CN112011575A - 一种Hspa12b基因敲除小鼠模型的构建方法 - Google Patents
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Abstract
本发明公开一种Hspa12b基因敲除小鼠模型的构建方法,根据小鼠Hspa12b基因第3‑7外显子序列,基于CRISPR/Cas9系统设计靶向基因sgRNA;sgRNA与Cas9核酸酶的mRNA经体外转录后,显微注射到小鼠的精卵中,注射后的受精卵胚胎移植到假孕母鼠体内,获得F0小鼠;提取F0代小鼠尾部DNA,经PCR扩增产物测序,鉴定基因型,获得阳性F0代小鼠;将F0小鼠分别与野生型小鼠交配,获得杂合子小鼠F1代,并进行鼠尾鉴定;将F1代小鼠杂交获得F2代纯合子小鼠。本发明小鼠模型的构建方法研究HSPA12B特异性表达于血管内皮细胞中,参与调控内皮细胞的迁移、增殖和血管新生,适合用Hspa12b基因条件性敲除动物模型研究心血管问题,为诊断、治疗提供理论基础。
Description
技术领域
本发明涉及一种小鼠模型的构建方法,具体是一种Hspa12b基因敲除小鼠模型的构建方法。
背景技术
热休克蛋白(Heat Shock Proteins,HSPs)是在几乎所有物种中均存在的进化上高度保守的蛋白质,作为分子伴侣在细胞周期调控、细胞信号转导、细胞结构维持等多种生命活动中发挥重要作用。HSP70是HSPs家族中最保守、含量最丰富、最重要的一类,正常情况下在细胞内呈基础表达,当机体受到有害刺激时,HSP70表达迅速增加,修复蛋白质损伤,抑制细胞损伤通路。糖尿病患者血清HSP70水平有明显升高,并且与内皮功能损伤和动脉粥样硬化指标密切相关。热休克蛋白A12B(Heat Shock Protein A12B,Hspa12b)是HSP70家族的新成员,由Han等首次从人胸主动脉粥样硬化斑块中克隆得到。后续研究证实,HSPA12B特异性表达于血管内皮细胞中,参与调控内皮细胞的迁移、增殖和血管新生。内皮细胞损伤模型中,Hspa12b表达有明显升高,过表达HSPA12B可以通过抑制内皮细胞的炎症反应,减轻血管内皮功能损伤,发挥心血管保护作用。因此,利用Hspa12b基因条件性敲除动物模型研究心血管问题,为诊断、治疗提供理论基础是必要的。
发明内容
本发明的目的在于提供一种Hspa12b基因敲除小鼠模型的构建方法,将CRI SPR/Cas9应用于小鼠Hspa12b基因的敲除,获得到Hspa12b基因敲除的小鼠,为进一步研究心血管等问题及基因治疗提供经济、简单、可靠的动物模型。
本发明的目的可以通过以下技术方案实现:
一种Hspa12b基因敲除小鼠模型的构建方法,所述方法包括如下步骤:
1)根据小鼠Hspa12b基因第3-7外显子序列,基于CRISPR/Cas9系统设计靶向基因sgRNA;
2)所述sgRNA与Cas9核酸酶的mRNA经体外转录后,显微注射到小鼠的精卵中,注射后的受精卵胚胎移植到假孕母鼠体内,获得F0小鼠;
3)提取F0代小鼠尾部DNA,经PCR扩增产物测序,鉴定基因型,获得阳性F0代小鼠;
ID | Gender | Color | DOB | Gen |
2 | ♂ | B | 2018/6/4 | F0 |
9 | ♂ | B | 2018/6/4 | F0 |
17 | ♀ | B | 2018/6/4 | F0 |
27 | ♀ | B | 2018/6/4 | F0 |
4)将F0小鼠分别与野生型小鼠交配,获得杂合子小鼠F1代,并进行鼠尾PCR和测序鉴定,37,40,42,43,44号小鼠均杂合子小鼠F1代。
5)将F1代小鼠杂交获得F2代纯合子小鼠,即为小鼠动物模型。
进一步地,所述用于敲除小鼠Hspa12b基因的CRISPR-Cas9系统,CRISPR-Cas9系统中sgRNA作用位点位于Hspa12b基因的第3-7外显子上,sgRNA作用位点的DNA序列为5'-GCTCTAGCC CAGAGA GGTCCC CGGTGC CCAGCC CTCCCG GCTCCC CGAGGA CCCAGG AAAGCT GTGGTATCGCTC CCCTCA CGCCTT CACAGT CTCCA-3'和5'-AAGC CAGAGG CCCGAG CTCTAC AGCAGGCTTCCT TCTCTG TGGTTG TGGCCA TCGACT TTGGAA CCACAT CCAGTG GCTATG CCTTCA GCTTTGCTAGTG ACCCTG AGGCCA TTCACA TGATGA G-3'和5'-GAA ATGGGA GGGAGG GGACCC AGGCGTGGCCCA CCAGAA GACCCC CACTTG CCTGCT GCTGAC CCCAGA AGGCAT CTTTCA CAGTTT TGGCTACACTGC CCGTGA CTACTA CCACGA CCTAGA CCCTGA GGAGGC TCGGGA CTGGCT CTACTT TGAGAAGTTTAA GATGAA GATTCA CAGTGC CACT-3'和5'-GAT CTCACC TTGAAG ACCCAG CTCGAGGCGGTA AATGGG AAGAAG ATGCTG GCTCTG GAAGTG TTTGCC CATGCC CTCCGT TTCTTC AAGGAGCACGCC CTTCAG-3'和5'-GGAGCT GAGAGA GCAGAG CGAGTG TATGCT TGAGAA GGGTGC TGTGCGCTGGGT GCTGAC AGTACC TGCCAT CTGGAA ACAACC GGCTAA ACAGTT CATGAG AGAGGC CGCCTACCTG-3'。
进一步地,所述基于CRISPR/Cas9系统的sgRNA表达载体为打靶载体,sgRNA作用位点位于小鼠Hspa12b基因的第3-7外显子,sgRNA作用位点的DNA序列为5'-CCAG GTGATATATAAG TCAGCA T-3'和5'-CCAGG GAGACG GTTACT TCTGGC TAATCC AGG-3'和5'-CCAAGATAAGGA GTACTG TTCAT-3'和5'-GGA CTCTTG GAATGG GACGAA GG-3'。
进一步地,所述CRISPR-Cas9和打靶载体在制备Hspa12b基因敲除的细胞系中的应用。
进一步地,所述Hspa12b基因敲除的细胞系是用CRISPR-Cas9系统和打靶载体转染细胞,获得的中靶阳性细胞克隆。
所述细胞系包括但不限于体细胞和生殖细胞。
进一步地,所述细胞系在制备Hspa12b基因敲除小鼠模型中的应用。
进一步地,选用F3代及以后获得稳定遗传性状的小鼠进行后续实验。
进一步地,所述用于PCR鉴定的特异性引物:
Hspa12b-F1:5'-GCTGTGGATATCCCGAGACACAGAG-3'
Hspa12b-R1:5'-ACAACCATAGCCACCCATCTGGC-3'
Hspa12b-F2:5'-AGCTAGAGAGCAGTCACTCCAACTC-3'
Hspa12b-R2:5'-GCCTATACCAGAATTTGAAGTCCCT-3'
所述PCR反应体系:
reaction component | Volume(μl) |
2×Taq Master Mix | 12.5 |
ddH2O | 9.5 |
Primer A(10pmol/μl) | 1 |
Primer B(10pmol/μl) | 1 |
Template(≈100ng/μl) | 1 |
所述PCR程序:
本发明的有益效果:
本发明小鼠模型的构建方法,根据小鼠Hspa12b基因第3-7外显子序列,基于CRISPR/Cas9系统设计靶向基因sgRNA,sgRNA与Cas9核酸酶的mRNA经体外转录,注射到小鼠精卵中,经多次交配,获得小鼠模型,Hspa12b基因敲除的小鼠模型,HSPA12B特异性表达于血管内皮细胞中,参与调控内皮细胞的迁移、增殖和血管新生。内皮细胞损伤模型中,Hspa12b表达有明显升高,过表达HSPA12B能通过抑制内皮细胞的炎症反应,减轻血管内皮功能损伤,发挥心血管保护作用,为进一步研究心血管疾病及基因治疗提供经济、简单、可靠的动物模型。
附图说明
下面结合附图对本发明作进一步的说明。
图1是本发明PCR策略示意图;
图2是本发明Hspa12b基因扩增产物琼脂糖凝胶电泳示意图;
图3是本发明DNA marker示意图;
图4是本发明野生型基因序列示意图;
图5是本发明野生型基因序列示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,实施本发明的过程、条件、实验方法等,除以下专门提及的内容外,均为本领域的普遍知识和常识。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例1:Hspa12b基因敲除小鼠模型的构建:
1、根据小鼠Hspa12b基因第3-7外显子序列,基于CRISPR/Cas9系统设计靶向基因sgRNA,CRISPR-Cas9系统中sgRNA作用位点位于Hspa12b基因的第3-7外显子上,如图4、图5所示。
2、sgRNA与Cas9核酸酶的mRNA经体外转录后,显微注射到小鼠的精卵中,注射后的受精卵胚胎移植到假孕母鼠体内,获得F0小鼠。
3、提取F0代小鼠尾部DNA,经PCR扩增产物测序,鉴定其基因型,获得如下阳性F0代小鼠。
ID | Gender | Color | DOB | Gen |
2 | ♂ | B | 2018/6/4 | F0 |
9 | ♂ | B | 2018/6/4 | F0 |
17 | ♀ | B | 2018/6/4 | F0 |
27 | ♀ | B | 2018/6/4 | F0 |
4、将F0小鼠分别与野生型小鼠交配,获得杂合子小鼠F1代,并进行鼠尾PCR和测序鉴定,如图2、图3所示,37,40,42,43,44号小鼠均杂合子小鼠F1代。
5、将F1代小鼠杂交获得F2代纯合子小鼠,即为小鼠动物模型。
6、本发明选用F3代及以后获得稳定遗传性状的小鼠进行后续实验。
二、小鼠基因型PCR鉴定结果,如图1所示:
野生型:①PCR反应未获得~668bp条带,②PCR反应能获得405bp条带;
杂合子:①PCR反应能获得~668bp条带,②PCR反应能获得405bp条带;
纯合子:①PCR反应能获得~668bp条带,②PCR反应未获得405bp条带。
用于PCR鉴定特异性引物包括:
PCR反应体系:
reaction component | Volume(μl) |
2×Taq Master Mix | 12.5 |
ddH2O | 9.5 |
Primer A(10pmol/μl) | 1 |
Primer B(10pmol/μl) | 1 |
Template(≈100ng/μl) | 1 |
PCR程序
在本说明书的描述中,参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。
序列表
<110> 南京医科大学附属逸夫医院
<120> 一种Hspa12b基因敲除小鼠模型的构建方法
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 98
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 1
gctctagccc agagaggtcc ccggtgccca gccctcccgg ctccccgagg acccaggaaa 60
gctgtggtat cgctcccctc acgccttcac agtctcca 98
<210> 2
<211> 125
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 2
aagccagagg cccgagctct acagcaggct tccttctctg tggttgtggc catcgacttt 60
ggaaccacat ccagtggcta tgccttcagc tttgctagtg accctgaggc cattcacatg 120
atgag 125
<210> 3
<211> 187
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 3
gaaatgggag ggaggggacc caggcgtggc ccaccagaag acccccactt gcctgctgct 60
gaccccagaa ggcatctttc acagttttgg ctacactgcc cgtgactact accacgacct 120
agaccctgag gaggctcggg actggctcta ctttgagaag tttaagatga agattcacag 180
tgccact 187
<210> 4
<211> 105
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 4
gatctcacct tgaagaccca gctcgaggcg gtaaatggga agaagatgct ggctctggaa 60
gtgtttgccc atgccctccg tttcttcaag gagcacgccc ttcag 105
<210> 5
<211> 118
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 5
ggagctgaga gagcagagcg agtgtatgct tgagaagggt gctgtgcgct gggtgctgac 60
agtacctgcc atctggaaac aaccggctaa acagttcatg agagaggccg cctacctg 118
<210> 6
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 6
ccaggtgata tataagtcag cat 23
<210> 7
<211> 32
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 7
ccagggagac ggttacttct ggctaatcca gg 32
<210> 8
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 8
ccaagataag gagtactgtt cat 23
<210> 9
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 9
ggactcttgg aatgggacga agg 23
<210> 10
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 10
gctgtggata tcccgagaca cagag 25
<210> 11
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 11
acaaccatag ccacccatct ggc 23
<210> 12
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 12
agctagagag cagtcactcc aactc 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 13
gcctatacca gaatttgaag tccct 25
<210> 14
<211> 98
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 14
gctctagccc agagaggtcc ccggtgccca gccctcccgg ctccccgagg acccaggaaa 60
gctgtggtat cgctcccctc acgccttcac agtctcca 98
<210> 15
<211> 125
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 15
aagccagagg cccgagctct acagcaggct tccttctctg tggttgtggc catcgacttt 60
ggaaccacat ccagtggcta tgccttcagc tttgctagtg accctgaggc cattcacatg 120
atgag 125
<210> 16
<211> 187
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 16
gaaatgggag ggaggggacc caggcgtggc ccaccagaag acccccactt gcctgctgct 60
gaccccagaa ggcatctttc acagttttgg ctacactgcc cgtgactact accacgacct 120
agaccctgag gaggctcggg actggctcta ctttgagaag tttaagatga agattcacag 180
tgccact 187
<210> 17
<211> 105
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 17
gatctcacct tgaagaccca gctcgaggcg gtaaatggga agaagatgct ggctctggaa 60
gtgtttgccc atgccctccg tttcttcaag gagcacgccc ttcag 105
<210> 18
<211> 118
<212> DNA
<213> 老鼠(C57/B6小鼠)
<400> 18
ggagctgaga gagcagagcg agtgtatgct tgagaagggt gctgtgcgct gggtgctgac 60
agtacctgcc atctggaaac aaccggctaa acagttcatg agagaggccg cctacctg 118
<210> 19
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 19
ccaggtgata tataagtcag cat 23
<210> 20
<211> 32
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 20
ccagggagac ggttacttct ggctaatcca gg 32
<210> 21
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 21
ccaagataag gagtactgtt cat 23
<210> 22
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 22
ggactcttgg aatgggacga agg 23
<210> 23
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 23
gctgtggata tcccgagaca cagag 25
<210> 24
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 24
acaaccatag ccacccatct ggc 23
<210> 25
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 25
agctagagag cagtcactcc aactc 25
<210> 26
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 26
gcctatacca gaatttgaag tccct 25
<210> 27
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 27
gctgtggata tcccgagaca cagag 25
<210> 28
<211> 23
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 28
acaaccatag ccacccatct ggc 23
<210> 29
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 29
agctagagag cagtcactcc aactc 25
<210> 30
<211> 25
<212> DNA
<213> 人工序列(C57/B6小鼠)
<400> 30
gcctatacca gaatttgaag tccct 25
Claims (8)
1.一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,所述方法包括如下步骤:
1)根据小鼠Hspa12b基因第3-7外显子序列,基于CRISPR/Cas9系统设计靶向基因sgRNA;
2)所述sgRNA与Cas9核酸酶的mRNA经体外转录后,显微注射到小鼠的精卵中,注射后的受精卵胚胎移植到假孕母鼠体内,获得F0小鼠;
3)提取F0代小鼠尾部DNA,经PCR扩增产物测序,鉴定基因型,获得阳性F0代小鼠;
4)将F0小鼠分别与野生型小鼠交配,获得杂合子小鼠F1代,并进行鼠尾鉴定;
5)将F1代小鼠杂交获得F2代纯合子小鼠,即为小鼠动物模型。
2.根据权利要求1所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,所述用于敲除小鼠Hspa12b基因的CRISPR-Cas9系统,CRISPR-Cas9系统中sgRNA作用位点位于Hspa12b基因的第3-7外显子上,sgRNA作用位点的DNA序列为5'-GCT CTAGCC CAGAGAGGTCCC CGGTGC CCAGCC CTCCCG GCTCCC CGAGGA CCCAGG AAAGCT GTGGTA TCGCTC CCCTCACGCCTT CACAGT CTCCA-3'和5'-AAGC CAGAGG CCCGAG CTCTAC AGCAGG CTTCCT TCTCTGTGGTTG TGGCCA TCGACT TTGGAA CCACAT CCAGTG GCTATG CCTTCA GCTTTG CTAGTG ACCCTGAGGCCA TTCACA TGATGA G-3'和5'-GAA ATGGGA GGGAGG GGACCC AGGCGT GGCCCA CCAGAAGACCCC CACTTG CCTGCT GCTGAC CCCAGA AGGCAT CTTTCA CAGTTT TGGCTA CACTGC CCGTGACTACTA CCACGA CCTAGA CCCTGA GGAGGC TCGGGA CTGGCT CTACTT TGAGAA GTTTAA GATGAAGATTCA CAGTGC CACT-3'和5'-GAT CTCACC TTGAAG ACCCAG CTCGAG GCGGTA AATGGGAAGAAG ATGCTG GCTCTG GAAGTG TTTGCC CATGCC CTCCGT TTCTTC AAGGAG CACGCC CTTCAG-3'和5'-GGAGCT GAGAGA GCAGAG CGAGTG TATGCT TGAGAA GGGTGC TGTGCG CTGGGT GCTGACAGTACC TGCCAT CTGGAA ACAACC GGCTAA ACAGTT CATGAG AGAGGC CGCCTA CCTG-3'。
3.根据权利要求2所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,打靶载体是基于CRISPR/Cas9系统的sgRNA表达载体,sgRNA作用位点位于小鼠Hspa12b基因的第3-7外显子,sgRNA作用位点的DNA序列为5'-CCAG GTGATA TATAAG TCAGCA T-3'和5'-CCAGG GAGACG GTTACT TCTGGC TAATCC AGG-3'和5'-CCAAGA TAAGGA GTACTG TTCAT-3'和5'-GGA CTCTTG GAATGG GACGAA GG-3'。
4.根据权利要求3所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,所述CRISPR-Cas9和打靶载体在制备Hspa12b基因敲除的细胞系中的应用。
5.根据权利要求4所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,所述Hspa12b基因敲除的细胞系是用CRISPR-Cas9系统和打靶载体转染细胞,获得的中靶阳性细胞克隆。
6.根据权利要求5所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,所述细胞系在制备Hspa12b基因敲除小鼠模型中的应用。
7.根据权利要求1所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,选用F3代及以后获得稳定遗传性状的小鼠进行后续实验。
8.根据权利要求1所述的一种Hspa12b基因敲除小鼠模型的构建方法,其特征在于,所述用于PCR鉴定的特异性引物:
Hspa12b-F1:5'-GCTGTGGATATCCCGAGACACAGAG-3'
Hspa12b-R1:5'-ACAACCATAGCCACCCATCTGGC-3'
Hspa12b-F2:5'-AGCTAGAGAGCAGTCACTCCAACTC-3'
Hspa12b-R2:5'-GCCTATACCAGAATTTGAAGTCCCT-3'。
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