CN110771573A - PirB基因敲入的小鼠动物模型及其构建方法 - Google Patents
PirB基因敲入的小鼠动物模型及其构建方法 Download PDFInfo
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Abstract
本发明公开了PirB基因敲入的小鼠动物模型,该模型包括确定PirB基因待敲入的特异性靶位点的gRNA1和gRNA2,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内,gRNA1的基因序列如SEQ ID NO.1所示,gRNA2的基因序列如SEQ ID NO.2所示。本发明还具体公开了上述小鼠动物模型的构建方法,本发明的小鼠动物模型对于PirB基因功能的研究和在体验证提供了良好的基础。通过PirB敲入动物模型与不同类型Cre小鼠的杂交,可以用于研究PirB在不同器官或不同细胞类型或不同疾病模型的功能。
Description
技术领域:
本发明属于遗传学和生物技术领域,具体涉及PirB基因敲入的小鼠动物模型,还涉及上述小鼠动物模型的构建方法。
背景技术
鼠源成对免疫球蛋白样受体B(paired immunoglobulin-like receptor B,PirB),是人类免疫球蛋白样受体B2(human leukocyte immunoglobulin(Ig)-likereceptor B2,LILRB2)的同源基因,PirB基因(NCBI reference sequence:NM_011095.2)位于小鼠的7号染色体近端,总大小为8.97kb,编码841个氨基酸,目前鉴定出15个外显子,外显子1起始密码为ATG,外显子15的终止密码子是TGA(转录本:Pirb-201ENSMUST00000078451.6)。PirB蛋白属于I型跨膜糖蛋白,包含由六个免疫球蛋白样结构域(domain,D)组成(D1-D6)的胞外段,一个疏水的跨膜段,三个免疫受体酪氨酸依赖的抑制序列(immunoreceptortyrosinebased inhibitory motifs,ITIMs)和一个ITIMs样序列组成的胞内段。理论上讲,PirB的分子量约为92kD,但是Western-blot分析中,PirB经常位于105kD处,因为PirB是糖蛋白。以往研究发现,PirB在免疫系统和中枢调节中均发挥重要作用。
在免疫系统中,PirB在许多造血细胞都有表达,包括B细胞、肥大细胞、巨噬细胞、粒细胞和树突细胞,但是在胸腺细胞、成熟的T细胞和自然杀伤细胞不表达,另外PirB在髓细胞和B细胞的表达随细胞分化和激活增加。在免疫系统,PirB作为主要组织相容性复合物1(class I major histocompatibility complex,MHC1)的受体,广泛参与免疫调节,包括中性粒细胞和巨噬细胞整合素通路、细胞毒性T淋巴细胞激活、B淋巴细胞激活和体液—细胞免疫应答等。PirB的前两个细胞外免疫球蛋白结构域(D1-D2)介导MHCI和PirB的结合。
在中枢神经系统(central nervous system,CNS),PirB在许多脑区包括皮层、海马、小脑和嗅球都有表达,但在成年脊髓不表达。在细胞层面,PirB不仅表达于神经元,也表达于星形胶质细胞。在许多病理条件下,如脑外伤、中风和CNS感染,PirB的mRNA和蛋白表达水平显著增加。在CNS,PirB是髓鞘抑制因子Nogo-A、MAG、OMgp的受体,参与神经元突起芽发和生长锥塌陷,抑制神经元轴突再生和突触可塑性。PirB的细胞外免疫球蛋白结构域(D3-D6)介导了PirB与NogoA的结合。近年来关于阿尔茨海默病(Alzheimer’s disease,AD)研究还发现,PirB是Aβ42寡聚体的高亲和力受体,亲和力达到纳摩尔水平。PirB的前两个细胞外免疫球蛋白结构域(D1-D2)介导了Aβ42寡聚体和PirB的相互作用,导致丝切蛋白(cofilin)信号通路增强。免疫组织化学染色结果发现,PirB在轴突生长锥表达,位于富含肌动蛋白的前缘和synapsin免疫阳性的囊泡中,在神经元突起的表达呈点状分布。文献表明,PirB表达随年龄增加,特别是在老龄认知损伤的小鼠海马中,PirB能够抑制轴突再生和突触可塑性。研究表明小鼠Aβ寡聚体对海马长时程增强的破坏作用需要PirB的参与,在AD转基因模型中,PirB不仅参与成年小鼠记忆缺失,而且介导幼年小鼠视皮层突触可塑性的丢失。这些研究提示我们,PirB参与突触可塑性,抑制PirB可能对AD起到治疗效应。但是在CNS,PirB的功能和下游抑制性信号通路仍然未被阐明,因此迫切需要PirB的细胞和动物模型。
目前对于PirB基因功能的研究,多采用可溶性的PirB的胞外段(PirBextracellular peptide,PEP)和抑制剂,或者采用慢病毒转染。前者受到是否能够透过血脑屏障和抑制剂效率的影响,后者慢病毒转染在原代细胞和在体的转染效率比较低,使研究PirB的功能受到极大的限制。为解决这些问题,我们通过CRISPR/Cas9技术建立了PirB基因敲入小鼠,将PirB基因敲入C57BL/6J的ROSA26位点,为研究PirB在免疫系统或者神经系统的作用和机制提供了很好的工具。
发明内容
本发明的目的在于提供PirB基因敲入的小鼠动物模型,该模型能够帮助我们研究PirB基因在免疫系统和神经系统的功能以及下游的调节机制。
本发明的另一目的在于提供上述小鼠动物模型的构建方法。
本发明所采用的第一种技术方案是:PirB基因敲入的小鼠动物模型,包括确定PirB基因的待敲入的特异性靶位点gRNA1和gRNA2,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内,所述gRNA1的基因序列如SEQ ID NO.1所示,gRNA2的基因序列如SEQ IDNO.2所示。
本发明所采用的第二种技术方案为:PirB基因敲入的小鼠动物模型的构建方法,具体按照以下步骤实施:
步骤1、基于CRISPR/Cas9技术构建针对C57BL/6J小鼠ROSA26基因的特异性gRNA1和gRNA2,gRNA1的基因序列如SEQ ID NO.1所示,gRNA2的基因序列如SEQ ID NO.2所示;
步骤2、构建“CAG promoter-loxP-Stop-loxP-Kozak-mouse PirbCDS-polyA”基因盒打靶载体,将打靶载体进行线性化处理;
步骤3、步骤2中将含有loxP位点打靶载体、步骤1中有活性的gRNA1和gRNA2与Cas9蛋白共同注射进入代孕小鼠受精卵中,获得F0代小鼠;
步骤4、将步骤3中性成熟的阳性F0小鼠分别与野生型鼠交配繁一代,获得F1代杂合子小鼠,通过PCR、测序和Southern杂交确定动物基因型;
步骤5、将步骤4获得的F1代杂合子小鼠近交获得F2代纯合子小鼠,即为本发明构建的一种PirB基因敲入的小鼠动物模型。
本发明所采用第二种技术方案的特点还在于,
步骤1中得到gRNA1和gRNA2后分别与trancrRNA在25℃孵育10min形成二级结构。
步骤3中gRNA1、gRNA2的浓度均为2~10pmol/ul,Cas9蛋白的注射浓度为30~100ng/μL。
步骤4中Southern杂交采用BamHI和AvrII核酸内切酶切割F1代杂合子小鼠的DNA。
本发明的有益效果是:本发明提供了PirB基因敲入的小鼠动物模型及其构建方法,本发明的小鼠动物模型对于PirB基因功能的研究和在体验证提供了良好的基础。分离自PirB基因敲入小鼠的细胞还可以用于研究PirB发挥调节作用的下游机制。通过PirB敲入动物模型与不同类型Cre小鼠的杂交,可以用于研究AD不同器官或不同细胞类型PirB的调节作用。
附图说明
图1为本发明PirB基因敲入的小鼠插入PirB基因CDS和loxP位点的打靶质粒载体的构建图;
图2为本发明PirB基因敲入的小鼠模型的构建方法的流程图;
图3为本发明F1代PirB敲入的小鼠的基因型PCR结果鉴定电泳图;
图4为本发明F1代PirB敲入的小鼠验证PirB基因敲入效果的测序峰图;
图5为本发明F1代PirB基因敲入的小鼠进行Southern鉴定的方法示意图;
图6为本发明F1代PirB基因敲入的小鼠进行Southern鉴定的结果图。
具体实施方式
下面结合附图和具体实施方式对发明作进一步阐述。
本发明构建了PirB基因敲入的小鼠动物模型,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内。具体来说,构建一个CAG promoter-loxP-Stop-loxP-Kozak-mouse PirB CDS-polyA的基因盒,将其克隆进入ROSA26基因的内含子1中。
ROSA26基因(NCBI Reference Sequence:NR_011095.2)位于小鼠的七号染色体。
本发明PirB基因敲入的小鼠动物模型的构建方法,具体按照以下步骤具体实施:
步骤1、根据小鼠ROSA26基因(Gene Bank:NR_027008.1))序列,利用Cas-Desigher软件在1号内含子设计gRNA靶序列,并搜索小鼠DBM-DB基因组数据库基因,采用CRISPR脱靶效应软件Cas-OFFinder检测潜在的脱靶位点:最终选取两个gRNA,gRNA1的基因序列如SEQID NO.1所示,gRNA2的基因序列如SEQ ID NO.2所示:
SEQ ID NO.1:GGCAGGCTTAAAGGCTAACCTGG
SEQ ID NO.2:CTCCAGTCTTTCTAGAAGATGGG
将gRNA1和gRNA2分别与trancrRNA在25℃孵育10min形成二级结构;
步骤2、利用C57BL/6小鼠文库BAC克隆,通过PCR扩增获得PirB基因CDS的同源臂,采用in-fusion方法构建含有CAG promoter-loxP-Stop-loxP-Kozak-mouse PirB CDS-polyA的基因盒的质粒打靶载体,通过酶切、PCR及测序对打靶质粒载体进行验证,图1为构建好的PirB基因打靶载体的示意图。
步骤3、PMSG处理C57BL/6雌性小鼠(6周龄,平均体重20g),46h后注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤1的gRNA1、gRNA2、Cas9蛋白以及线性化后的打靶载体一起注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即F0代小鼠。
上述步骤中gRNA1、gRNA2的浓度均为2~10pmol/ul,Cas9蛋白的注射浓度为30~100ng/μL,Cas9蛋白购自New England Biolabs,货号为M0386M。
步骤4、提取F0代小鼠尾部DNA,PCR扩增产物测序,鉴定是否为嵌合体。
本发明用于F0代小鼠PirB基因PCR鉴定的特异性引物如下:
PCR Primers 1(Annealing Temperature 60.0℃):
5’arm forward primer(F1):
5’-TACGCCACAGGGAGTCCAAGAATG-3’
5’KI reverse primer(R1):
5’-GATGGGGAGAGTGAAGCAGAACG-3’
PCR Primers 2(Annealing Temperature 60.0℃):
3’KI forward primer(F2):
5’-CTGCTGTCCATTCCTTATTCCATAG-3’
3’arm reverse primer(R2):
5’-CTGGAAATCAGGCTGCAAATCTC-3’。
Primers1对应的扩增产物大小为2.7kb,Primers2对应的扩增产物为2.5kb。
用于F0代小鼠测序的引物如下:
Sequencing Primer for PCR product 1:
5’Sequence primer(F3):
5’-CACTTGCTCTCCCAAAGTCGCTC-3’
Sequencing Primer for PCR product 2:
3’Sequence primer(R3):5’-ATACTCCGAGGCGGATCACAA-3’
步骤4、鉴定为PirB基因敲入的F0代雄性小鼠7周龄、雌性小鼠6周龄分别与野生型异性小鼠交配获得F1代杂合子小鼠,图2为本发明F1代杂合子小鼠PirB基因敲除小鼠的流程图,小鼠出生后20天进行PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞。
(1)通过PCR方法对获得的F1代小鼠进行基因型的鉴定:
(A)DNA的提取:
方法1:采用TaKaRaMiniBEST Universal Genomic DNA Extraction kit(Ver.5.0_Code No.9765)来获得高纯度的基因组DNA。
步骤a.每只小鼠剪下一段尾巴(2~5mm),放入离心管中,加入180μLBufferGL,20μL蛋白激酶K和10μLRNase A。
步骤b.将步骤a离心管内于56℃孵育过夜。
步骤c.过夜后12000rpm离心2min去除杂质。
步骤d.加入200μLBuffer GB和200μL无水乙醇,充分混匀。
步骤e.将吸附柱放在收集管上,将步骤d得到的样品加到吸附柱里,12000rpm离心2min,弃掉收集管中液体。
步骤f.弃去液体后在吸附柱中加入500μLBuffer WA,12000rpm离心1min,弃掉收集管中液体。
步骤g.在收集管中加入700μL Buffer WB,12000rpm离心1min。弃掉收集管中液体。
注意:BufferWB需要与100%乙醇预混,沿管壁加入Buffer WB冲走残余的盐。
步骤h.重复步骤g一次。
步骤i.将步骤h的吸附柱放入收集管,12000rpm离心2min。
步骤j.吸附柱放到一个新的1.5mL的离心管,在吸附柱膜中央加入50~200μL灭菌水或者洗脱液,停留5min。(将无菌水或者洗脱液加热到65℃能够增加洗脱的得率)。
步骤i.基因组DNA定量,洗脱的基因组DNA可以通过电泳鉴定。
方法2:一种低成本基因组DNA的粗提方法:
步骤a.每只小鼠剪下一段尾巴(2-5mm),放入离心管中,加入100μL尾部消化缓冲液,注意不要剪尾太长;
步骤b.将离心管及其内容物于56℃孵育过夜;
步骤c.过夜后将离心管于98℃孵育13min,使蛋白酶K失活;
步骤d.在微型离心机以最大转速旋转15min,上清直接用于PCR体系(每50μLPCR体系加入上清2μL)。
尾消化缓冲液成分:
50mM KCl
10mM Tris-HCl(pH9.0)
0.1%TritonX-100
0.4mg/mL Proteinase K
(B)长链PCR反应:
长链引物:
PCR Primers 1(Annealing Temperature 60.0℃):扩增片段:2.7kb
5’arm forward primer(F1):
5’-TACGCCACAGGGAGTCCAAGAATG-3’
5’KI reverse primer(R1):
5’-GATGGGGAGAGTGAAGCAGAACG-3’
PCR Primers 2(Annealing Temperature 60.0℃):扩增片段:2.5kb
3’KI forward primer(F2):
5’-CTGCTGTCCATTCCTTATTCCATAG-3’
3’arm reverse primer(R2):
5’-CTGGAAATCAGGCTGCAAATCTC-3’
PirB基因敲除型有上述两条扩增片段,野生型等位基因没有扩增产物。
PCR Mix:
反应条件:
PCR扩增产物的电泳鉴定结果如图3所示,从图3中可以看出,6、8、9号为PirB基因敲入小鼠在2.7kb、2.5kb有特异性扩增产物,WT小鼠PCR产物没有这两个扩增产物。
(C)短链PCR反应,引物如下:
Primers(Annealing Temperature 60.0℃):
Forward primer(F4):5’-AACAATCAGGCTGCCGAATCTG-3’
Reverse primer(R4):5’-CTTTATTAGCCAGAAGTCAGATGC-3’
Expected PCR Product:
Targeted allele:344bp
PirB基因敲入型小鼠能够扩增出344bp的产物,而野生型没有。
PCR体系:
反应条件:
(2)F1代小鼠测序:
通过PCR扩增后测序鉴定小鼠基因型,如图4所示,为6号PirB基因敲入小鼠测序峰图,PCR所用引物如下:
Sequencing Primer for PCR product 1:
5’Sequence primer(F3):
5’-CACTTGCTCTCCCAAAGTCGCTC-3’
Sequencing Primer for PCR product 2:
3’Sequence primer(R3):
5’-ATACTCCGAGGCGGATCACAA-3’
(3)Southern杂交检测F1代小鼠基因型:
采用BamHI和AvrII核酸内切酶切割DNA分子,切割示意图如图5。具体的Southern杂交检测过程如下:
步骤a、提取F1代小鼠尾部DNA;
步骤b、制备探针,通过PCR方法将Dig-dUTP渗入到步骤a的DNA分子中;
F1代小鼠southern印记的5’探针引物如下:
Primers for 5’Probe:
5’Probe forward primer:
5’-AAACGTGGAGTAGGCAATACCCAGG-3’
5’Probe reverse primer:
5’-AAAGAAGGGTCACCTCAGTCTCCCT-3’
Primers for 3’Probe:
3’Probe forward primer:
5’-TTCTGGGCAGGCTTAAAGGCTAAC-3’
3’Probe reverse primer:
5’-AGGAGCGGGAGAAATGGATATGAAG-3’
Southern印记的目标片段大小如下:
5’Probe-BamHI:5.83kb-WT,4.80kb-MT
3’Probe-AvrII:5.27kb-WT,11.12kb-MT。
步骤c、采用限制性内切酶BamHI和AvrII对DNA进行切割;琼脂糖凝胶电泳分离DNA;
步骤d、将DNA转到尼龙膜上;
步骤e、探针变性后,与DNA分子进行杂交,NBT/BCIP化学显色法显色,拍照观察。Southern杂交结果如图6所示,可以看到:6、8、9号PirB基因敲除小鼠如果被BamHI切割,在5.83kb和3.56kb有两个片段,WT小鼠只在5.83kb处有一个切割片段,如果被AvrII切割,PirB基因敲除小鼠在11.12kb和5.27kb有两个片段,WT小鼠只在5.27kb处有一个切割片段。
步骤6、将F1代杂合子小鼠近交获得F2代纯合子PirB基因敲入小鼠,即为本发明所述小鼠PirB基因敲入的动物模型。
将本发明获得的动物模型F2代纯合子小鼠与同品系小鼠组织/细胞特异性Cre表达的小鼠杂交,获得F3代小鼠,可以实现在不同组织或者细胞类型中敲入PirB基因。
对F3代小鼠进行基因型的鉴定:含有无(PirB-cWT)、一个(PirB-cHET)或者两个(PirB-cKI)PirB基因敲入的小鼠,PirB基因敲入到特异性组织或细胞中。其中PirB-cWT小鼠表达Cre,但不含有loxP-PirB等位基因,PirB-cHET and PirB-cKI小鼠表达Cre并分别含有一个或者两个loxP-PirB等位基因。
其中F3代杂交小鼠纯合子/杂合子鉴定所用的PCR引物如下:
F3:5’-CACTTGCTCTCCCAAAGTCGCTC-3’
R3:5’-ATACTCCGAGGCGGATCACAA-3’
F5:5’-GCATCTGACTTCTGGCTAATAAAG-3’
Homozygotes:644bp
Heterozygotes:644bp/453bp
Wildtype allele:453bp
组织特异性的基因敲入也可以通过加入另一对引物,用PCR来验证:
PCR for constitutive KI allele:
F6:5’-GGCAACGTGCTGGTTATTGTG-3’
R6:5’-GGCTGTACTCTGAGGATAGGCTTAG-3’
F7:5’-AGATCTGCAAGCTAATTCCTGC-3’
With CKI:1180bp/215bp
Constitutive KI allele:303bp
注意:如果DNA的样本不够纯,或者PCR的延伸时间不够长,可能扩增不出来1180bp的产物。
Cre阳性PCR鉴定所需的引物如下:
Forward:5’-GAACGCACTGATTTCGACCA-3’
Reverse:5’-GCTAACCAGCGTTTTCGTTC-3’
Creamplicon:204bp。
序列表
<110> 西安医学院
<120> PirB基因敲入的小鼠动物模型及其构建方法
<130> 2019
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggcaggctta aaggctaacc tgg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctccagtctt tctagaagat ggg 23
Claims (5)
1.PirB基因敲入的小鼠动物模型,其特征在于,所述小鼠动物模型包括确定PirB基因的待敲入的特异性靶位点gRNA1和gRNA2,将PirB基因敲入C57BL/6J小鼠的ROSA26基因的内含子1内,所述gRNA1的基因序列如SEQ ID NO.1所示,所述gRNA2的基因序列如SEQ ID NO.2所示。
2.一种权利要求1所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,按照以下步骤进行实施:
步骤1、基于CRISPR/Cas9技术构建针对C57BL/6J小鼠ROSA26基因内含子1的特异性gRNA1和gRNA2,所述gRNA1的基因序列如SEQ ID NO.1所示,所述gRNA2的基因序列如SEQ IDNO.2所示;
步骤2、构建“CAG promoter-loxP-Stop-loxP-Kozak-mouse PirbCDS-polyA”基因盒打靶载体,将打靶载体进行线性化处理;
步骤3、步骤2中将含有loxP位点打靶载体、步骤1中有活性的gRNA1和gRNA2和Cas9蛋白共同注射进入代孕小鼠受精卵中,获得F0代小鼠;
步骤4、将步骤3中性成熟的阳性F0小鼠分别与野生型鼠交配繁一代,获得F1代杂合子小鼠,通过PCR、测序和Southern杂交鉴定动物基因型;
步骤5、将步骤4获得的F1代杂合子小鼠近交获得F2代纯合子小鼠,即为本发明构建的一种PirB基因敲入的小鼠动物模型。
3.根据权利要求2所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,所述步骤1中得到gRNA1和gRNA2后需要将gRNA1和gRNA2分别与trancrRNA在25℃孵育10min形成二级结构。
4.根据权利要求2所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,所述步骤3中gRNA1、gRNA2的浓度均为2~10pmol/ul,Cas9蛋白的注射浓度为30~100ng/μL。
5.根据权利要求2所述的PirB基因敲入的小鼠动物模型的构建方法,其特征在于,所述步骤4中Southern杂交采用BamHI和AvrII核酸内切酶切割F1代杂合子小鼠的DNA。
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