有益效果如下：本发明首次用CRISPR/Cas9技术精准编辑构建点突变血小板无力症小鼠模型，该模型精准编辑小鼠的基因，比以往通过抗体注射诱导或者基因敲除技术构建的GT小鼠模型更为精准。为深入研究GT的发病机制及探索新的治疗方法提供了很好的模型。
附图说明
图1为本发明中靶向策略设计图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
本发明提供一种点突变血小板无力症小鼠模型的构建方法，包括如下步骤：
(1)载体设计和构建、体外转录
(1-1)根据基因信息及实验要求，设计gRNA和Donor Oligo的序列；
其中，基因信息如下：
(1-1-1)小鼠的ITGA2B基因gene位于小鼠的11号染色体上(GenBankaccessionnumber:NM_010575.2；Ensembl:ENSMUSG00000034664)；
(1-1-2)步骤(1-1-1)的基因共具有30个外显子，起始密码子ATG位于1号外显子，终止密码子TGA位于30号外显子，Q887位于26号外显子；
(1-1-3)选择外显子26为靶向位点。
本发明中，小鼠Itga2b基因座的基因组区域如图1所示的靶向策略设计图(基因从左向右，总大小为16.59kb)。实心条表示ORF；开放条表示UTR。
注:野生型等位基因中括号内的序列在构建成功后将被突变等位基因中括号内的序列所取代。
本步骤中，gRNA靶向序列如下：
gRNA1(匹配基因的反向链)：ATGCCTGTCTGCGCTCACGCTGG；
gRNA2(匹配基因的反向链)：GCCCTGGCTTGGGCCCCTGCAGG。
Note：Color schemes are employed to facilitate tracking of sequences。下划线为PAM序列。
gRNA载体在VectorBuilder上的链接如下：
gRNA1:https://en.vectorbuilder.com/vector/VB190829-1209qve.html；
gRNA2:https://en.vectorbuilder.com/vector/VB190830-1077xnt.html。
Donor Oligo序列如下：
供者寡核苷酸序列1：
GGTGGACTGGAAACTATCCACGCCCAGCCCTTCTTCCATTCGCCCCGTCCATCACCAACGTGAGCGCAGATAG；
其中，CAA为沉默突变，TAG为突变序列；
GCATTCCTGCAGGGGCCCAAGCCAGGGCAGCAGGACCCAGTTCTGGTGGTGAGAAGGCTC；
供者寡核苷酸序列2：
AAACTATCCACGCCCAGCCCTTCTTCCATTCGCCCCGTCCATCACCAGCGTGAGCGCAGATAGGCATTCTTAC；
其中，TAG为突变序列，TTA为沉默突变。
(1-2)合成Donor Oligo，构建gRNA载体；具体步骤为：
(1-2-1)设计靶向载体和供者寡核甘酸的gRNA，左右侧翼分别结合130bp或120bp的同源序列；
(1-2-2)供者寡核苷酸的Q887X(CAG to TAG)突变位点通过同源配对引进26号外显子；同时引进一个无义突变(CAG to CAAor CTG to TTA)来防止同源配对后gRNA结合并重新切割序列。
(1-3)将gRNA载体和Cas9载体进行体外转录；
(2)显微注射及F0代鼠的鉴定
(2-1)将步骤(1)得到的gRNA和Cas9 mRNA与Donor Oligo共注射到受精卵；
(2-2)将显微注射后的受精卵送回到代孕鼠输卵管中；
(2-3)鼠出生后进行PCR基因分型和序列分析，获得阳性F0鼠；
(3)F1代鼠的繁殖和鉴定
将步骤(2)中性成熟的阳性F0鼠分别与野生型鼠配繁一代，最终提供至少3只经PCR和测序验证的F1代点突变GT小鼠模型；
本步骤中，测序验证包括：
3.1、用特异性引物PCR扩增小鼠ITGA2B的基因八点。通过PCR后测序来确定其靶向性。
引物序列：
Mouse Itga2b(Q887X)-F:CCCTCGGATCTGCTCTACATCCTG；
Mouse Itga2b(Q887X)-R:AGCGACACACACAGAGAACCTACGTG。
预期的PCR产物大小：
Wildtype allele:431bp；
Mutant allele:431bp。
3.2、脱靶分析
3.2.1、在所有可能的gRNAs中,根据其位点来预测其脱靶可能，选择两个不容易脱靶的gRNA。
3.2.2、脱靶分析基于GRCm38/mm10 assembly(2011)。
表1和表2列出了预测在种子序列最多一个错配以及总错配最多3个或4个的脱靶位点。小写字母表示与目标位点不匹配(表1+2)。
表1:gRNA1的脱靶分析
表2:gRNA2的脱靶分析
3.2.3、脱靶位点的说明：E＝exonic：I＝intronic：-＝intergenic；CRISPRater分数的说明；低效(score<0.56)；中效(0.56<＝score<＝0.74)；高效(score>0)。
(4)冷冻保种。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南通大学附属医院
<120> 一种点突变血小板无力症小鼠模型的构建方法
<141> 2020-09-17
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工合成(Synotype)
<400> 1
atgcctgtct gcgctcacgc tgg 23
<210> 2
<211> 23
<212> DNA
<213> 人工合成(Synotype)
<400> 2
gccctggctt gggcccctgc agg 23
<210> 3
<211> 73
<212> PRT
<213> 未知(Unknown)
<400> 3
Gly Gly Thr Gly Gly Ala Cys Thr Gly Gly Ala Ala Ala Cys Thr Ala
1 5 10 15
Thr Cys Cys Ala Cys Gly Cys Cys Cys Ala Gly Cys Cys Cys Thr Thr
20 25 30
Cys Thr Thr Cys Cys Ala Thr Thr Cys Gly Cys Cys Cys Cys Gly Thr
35 40 45
Cys Cys Ala Thr Cys Ala Cys Cys Ala Ala Cys Gly Thr Gly Ala Gly
50 55 60
Cys Gly Cys Ala Gly Ala Thr Ala Gly
65 70
<210> 4
<211> 60
<212> PRT
<213> 未知(Unknown)
<400> 4
Gly Cys Ala Thr Thr Cys Cys Thr Gly Cys Ala Gly Gly Gly Gly Cys
1 5 10 15
Cys Cys Ala Ala Gly Cys Cys Ala Gly Gly Gly Cys Ala Gly Cys Ala
20 25 30
Gly Gly Ala Cys Cys Cys Ala Gly Thr Thr Cys Thr Gly Gly Thr Gly
35 40 45
Gly Thr Gly Ala Gly Ala Ala Gly Gly Cys Thr Cys
50 55 60
<210> 5
<211> 73
<212> PRT
<213> 未知(Unknown)
<400> 5
Ala Ala Ala Cys Thr Ala Thr Cys Cys Ala Cys Gly Cys Cys Cys Ala
1 5 10 15
Gly Cys Cys Cys Thr Thr Cys Thr Thr Cys Cys Ala Thr Thr Cys Gly
20 25 30
Cys Cys Cys Cys Gly Thr Cys Cys Ala Thr Cys Ala Cys Cys Ala Gly
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Cys Gly Thr Gly Ala Gly Cys Gly Cys Ala Gly Ala Thr Ala Gly Gly
50 55 60
Cys Ala Thr Thr Cys Thr Thr Ala Cys
65 70
<210> 6
<211> 24
<212> DNA
<213> 未知(Unknown)
<400> 6
ccctcggatc tgctctacat cctg 24
<210> 7
<211> 26
<212> DNA
<213> 未知(Unknown)
<400> 7
agcgacacac acagagaacc tacgtg 26
Claims (1)
1.一种点突变血小板无力症小鼠模型的构建方法，其特征在于，包括如下步骤：
(1)载体设计和构建、体外转录
(1-1)根据基因信息及实验要求，设计gRNA和Donor Oligo的序列；
步骤(1-1)中的基因信息如下：
(1-1-1)小鼠的ITGA2B基因gene位于小鼠的11号染色体上；
(1-1-2)步骤(1-1-1)的基因共具有30个外显子，起始密码子ATG位于1号外显子，终止密码子TGA位于30号外显子，Q887位于26号外显子；
(1-1-3)选择外显子26为靶向位点；
步骤(1)中的gRNA靶向序列如下：
gRNA1：ATGCCTGTCTGCGCTCACGCTGG；
gRNA2：GCCCTGGCTTGGGCCCCTGCAGG；
步骤(1-1)中，Donor Oligo序列如下：
供者寡核苷酸序列1：
GGTGGACTGGAAACTATCCACGCCCAGCCCTTCTTCCATTCGCCCCGTCCATCACCAACGTGAGCGCAGATAG；
其中，CAA为沉默突变，TAG为突变序列；
GCATTCCTGCAGGGGCCCAAGCCAGGGCAGCAGGACCCAGTTCTGG TGGTGAGAAGGCTC；
供者寡核苷酸序列2：
AAACTATCCACGCCCAGCCCTTCTTCCATTCGCCCCGTCCATCACCAGCGTGAGCGCAGATAGGCATTCTTAC；
其中，TAG为突变序列，TTA为沉默突变；
(1-2)合成Donor Oligo，构建gRNA载体；
步骤(1-2)的具体步骤为：
(1-2-1)设计靶向载体和供者寡核甘酸的gRNA，左右侧翼分别结合130bp或120bp的同源序列；
(1-2-2)供者寡核苷酸的Q887X突变位点通过同源配对引进26号外显子；同时引进一个无义突变来防止同源配对后gRNA结合并重新切割序列
(1-3)将gRNA载体和Cas9载体进行体外转录；
(2)显微注射及F0代鼠的鉴定
(2-1)将步骤(1)得到的gRNA和Cas9 mRNA与Donor Oligo共注射到受精卵；
(2-2)将显微注射后的受精卵送回到代孕鼠输卵管中；
(2-3)鼠出生后进行PCR基因分型和序列分析，获得阳性F0鼠；
(3)F1代鼠的繁殖和鉴定
将步骤(2)中性成熟的阳性F0鼠分别与野生型鼠配繁一代，最终提供至少3只经PCR和测序验证的F1代点突变GT小鼠模型；
(4)冷冻保种。
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