CN106480099A - 条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型及其构建方法 - Google Patents
条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型及其构建方法 Download PDFInfo
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Abstract
本发明公开了一种条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型及其构建方法,构建方法为:先获得可诱导型Spp1过表达转基因载体;再获得转基因F0代小鼠;与野生型小鼠交配,传代建系,获得F1代小鼠;选择在骨髓、脾脏、淋巴结以及胸腺均高表达的F1代小鼠作为后续的实验品系,获得转基因工具小鼠;将转基因工具小鼠与Cre转基因小鼠交配,即得。本发明构建得到的转基因小鼠模型为可诱导的白血病模型,在没有诱导剂的情况下Spp1不表达不发病,在诱导剂作用的情况下Spp1过表达,引发白血病的发生,较常规的转基因小鼠模型具有更好的可控性,可根据实验需要调整需维持的种群数量。
Description
技术领域
本发明属于基因工程技术领域,更具体地,本发明涉及一种条件性过表达SPP1基因的H11定点敲入杂合子小鼠模型及其构建方法。
背景技术
白血病是一类发生于造血器官,以血液和骨髓中的白细胞及其前体细胞的增殖和发育异常为特征的恶性疾病。根据世界卫生组织的报告,2000年全球约有21万人死于白血病,其中90%为成年人。小鼠造血系统的特点与人类类似,小鼠白血病与人类白血病在许多方面极为相似。小鼠白血病模型的研究和开发已成为研究人类白血病发病机制、生化免疫特征、病理生理改变、细胞和分子生物学特性及进行实验治疗的有力工具,具有重要意义。
近年研究表明,白血病的发生与细胞原癌基因的激活和抑癌基因的缺失或突变有着密切的关系。其中原癌基因SPP1是目前研究最为广泛的癌基因之一。SPP1蛋白为basic-helix-loop-helix zipper(bHLHZ)类转录因子,其可以与Max形成异二聚体结合到特异的CAC(G/A)TG‘E-box’序列,通过与各种其他因子相结合调控大量靶基因的表达。SPP1被认为参与最基本的细胞活动过程,如增殖,生长,分化和凋亡等,在胚胎发育和肿瘤发生过程中发挥重要作用。
在造血系统中,SPP1基因不仅在造血细胞生成的各个阶段起调节作用,其表达异常与血液系统疾病的发生,特别是各类型白血病的发生密切相关。研究表明,几乎在所有的男性Burkitt’s B细胞淋巴瘤病例中均可发现因染色体重排导致的SPP1基因组成型表达;在急性淋巴细胞白血病和急性髓细胞白血病中,虽未发现SPP1基因的易位,但仍可发现通过其他染色体重排、对SPP1异常的磷酸化修饰、SPP1上游正调控基因异常激活等引发的SPP1基因功能上的过表达;在慢性粒细胞白血病整个发病过程中,均可发现因SPP1上游调控激酶bcr/abl的表达引发的SPP1的过表达,并且发现SPP1的mRNA水平随着慢性粒细胞白血病的进展过程,从慢性期、加速期至急性变期不断升高;最近的研究表明SPP1的泛素化调节在慢性髓系白血病的起始和发展过程中发挥重要作用。
SPP1转基因小鼠的研究表明:受Ig重链增强子驱动的SPP1基因在B淋巴细胞中的高表达可导致淋巴瘤和早期B细胞白血病的发生。该转基因小鼠模型已经作为自发性白血病的经典模型,广泛应用于白血病发病机制,相关药物筛选等研究。通过病毒感染在骨髓细胞中过表达SPP1的研究表明,骨髓细胞过表达SPP1可导致急性髓性白血病的发生。这些证据表明SPP1基因在多种类型白血病的发生、发展过程中发挥重要作用。该基因相关转基因模型的建立,模拟了人类白血病的发生过程,促进了对SPP1基因功能的了解,加速了对白血病药物的研发进程。
然而,这些模型也有其局限性:以Ig重链增强子驱动的SPP1转基因小鼠(Eμ-myc)为例,SPP1的过表达始于胚胎期,出生后小鼠中即可观察到B细胞体积增大等白血病症状,阻碍了对于SPP1导致的白血病发生过程中myc基因功能的了解,在该转基因小鼠模型中,myc从早期持续的高表达导致Eμ-myc转基因小鼠的平均寿命只有12周,在研究过程中需要维持大量的种群。
综上所述,由于目前常用的SPP1转基因白血病小鼠模型具有SPP1持续高表达,发病时间不可控等缺陷,迫切需要建立一种可诱导的自发的白血病转基因模型。
发明内容
基于此,为了克服上述现有技术的缺陷,本发明提供了一种条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型及其构建方法。
为了实现上述发明目的,本发明采取了以下技术方案:
一种条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型的构建方法,包括以下步骤:
A、根据Spp1基因的序列,采用常规方法获得gRNA;
B、以pLVX-EF1a-ires-puro质粒为模板,PCR扩增获得EF1a启动子片段,BamHI和EcoRI酶切EF1a启动子片段和pcDNA3-Loxp-Stop-Loxp-Spp1质粒,连接获得可诱导型Spp1过表达转基因载体;
C、SmaI和PvuI酶切上述可诱导型Spp1过表达转基因载体,凝胶回收包含EF1apromoter-Loxp-Stop-Loxp-Spp1的片段,受精卵供体雌鼠超排卵后雌鼠雄鼠合笼,将见栓的雌鼠处死收集处于单细胞期的受精卵,进行雄原核显微注射;将正常发育为两细胞的受精卵移植到假孕雌鼠子宫,假孕雌鼠所生小鼠即为转基因F0代小鼠;
D、将上述转基因F0代小鼠与野生型小鼠交配,传代建系,获得F1代小鼠;选择在骨髓、脾脏、淋巴结以及胸腺均高表达的F1代小鼠作为后续的实验品系,获得转基因工具小鼠;
E、将上述转基因工具小鼠与Cre转基因小鼠交配,获得条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型。
本发明还提供了上述构建方法构建得到的条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型。
与现有技术相比,本发明具有以下有益效果:
1、本发明构建得到的转基因小鼠模型为可诱导的白血病模型,在没有诱导剂的情况下Spp1不表达不发病,在诱导剂作用的情况下Spp1过表达,引发白血病的发生,较常规的转基因小鼠模型具有更好的可控性,可根据实验需要调整需维持的种群数量;
2、本发明建立的白血病转基因小鼠模型通过控制给予诱导剂的时间,控制Spp1的过表达时机,从而控制白血病的发病时间,研究Spp1在白血病发病不同时期的功能;以该模型为基础建立动物整体水平的白血病动物模型,为筛选针对不同时期白血病的药物提供了支持;本发明建立的白血病小鼠模型为自发型的白血病模型,较肿瘤细胞移植模型等更能模拟体内的发病状况;
3、本发明建立的药物筛选模型,以Spp1原癌基因为靶点,该基因与多种类型的白血病的发生、发展密切相关,为白血病药物筛选提供了新的选择。
附图说明
图1为体外转录Cas9、gRNA的电泳结果图;
图2为同源重组载体质粒的图谱;
图3为同源重组载体酶切鉴定的电泳图,其中,S:SacI酶切鉴定结果,理论条带大小为5884bp、14780bp;M:1kb DNA ladder;
图4为F0代小鼠鉴定策略的示意图;
图5为同源重组阳性F0代小鼠5’同源臂鉴定结果图,其中2、4、6、9号为阳性,M为1kb DNA marker,有较小的阳性条带存在的情况下,较长的野生型条带较难扩增出;
图6为同源重组阳性F0代小鼠3’同源臂鉴定结果图,其中2、4、6、9号为阳性,M为1kb DNA marker,有较小的阳性条带存在的情况下,较长的野生型条带较难扩增出;
图7为F1代小鼠鉴定策略的示意图;
图8为同源重组阳性F1代小鼠5’同源臂的PCR鉴定电泳图,其中2,4,9,10,15号为阳性,M为1kb DNA marker;
图9为同源重组阳性F1代小鼠3’同源臂的PCR鉴定电泳图,其中2,4,9,10,15号为阳性,M为1kb DNA marker;
图10为F1代小鼠PCR产物的测序验证示意图。
具体实施方式
以下结合附图和具体实施例来详细说明本发明,以下实施例中如无特殊说明,所使用原料均来源于市售,所采用方法均为本领域技术人员公知的常规操作方法。
实施例1条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型的构建方法
包括以下步骤:
(1)、获得gRNA
gRNA1:5’-ATGATGGCATCTAATGAGCTTGG-3’
(2)、构建同源重组质粒
同源重组质粒的图谱见图2,其包含了表1的所有元件。使用SacI对同源重组载体进行酶切鉴定,鉴定结果如图3所示,理论条带大小为5884bp、14780bp,说明同源重组质粒构建成功。
表1同源重组质粒的元件
(3)、构建F0代小鼠
经受精卵显微注射,共获得15只F0代小鼠。
对F0代小鼠的基因型进行鉴定,鉴定策略如图4所示,5’臂同源重组阳性基因组应扩增出5.4kb片段,野生型基因组产物大小为10.2kb;3’臂同源重组阳性基因组应扩增出6.9kb片段,野生型基因组产物大小为11.5kb。
通过长片段PCR的方式,对双臂同源重组阳性的F0代小鼠进行鉴定,其中:5’同源臂重组阳性F0代小鼠PCR鉴定方法的引物信息如表2所示,反应体系如表3所示,PCR反应程序为表4所示,PCR结果如图5所示;3’同源臂重组阳性F0代小鼠PCR鉴定方法的引物信息如表5所示,反应体系如表6所示,PCR反应程序为表7所示,PCR结果如图6所示,经测序确认,共获得4只正确同源重组的阳性F0代小鼠为2、4、6、9号。
表2
Primer | Sequence 5'-->3' | Primer Type |
I | Cttgtgagggcctactgtgac | Forward |
II | ctttccggagatagggtgtta | Reverse |
表3
Reaction Component | Volume(μl) |
ddH2O | 31 |
PrimeStar GXL PCR Buffer | 10 |
2.5mM dNTP | 4 |
Primer I(20pmol/μl) | 1 |
Primer II(20pmol/μl) | 1 |
PrimeStar GXL DNA Polymerase* | 2 |
genomic DNA | 1 |
Total | 50 |
注:*PrimeStar GXL(TaKaRa,Code No:R050A)
表4
Temp℃ | Time | Note |
98 | 2min | - |
98 | 10sec | - |
68 | 15sec | - |
68 | 5min | repeat steps 2-4 for34cycles |
68 | 10min | - |
12 | - | hold |
表5
Primer | Sequence 5′-->3′ | Primer Type |
III | Ttgcccctttgtgttctcttgtag | Forward |
IV | atcgtgggcatgtgacctctc | Reverse |
表6
Reaction Component | Volume(μl) |
ddH2O | 31 |
PrimeStar GXL PCR Buffer | 10 |
2.5mM dNTP | 4 |
Primer III(20pmol/μl) | 1 |
Primer IV(20pmol/μl) | 1 |
PrimeStar GXL DNA Polymerase* | 2 |
genomic DNA | 1 |
Total | 50 |
注:*PrimeStar GXL(TaKaRa.Code No:R050A)
表7
Step# | Temp℃ | Time | Note |
1 | 98 | 2min | - |
2 | 98 | 10sec | - |
3 | 68 | 15sec | - |
4 | 68 | 5min | repeat steps 2-4 for 34cycles |
5 | 68 | 10min | - |
6 | 12 | - | hold |
(4)、获得F1代小鼠
FO代阳性小鼠与野生型C57BL/6J小鼠交配,繁育获得F1代小鼠。
对F1代小鼠进行鉴定,鉴定策略如图7所示,5’臂同源重组阳性基因组应扩增出5.4kb片段,野生型基因组产物大小为10.2kb;3’臂同源重组阳性基因组应扩增出6.6kb片段,野生型基因组无产物。
通过长片段PCR的方式,对双臂同源重组阳性的F1代小鼠进行鉴定,其中:5’同源臂重组阳性F1代小鼠PCR鉴定方法的引物信息如表8所示,反应体系如表9所示,PCR反应程序为表10所示,PCR结果如图8所示;3’同源臂重组阳性F1代小鼠PCR鉴定方法的引物信息如表11所示,反应体系如表12所示,PCR反应程序为表13所示,PCR结果如图9所示。
对PCR产物共进行了4个测序反应。测序反应对应的区域,如图10所示。其中,5’同源臂鉴定,PCR产物测序共进行了2个测序反应,分别标注为:1、2;3’同源臂鉴定,PCR产物测序共进行了2个测序反应,分别标注为:3、4。经测序确认,共获得5只正确同源重组的阳性F1代小鼠,号码为:2,4,9,10,15号。
表8
Primer | Sequence5′-->3′ | Primer Type |
I | Cttgtgagggcctactgtgac | Forward |
II | ctttccggagatagggtgtta | Reverse |
表9
Reaction Component | Volume(μl) |
ddH2O | 31 |
PrimeStar GXL PCR Buffer | 10 |
2.5mM dNTP | 4 |
Primer I(20pmol/μl) | 1 |
PrimerII(20pmol/μl) | 1 |
PrimeStar GXL DNA Polymerase* | 2 |
genomic DNA | 1 |
Total | 50 |
注:*PrimeStar GXL(TaKaRa.Code No:R050A)
表10
Step# | Temp℃ | Time | Note |
1 | 98 | 2min | - |
2 | 98 | 10sec | - |
3 | 68 | 15sec | - |
4 | 68 | 5min | repeat steps 2-4 for 34cycles |
5 | 68 | 10min | - |
6 | 12 | - | hold |
表11
Primer | Sequence 5′-->3′ | Primer Type |
III | GGGGATGCGGTGGGCTCTATGGT | Forward |
IV | TGTATGTATGCTTGCCTCGGTGTG | Reverse |
表12
Reaction Component | Volume(μl) |
ddH2O | 31 |
PrimeStar GXL PCR Buffer | 10 |
2.5mM dNTP | 4 |
Primer III(20pmol/μl) | 1 |
Primer IV(20pmol/μl) | 1 |
PrimeStar GXL DNA Polymerase* | 2 |
genomic DNA | 1 |
Total | 50 |
注:*PrimeStar GXL(TaKaRa,Code No:R050A)
表13
Step# | Temp℃ | Time | Note |
1 | 98 | 2min | - |
2 | 98 | 10sec | - |
3 | 68 | 15sec | - |
4 | 68 | 3min | repeat steps 2-4 for 34cycles |
5 | 68 | 10min | - |
6 | 12 | - | hold |
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (2)
1.一种条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型的构建方法,其特征在于,包括以下步骤:
A、根据Spp1基因的序列,采用常规方法获得gRNA;
B、以pLVX-EF1a-ires-puro质粒为模板,PCR扩增获得EF1a启动子片段,BamHI和EcoRI酶切EF1a启动子片段和pcDNA3-Loxp-Stop-Loxp-Spp1质粒,连接获得可诱导型Spp1过表达转基因载体;
C、SmaI和PvuI酶切上述可诱导型Spp1过表达转基因载体,凝胶回收包含EF1apromoter-Loxp-Stop-Loxp-Spp1的片段,受精卵供体雌鼠超排卵后雌鼠雄鼠合笼,将见栓的雌鼠处死收集处于单细胞期的受精卵,进行雄原核显微注射;将正常发育为两细胞的受精卵移植到假孕雌鼠子宫,假孕雌鼠所生小鼠即为转基因F0代小鼠;
D、将上述转基因F0代小鼠与野生型小鼠交配,传代建系,获得F1代小鼠;选择在骨髓、脾脏、淋巴结以及胸腺均高表达的F1代小鼠作为后续的实验品系,获得转基因工具小鼠;
E、将上述转基因工具小鼠与Cre转基因小鼠交配,获得条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型。
2.权利要求1所述的构建方法构建得到的条件性过表达Spp1基因的H11定点敲入杂合子小鼠模型。
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