CN110250108B - Rprm基因敲除小鼠模型及其构建方法与应用 - Google Patents
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Abstract
本发明揭示了一种RPRM基因敲除小鼠模型,所述小鼠模型是被敲除了RPRM(又名Reprimo)基因的小鼠,同时揭示了一种RPRM基因敲除小鼠模型的构建方法,通过对小鼠待敲除基因的靶位点的确定及sgRNA的设计,并进行扩增、活性测试后,将有活性的sgRNA和Cas9注入小鼠体内进行小鼠模型的建立。本发明获得的RPRM基因敲除小鼠为RPRM在胚胎发育中的功能、对组织和个体放射敏感性的影响及其在肿瘤发生发展中的作用等方面的研究提供可靠的动物模型。
Description
技术领域
本发明涉及一种RPRM基因敲除小鼠模型及其构建方法与应用,属于生物技术领域。
背景技术
RPRM基因家族包括RPRM、RPRML、RPRM3,其中RPRM和RPRML 在大多数脊椎动物中都有表达。实验和临床证据表明,只有RPRM在人类肿瘤发生过程中具有重要的生物学功能。RPRM是含有单一外显子不含有内含子的基因,其基因组DNA位于染色体2q23.2的负股链上,有1.4kb,编码109个氨基酸,表达产物长度为11.774kDa。RPRM是一个高度糖基化蛋白,在第7和第18个氨基酸具有两个N端糖基化位点,在第82个氨基酸上有一个预测的 sumo化位点,在第98个丝氨酸上具有一个可能的磷酸化位点。RPRM主要定位于细胞质中。
RPRM基因在人体多种组织器官中均有表达,尤其是在血管和脑组织、内分泌组织、肌肉组织、生殖***及消化***组织中表达较高,提示其在人类胚胎组织发育中可能发挥重要功能。RPRM最初是从经过X射线照射后的含有野生型p53的小鼠胚胎成纤维细胞中首次分离发现的。功能学研究表明,RPRM 是p53的一个转录调控靶点,其诱导表达后通过抑制Cdc2·CyclinB1的核转运而引起细胞G2/M期阻滞。进一步的研究发现RPRM通过引发细胞周期阻滞、抑制细胞增殖、促进细胞凋亡等多重机制参与调控细胞生长,在调节细胞和组织的动态平衡包括增殖、细胞存活等的信号通路中起着重要作用。并且,在肿瘤细胞中外源性高表达RPRM之后,肿瘤细胞增殖受到明显抑制,表现为克隆形成能力降低。除此之外,高表达RPRM的肿瘤细胞侵袭、迁移能力减弱,凋亡增加。在联合DNA损伤诱导剂或者电离辐射的情况下,RPRM促进肿瘤细胞凋亡,抑制细胞生长的作用更加明显,肿瘤生长受限,肿瘤体积明显缩小,提示RPRM具有增加肿瘤细胞DNA损伤和放射敏感性的作用。另外,对临床样本的研究显示RPRM在胃癌、乳腺癌、头颈部肿瘤、结直肠癌等多种肿瘤中由于其启动子异常高度甲基化而导致RPRM在这些肿瘤组织中低表达甚至缺失。这些研究结果提示RPRM是一个抑癌基因。尽管已有以上研究结果,但是目前对于RPRM及其家族尚存在很多未知功能及其调控机制需要进一步探索。
对RPRM的功能及相关机制的研究对于更好地理解人类胚胎发育、组织和个体对电离辐射的敏感性和肿瘤的发生发展等具有重要的理论意义,同时,对于临床肿瘤病人的诊断和治疗也具有非常重要的现实意义。而由于伦理学限制,很多研究无法在人体中进行,为了更深入地研究这一抑癌基因的重要生理学功能需要借助一定的动物模型来模拟人体的情况。而最常见的疾病动物模型和药理实验动物模型就是啮齿类的小鼠。RPRM氨基酸序列在人和小鼠中的相似度高达97.25%(www.ncbi.nlm.nih.gov),具有高度保守性,可作为研究 RPRM功能的良好的动物模型。且目前还没有关于RPRM基因敲除小鼠模型的报道。
发明内容
鉴于现有技术存在上述缺陷,本发明采用第三代基因编辑技术 CRISPR/Cas9***,通过Cas9蛋白结合成熟的crRNA形成复合物,实现对 RPRM基因序列的识别和切割,构建RPRM基因敲除小鼠。由于CRISPR/Cas9 ***的高精准和易操作等优势已成为实现基因编辑和动物模型构建的最好方法。
本发明的目的,将通过以下技术方案得以实现:
一种RPRM基因敲除小鼠模型,所述小鼠模型是被敲除了RPRM基因的小鼠。
优选地,一种RPRM基因敲除小鼠模型的构建方法,包括如下步骤:
S1、确定小鼠待敲除基因的靶位点,设计针对RPRM-s1、RPRM-s2、RPRM-s3、RPRM-s4的sgRNA,所述RPRM-s1的sgRNA序列如SEQ ID NO.1 所示;所述RPRM-s2的sgRNA序列如SEQID NO.2所示;所述RPRM-s3的 sgRNA序列如SEQ ID NO.3所示;所述RPRM-s4的sgRNA序列如SEQ ID NO.4所示;
S2、将S1中设计的sgRNA进行体外扩增构建表达sgRNA的质粒;
S3、sgRNA体外活性测试;
S4、将有活性的sgRNA和Cas9通过显微注射直接注入小鼠受精卵中;取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0代小鼠;
S5、F0代小鼠与正常野生型雌鼠回交后得到F1代敲除RPRM基因的小鼠。
优选地,所述S2包括如下步骤:
S21、将S1中设计好的sgRNA以梯度降温的方式退火成双链后,与质粒连接,以DH5α大肠杆菌为载体进行转化操作,进行平板涂板;
S22、将S21中的平板在培养箱中孵育过夜,选取单克隆接种于培养液中,并收集菌液进行质粒提取,进行PCR鉴定阳性克隆。
优选地,所述S3中采用Precut pSG-target Cloning kit&SSA assay试剂盒对sgRNA的剪切活性进行体外检测。
优选地,所述S4中的小鼠采用C57BL/6J品系小鼠。
优选地,一种RPRM基因敲除小鼠模型在抑癌基因模型中的应用。
本发明产生的技术效果是:本发明获得的RPRM基因敲除小鼠为RPRM 在胚胎发育中的功能、对组织和个体放射敏感性的影响及其在肿瘤发生发展中的作用等方面的研究提供可靠经济的动物模型。
以下便结合实施例附图,对本发明的具体实施方式作进一步的详述,以使本发明技术方案更易于理解、掌握。
附图说明
图1:本发明基因敲除策略图。
图2:本发明中实施例中基因敲除策略图。
图3:对F0代雄鼠与正常野生型雌鼠回交后得到F1代小鼠进行电泳鉴定图。
具体实施方式
下面通过具体实施例对本发明的方法进行说明,以使本发明技术方案更易于理解、掌握,但本发明并不局限于此。下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
本发明揭示了一种RPRM基因敲除小鼠模型,所述小鼠模型是被敲除了 RPRM(又名Reprimo)基因的小鼠。该小鼠模型基因敲除策略如图1所示,所述小鼠为C57BL/6J品系,敲除基因名称(NCBI号):Reprimo (67874),敲除基因名称(MGI号):Rprm(1915124)。敲除的外显子 (exon):exon1。
本发明还揭示了一种RPRM基因敲除小鼠模型的建立方法,包括如下步骤:
首先,确定待敲除基因的靶位点和sgRNA的设计:设计针对RPRM-s1、 RPRM-s2、RPRM-s3、RPRM-s4的sgRNA,所述RPRM-s1的sgRNA序列如 SEQ ID NO.1所示;所述RPRM-s2的sgRNA序列如SEQ ID NO.2所示;所述 RPRM-s3的sgRNA序列如SEQ ID NO.3所示;所述RPRM-s4的sgRNA序列如SEQ ID NO.4所示。其中RPRM-s1的序列为反向序列。
野生型RPRM基因序列为SEQ ID NO.5,其下划线部分为外显子,外显子序列为SEQID NO.6。
SEQ ID NO.5:
然后,对设计的sgRNA体外扩增后构建表达sgRNA的质粒:将设计好的 sgRNA以梯度降温的方式退火成双链后与质粒连接,所述质粒为大肠杆菌。以 DH5α大肠杆菌为载体进行转化操作,进行LB平板涂板。平板置于37℃细菌培养箱中孵育过夜。随后在无菌操作台挑取平板上的单克隆10个,接种于LB 培养液中,在37℃,160rpm恒温摇床中培养过夜。之后收集菌液进行质粒提取,同时进行PCR鉴定阳性克隆。
接着,对sgRNA进行体外活性测试:采用Precut pSG-target Cloning kit& SSAassay试剂盒对sgRNA的剪切活性进行体外检测。SSA胞报告质粒中的荧光素酶基因由于终止密码子的作用被提前终止,这种截短的荧光素酶没有生物活性。而将Cas9/sgRNA的靶点序列***到终止密码子之后,在Cas9/sgRNA 的作用下,靶点序列被有效剪切形成DNA双链断裂位点,通过细胞同源重组修复途径形成没有终止密码子的有活性的荧光素酶,荧光素酶的活性升高即反应Cas9/sgRNA的剪切活性。
将有活性的sgRNA和Cas9通过显微注射直接注入C57BL/6J小鼠受精卵中,待F0代小鼠出生后进行基因型鉴定。
所述F0代小鼠的基因鉴定包括如下步骤:
1)剪尾提取小鼠基因组DNA:
原理:DNA 是染色体的组成成分,遗传的物质基础,是研究遗传、疾病发生的基础。所以研究的前提即为DNA的提取。在蛋白酶K及SDS在 EDTA存在的条件下,裂解细胞、消化蛋白质,使核蛋白解聚、细胞内存在的 DNA酶失活,在酚-氯仿有机溶剂抽提以后,用乙醇沉淀得到纯化的DNA。保证核酸一级结构的完整性;排除其它分子的污染,将蛋白质/糖/RNA的污染降到最低。
设备:水平离心机、恒温干燥箱、移液器。
试剂耗材:裂解液Tail Digestion Buffer(500ml):1M Tris-HCl(pH 8.0) 25ml、0.5M EDTA100ml、5M NaCl 10ml、20%SDS 25ml、MillQ 340ml,含蛋白酶K(简称PK,10mg/ml),100%乙醇,70%乙醇,TE Buffer (500ml):1M Tris-HCl(pH 8.0)5ml、0.5M EDTA(pH8.0)1ml、MillQ 494ml,酚和氯仿1:1混合溶液(4℃保存)。
操作步骤:
S1、取小鼠尾巴,长不超过0.3cm(同样适用于脚趾,胚胎组织,卵黄膜等),放入1.5ml EP管中。
S2、准备裂解液:每500μl裂解液加5μl蛋白酶K,每个装有尾巴的EP 管分装500μl。
S3、将加好裂解液的EP管放在EP管架上并用保鲜膜将其包裹好放入 55℃的干燥箱中(使用保鲜膜防止EP管受热打开裂解液减少),进行过夜消化。
S4、第二天,用力摇晃EP管使消化好的组织均匀。
S5、每管加入等体积的酚/氯仿混合溶液(500μl)。
S6、用力混匀后,以12,000rpm离心15min。
S7、用1000μl移液枪从离心好的EP管中吸取约300μl上清液到新的EP 管中并将号码标示清楚,在吸取上清液时需要注意一定不要吸到中间层或者下层,如不好操作可将大枪头的尖端剪去,减少吸力可避免将中间蛋白层吸起。
S8、每管中加入2倍于上清体积(400μl)的100%乙醇,上下颠倒混匀,此时应可看到白色丝状的DNA,以12,000rpm离心10min。
S9、弃去上清,再加入800μl 70%乙醇,以12,000rpm离心5min。
S10、弃去上清,小心吸取残留乙醇,晾干(时间大于30min),无酒精味即可。为避免基因组织DNA难溶,不采用DNA-Plus真空抽干。
S11、加入100μl TE溶液溶解DNA,可在55℃或37℃助溶。短期4℃保存,长期-20℃保存。
2)PCR鉴定:
根据图2中的PCR鉴定策略示意图进行PCR鉴定,其结果为,
野生型:①PCR反应未获得~625bp条带,②PCR反应可以获得~532bp条带;
杂合子:①PCR反应可以获得~625bp条带,②PCR反应可以获得~532bp 条带;
纯合子:①PCR反应可以获得~625bp条带,②PCR反应未获得~532bp条带;
具体的,所述PCR鉴定所需信息如表1-表3所示。
表1:PCR引物信息
测序方案:先用KO引物对DNA进行扩增,然后通过DNA琼脂糖凝胶电泳进行鉴定,所述KO引物为GPS00000422-Rprm-KO-tR1,其序列为:
TCTAGCAAGGTTGACTCTGAGAGG,为了确保KO产物的正确性,在切胶回收用测序方案的引物进行扩增,若能扩增出,则就说明KO产物正确。
表2:测序方案所提及引物及引物序列
表3:PCR反应具体条件
3)获得的F0代阳性小鼠信息如表4所示:
表4:F0代小鼠具体信息
编号 | 性别 | 颜色 | 基因型 | 出生日期 | 代数 |
1 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
3 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
4 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
13 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
14 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
19 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
20 | 雄 | 黑 | 阳性 | 2018/10/8 | F0 |
4)F1代小鼠基因敲除鉴定
对F0代雄鼠与正常野生型雌鼠回交后得到F1代小鼠进行电泳鉴定,
鉴定结果如图3所示:
综上,根据PCR策略示意图,经PCR和测序确认,得到两条KO Line小鼠分别为:
Line1:25#,30#,32#:-2636bp/wt,E1完全删除,KO阳性。
Line2:33#,35#,37#,38#,40#:-2741bp/wt,E1完全删除,KO阳性。
阳性F1代小鼠信息如下:
本发明尚有多种具体的实施方式。凡采用等同替换或者等效变换而形成的所有技术方案,均落在本发明要求保护的范围之内。
序列表
<110> 苏州大学
<120> RPRM基因敲除小鼠模型及其构建方法与应用
<160> 6
<170> SIPOSequenceListing 1.0
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<211> 16
<212> DNA
<213> 小向导核糖核酸(RPRM)
<400> 1
gcgctggtca aggtac 16
<210> 2
<211> 20
<212> DNA
<213> 小向导核糖核酸(RPRM)
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cgaaggacac acttaagaga 20
<210> 3
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<212> DNA
<213> 小向导核糖核酸(RPRM)
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gctatagatt agatgg 16
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<213> 小向导核糖核酸(RPRM)
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tcagccatcc attggcct 18
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<212> DNA
<213> 小向导核糖核酸(RPRM)
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gccagtacct tgaccagcgc catctggtac ctgtccacct tacatctgct cagaggtgat 60
agggtggctt cgaaggacac acttaagaga gggtgtgatc cgggtcctct gcaactttca 120
gcctttctct ccaagttctt tacttgctct tcagatcagg agtgcatagt gaaccctgcc 180
tagcccttgg aaatgcagaa ggcacaaagc ctgctgctag gttagctcct atcttagccg 240
cccctctaag ccgagcgggc gcagcaaggc tctatcacca gcaggggccg ctgttcgctt 300
ggagcaggag ccgggccacc gccaaggccc gcgcctccca cggcccctcc ggcccaaccc 360
gccaagttcc aagagctccc agtcgtgcga gcgccgggaa cgagcactgc ctgggcagaa 420
cacagcgcac aggagccagc ttgcggcgcg ccctgaagag gttcttgaga cacttacgga 480
cctgggactt tgaaagggag actgaggagc cctgcgcgca caagtgatca agccagcgct 540
ccgccgggca cctgttcgtc ccgcgctaca gccactctgg tccccacccc tccgatcctc 600
gccttcccgg ctcccgcttg ctctcgtgat gaattcagtg ctgggcaacc agaccgacgt 660
ggctggcctg ttcctggtca acagtagcga ggcgctggag cgcgcggtgc gctgctgcac 720
ccaggcgtcg gtggtgaccg acgatggctt cgccgaggga ggccccgacg agcgcagcct 780
gtacatcatg cgtgtggtgc agatcgcagt catgtgtgtg ctctcgctca ctgtggtttt 840
tggcatcttc ttccttggct gcaacctgct catcaagtcc gaaggcatga tcaacttcct 900
agtgaaggac cggaggccgt ctaaagaggt ggaggcagtg gtcgtgggac cctactgagt 960
ggccctcggg tccccctcgg cgggtgcctc ccttctggca cctccagtac tccgccagcc 1020
gcgatgcacc cctcaccatc acggaccgag caacgcaaac ctgtcggagt cagttttttt 1080
ctcttgactt ctgcctttgg ggaaaaagtg accgatttct gatagctctt cgaaaggccc 1140
caagcaagac tgtacaggag gagcgcccag acttcgggat acagcagcag gtggcaaaag 1200
cagggcgcag aactttggga gtggctgctg gcgtgggata ctgggagatg aaccaggcgg 1260
cgaaggcccc tttccacctg caggtgggct cagcactggg gacagcttga gaggccgagc 1320
aaggcggggt gtggatgggt gtgtattcag aggcccagtg accggaaaaa gtgactgttt 1380
attcgccacg ctgcagacct aattgggaag gaacatagat gcgtgtgggt tttgcgacct 1440
actaagagag tgaaactttg atcgatagta acctgtggtt ttaggggatt tgtgttagtt 1500
tgcttgaata caaatatttg ataagtcttt tgtgtcctag ggcctgtttg cctgcctgcg 1560
ggttagagtt ttttgtcatt ctgggagaac gggtgggggg aggggagtct gagactgtga 1620
atggggtaag tgctttcttt tagtgcattt ccagctgggt ctttatggga ggctagcgct 1680
ccagctacgc ccgggacaaa gatccagagt agaattctta gctcctgtct gcacggttta 1740
cgccacaaaa gtgctcttga tatccgtgac accgtttgac tctttttctt atttaacatt 1800
tccttaataa atgcaacatt taagtgtttt gtttctggct tcctggtagt cattctgttc 1860
tcctggagga ggggaaggtc ggctgcgcag cttctgggtc ccagccagaa ggtttcgcct 1920
ggccatagct gcgattctac ttcatagatc cttagatcca tggaggggag gaggggctgg 1980
acagagagcc cagggatttg tgacacacac gaagccccag gcataaaagc catctcgaaa 2040
acagcggcct ttgttttctc gcatatttat gatttaaaat ttcatttcgt ttcgaatgtc 2100
aggtaggttc tcgcgggaaa ttacacttcc gtgccaggct cctcggggaa ctctaggtta 2160
ccccgctaat gaaaggagga gacaccaagc cagtctttag ggagagcatc ttttccccta 2220
cccacgtcag caggaaggca aggggatgtt gccccagaaa aagaaaagta cagattccca 2280
ggaaaagtag tggtcaaagt tctcaatacc agccagggta agcattgtct ccaagtcaga 2340
agggcttagt tgtttttgac gtattatggt ttctagagaa aggaagataa gcgagcgtga 2400
agcaatgaga aatgtttgag gtgatgaatt aatcatgggt acatgtatta aagcattata 2460
ttgtgtcctg ttaacatatg cagtccatca attaagtaaa acacacacac acacacacac 2520
acacacacac agagagagag agagagagag agagagagag agagagagag actccaaaca 2580
tctgtcttca tcttaagtca gccatccatt ggcctgggcc aagcagaggg ctcaaaggag 2640
ttagctgcag actactacaa accaaactag aacacatggc tggcatggct atagattaga 2700
tgggggccac acagacacat ttagggccca gaaactgaac 2740
<210> 6
<211> 1460
<212> DNA
<213> 小向导核糖核酸(RPRM)
<400> 6
agtcgtgcga gcgccgggaa cgagcactgc ctgggcagaa cacagcgcac aggagccagc 60
ttgcggcgcg ccctgaagag gttcttgaga cacttacgga cctgggactt tgaaagggag 120
actgaggagc cctgcgcgca caagtgatca agccagcgct ccgccgggca cctgttcgtc 180
ccgcgctaca gccactctgg tccccacccc tccgatcctc gccttcccgg ctcccgcttg 240
ctctcgtgat gaattcagtg ctgggcaacc agaccgacgt ggctggcctg ttcctggtca 300
acagtagcga ggcgctggag cgcgcggtgc gctgctgcac ccaggcgtcg gtggtgaccg 360
acgatggctt cgccgaggga ggccccgacg agcgcagcct gtacatcatg cgtgtggtgc 420
agatcgcagt catgtgtgtg ctctcgctca ctgtggtttt tggcatcttc ttccttggct 480
gcaacctgct catcaagtcc gaaggcatga tcaacttcct agtgaaggac cggaggccgt 540
ctaaagaggt ggaggcagtg gtcgtgggac cctactgagt ggccctcggg tccccctcgg 600
cgggtgcctc ccttctggca cctccagtac tccgccagcc gcgatgcacc cctcaccatc 660
acggaccgag caacgcaaac ctgtcggagt cagttttttt ctcttgactt ctgcctttgg 720
ggaaaaagtg accgatttct gatagctctt cgaaaggccc caagcaagac tgtacaggag 780
gagcgcccag acttcgggat acagcagcag gtggcaaaag cagggcgcag aactttggga 840
gtggctgctg gcgtgggata ctgggagatg aaccaggcgg cgaaggcccc tttccacctg 900
caggtgggct cagcactggg gacagcttga gaggccgagc aaggcggggt gtggatgggt 960
gtgtattcag aggcccagtg accggaaaaa gtgactgttt attcgccacg ctgcagacct 1020
aattgggaag gaacatagat gcgtgtgggt tttgcgacct actaagagag tgaaactttg 1080
atcgatagta acctgtggtt ttaggggatt tgtgttagtt tgcttgaata caaatatttg 1140
ataagtcttt tgtgtcctag tggcctgttt gcctgcctgc gggttagagt tttttgtcat 1200
tctgggagaa cgggtggggg gaggggagtc tgagactgtg aatggggtaa gtgctttctt 1260
ttagtgcatt tccagctggg tctttatggg aggctagcgc tccagctacg cccgggacaa 1320
agatccagag tagaattctt agctcctgtc tgcacggttt acgccacaaa agtgctcttg 1380
atatccgtga caccgtttga ctctttttct tatttaacat ttccttaata aatgcaacat 1440
ttaagtgttt tgtttctggc 1460
Claims (6)
1.一种RPRM基因敲除小鼠模型的构建方法,其特征在于:包括如下步骤:
S1、确定小鼠待敲除基因的靶位点,设计针对RPRM-s1、RPRM-s2、RPRM-s3、RPRM-s4的sgRNA,所述RPRM-s1的sgRNA序列如SEQ ID NO.1所示;所述RPRM-s2的sgRNA序列如SEQ IDNO.2所示;所述RPRM-s3的sgRNA序列如SEQ ID NO.3所示;所述RPRM-s4的sgRNA序列如SEQID NO.4所示;
S2、将S1中设计的sgRNA进行体外扩增构建表达sgRNA的质粒;
S3、sgRNA体外活性测试;
S4、将有活性的sgRNA和Cas9通过显微注射直接注入小鼠受精卵中;取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0代小鼠;
S5、F0代小鼠与正常野生型雌鼠回交后得到F1代敲除RPRM基因的小鼠模型。
2.根据权利要求1所述的RPRM基因敲除小鼠模型的构建方法,其特征在于:所述S2包括如下步骤:
S21、将S1中设计好的sgRNA以梯度降温的方式退火成双链后,与质粒连接,以DH5α大肠杆菌为载体进行转化操作,进行平板涂板;
S22、将S21中的平板在培养箱中孵育过夜,选取单克隆接种于培养液中,并收集菌液进行质粒提取,进行PCR鉴定阳性克隆。
3.根据权利要求1所述的RPRM基因敲除小鼠模型的构建方法,其特征在于:所述S3中采用Precut pSG-target Cloning kit & SSA assay试剂盒对sgRNA的剪切活性进行体外检测。
4.根据权利要求1所述的RPRM基因敲除小鼠模型的构建方法,其特征在于:所述S4中的小鼠采用C57BL/6J品系小鼠。
5.一种如权利要求1所述构建方法得的RPRM基因敲除小鼠模型。
6.一种如权利要求5所述的RPRM基因敲除小鼠模型在放射敏感性和抑癌基因模型中的应用。
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