WO2019114439A1 - 一种化合物皮肤致敏体外评估细胞模型及其构建方法 - Google Patents

一种化合物皮肤致敏体外评估细胞模型及其构建方法 Download PDF

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WO2019114439A1
WO2019114439A1 PCT/CN2018/112411 CN2018112411W WO2019114439A1 WO 2019114439 A1 WO2019114439 A1 WO 2019114439A1 CN 2018112411 W CN2018112411 W CN 2018112411W WO 2019114439 A1 WO2019114439 A1 WO 2019114439A1
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gene
hmox1
cell model
cas9
crispr
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French (fr)
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黄黎珍
钟国瑞
李浩健
谢水林
庞士慧
白静
何昌生
都心怡
张琪霄
王海杰
杜红丽
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华南理工大学
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Definitions

  • the invention relates to the field of genetic engineering, in particular to a compound skin sensitization in vitro evaluation cell model and a construction method thereof, in particular to a cell model of a CRISPR/CAS9-mediated HMOX1 gene targeted knock-in reporter gene and a method thereof.
  • Skin sensitization is a delayed hypersensitivity reaction caused by skin contact inducers.
  • the sensitizing compound binds to the endogenous protein to form a hapten, which causes activation of dendritic cells and a series of keratinocyte reactions, which in turn activate T cells. Lymph node hyperplasia and skin inflammatory response, this process is also called skin sensitization and harmful outcome path AOP.
  • Skin-sensitized animal experiments mainly include guinea pig skin test and mouse local lymph node assay (LLNA), in which tracer modified version of LLNA: BrdU ELISA has become the most widely used global standard method, and is currently the skin of our country.
  • the main method of sensitization evaluation is the main method of sensitization evaluation.
  • the development of in vitro skin sensitization evaluation methods for replacement animals is imminent. Since the skin sensitization and harmful outcome path AOP has been relatively clear, the development of a system model for simulating in vivo sensitization processes based on the key steps of AOP is the basic strategy for the development of in vitro alternative methods at home and abroad.
  • the establishment of luciferase reporter cell model is an important means to estimate the accuracy and efficiency of sensitization and achieve high throughput.
  • Regulatory element KeratinoSens TM cells are combined with a human SV40 ARE element AKR1C2 gene, the ARE LuSens same regulatory elements of NQO1 cells from rats, an accurate assessment of both cell models were up to 75 to 96% and 71 to 85% .
  • a similar method to track the expression of THP-G8 cells expressed by IL-8 cells was as high as 82%. The detection accuracy of some cell models is even higher than that of mouse local lymph node experiments, indicating that AOP-based reporter cells are an effective alternative to LLNA in vitro.
  • reporter cell models only indirectly reflect target gene expression, rather than real-time, real-time reporter gene expression.
  • the model is essentially a transgenic cell in which the regulatory element-luciferase is randomly inserted. Although the cell can indirectly evaluate the expression of the target gene by luciferase expression to some extent, it is not a true reporter cell that simultaneously tracks the expression of the endogenous gene. Leading the model to only assess whether the compound is sensitized, it is difficult to quantitatively assess the sensitization strength.
  • Another object of the present invention is to provide a cell model of the above CRISPR/CAS9-mediated HMOX1 gene-targeted knock-in reporter gene.
  • a further object of the present invention is to provide the use of the above described CRISPR/CAS9-mediated cellular model of the HMOX1 gene-targeted knock-in reporter gene.
  • a method for constructing a cell model of CRISPR/CAS9-mediated HMOX1 gene targeted knock-in reporter gene comprising the following steps:
  • sequence of the sgRNA described in the step (1) is represented by SEQ ID NO: 1 or SEQ ID NO: 3.
  • the sgRNA is expressed by a U6 promoter, and the designed sgRNA sequence is synthesized into Oligo to construct an sgRNA expression vector;
  • the reporter gene described in the step (2) is a luciferase gene, a chloramphenicol acetyltransferase gene (cat), a ⁇ -galactosidase gene (LacZ) or a dihydrofolate reductase gene;
  • the self-cleaving peptide described in the step (2) is a T2A peptide, an E2A peptide, an F2A peptide or a P2A peptide or the like.
  • the fixed point described in the step (2) is located between the 17529 position and the 17530 base of the HMOX1 gene (NG_023030), but is not limited to this position.
  • the HMOX1 gene refers to the HMOX1 gene with a non-coding region, NCBI accession number NG_023030.
  • the sequence of the homologous recombination vector described in the step (2) is shown in SEQ ID NO: 4.
  • the dendritic cells of the mammal described in the step (3) are CD34-derived dendritic cells, but are not limited thereto.
  • the mammalian monocyte described in the step (3) is a THP-1 cell, but is not limited thereto.
  • a CRISPR/CAS9-mediated cellular model of the HMOX1 gene-targeted knock-in reporter gene was constructed by the above construction method.
  • the mechanism of the invention is:
  • the HMOX1 gene was selected as a target gene for the keratinocyte level response of the AOP pathway.
  • the efficiency of genome-based editing has been significantly improved. Therefore, the luciferase reporter gene can be inserted into the endogenous target gene expression frame with the luciferase gene accompanied by the target. Gene expression and expression can achieve true real-time reporting of endogenous target gene expression, thereby more accurately and scientifically assessing skin sensitization response.
  • the present invention obtains a HaCaT cell model in which a luciferase gene is knocked in before the stop codon of the HMOX1 gene by CRISPR/CAS9 in combination with a cell monoclonal technique.
  • the cell model realizes the simultaneous expression of the luciferase gene and the HMOX1 gene, thereby effectively distinguishing the sensitizing compound from the non-sensitizing compound, and providing a more specific and sensitive cell model for compound sensitization research.
  • Figure 1 is a graph showing the results of T7E1 digestion of sgRNA target cleavage.
  • FIG. 2 is a schematic representation of the HaCaT cell monoclonal (No. 6).
  • FIG. 3 is a schematic representation of the HaCaT cell monoclonal (No. 8).
  • FIG. 4 is a schematic representation of the HaCaT cell monoclonal (No. 10).
  • FIG. 5 is a schematic representation of the HaCaT cell monoclonal (No. 38).
  • Figure 6 is a schematic representation of the HaCaT cell monoclonal (No. 43).
  • Figure 7 is a graph showing the results of knockdown PCR identification of monoclonal luciferase genes No. 6 and No. 10.
  • Figure 8 is a graph showing the results of PCR identification of the monoclonal luciferase gene No. 8.
  • Figure 9 is a graph showing the results of PCR identification of the monoclonal luciferase gene No. 38.
  • Figure 10 is a graph showing the results of PCR identification of the No. 43 monoclonal luciferase gene.
  • Figure 11 is a graph showing the results of knockdown sequencing of the monoclonal luciferase gene No. 38.
  • Figure 12 is a graph showing the results of sensitization of monoclonal antibody No. 38 for detecting cinnamyl alcohol, 2-mercaptobenzothiazole, and sulfonamide.
  • test methods for the specific experimental conditions are not indicated in the following examples, and are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer.
  • a method for constructing a HaCaT cell model in which CRISPR/CAS9 mediates the HMOX1 gene-targeted knock-in luciferase gene, which is used for non-diagnostic or therapeutic purposes includes the following steps:
  • Target-specific sgRNA was designed for HMOX1 gene, its expression vector was constructed, and target cutting efficiency was detected.
  • a specific sgRNA was designed in the vicinity of the gene stop codon of HMOX1 (NCBI accession number: NG_023030HMOX1), and off-target analysis was performed to screen three sgRNAs with good specificity and low off-target. The design results are shown in Table 1.
  • the sgRNA was expressed by the U6 promoter, and the designed sgRNA sequence was synthesized into Oligo to construct the sgRNA expression vector pU6-sgRNA.
  • the sequencing analysis was successful.
  • the specific method is as follows:
  • the sequence of the sp6 primer was 5'-GATTTAGGTGACACTATAG-3' (SEQ ID NO: 5).
  • sgRNA-1, sgRNA-10, sgRNA-13 plasmid and hCas9 plasmid were co-transfected into 293T cells respectively.
  • the genome was extracted with the primers of Table 2 for amplification of the target site, and the PCR product was identified by T7EI digestion.
  • Electrophoresis showed that sgRNA-1 has four bands, which are lighter; sgRNA-10 has two bands, which are clearer; sgRNA-13 has four bands, which are shallow, as shown in Figure 1, where sg1, sg10, and sg13 are In the sgRNA cleavage group, WT is an uncut wild type and M is a DL2000 marker.
  • Figure 1 shows that both sgRNA-1 and sgRNA-13 were designed to efficiently cleave targets.
  • Genomic DNA was extracted by genomic extraction kit, and finally eluted with 40 ⁇ L of DDH 2 O;
  • PCR amplification of sgRNA-1, sgRNA-10, and sgRNA-13 was carried out using the genome prior to transfection as a template; PCR amplification was performed using wild type (WT) as a template, and primers SEQ ID NO: 6 were amplified. 7 as shown in Table 2.
  • PCR amplification reaction procedure 95 ° C for 3 min; 95 ° C for 45 s, 61 ° C for 45 s, 72 ° C for 30 s, 30 cycles; 72 ° C for 5 min; 4 ° C storage.
  • the luciferase gene sequence is the 1488th to 3137th bases from the 5' end of SEQ ID NO: 4) to the T2A peptide sequence (SEQ ID NO:4 from the 5' end 1434 to 1487 bases) homologous recombination to the left arm of the homology arm and the right arm of the homology arm;
  • the constructed sgRNA-13 plasmid, luciferase gene homologous recombination vector and hCas9 plasmid were co-transfected into HaCaT cells. After 72 hours, 800 ⁇ g/mL G418 (geneticmycin) was added for screening. The drug was changed once every 2 days, and digested and digested after 7 days. The cells were diluted by limiting dilution method, and 5 10 cm plates were plated at a density of 100 cells per well, and the cells were picked up for about 15 days until the monoclonal cells were grown to about 0.5 cm. 45 cell monoclonals were picked and cultured in 24-well plates.
  • PCR amplification and sequencing were performed using the primers SEQ ID NOs: 10 to 11 of Table 4, respectively.
  • the results of the identification are shown in Fig. 7 to Fig. 10. Electrophoresis shows that the clones No. 6, No. 8, No. 10, No. 38, No. 43 have a target band, and the luciferase gene was inserted into the genome; Fig. 11 is 38 The monoclonal genomic PCR product sequencing map identification map, sequencing verification is correct.
  • the sensitizing compound 2-mercaptobenzothiazole was selected for the experiment.
  • the results of luciferase expression assay and MTT activity assay are shown in Figure 12.
  • the cell model was constructed. Compared with the negative control group, the expression of luciferase was increased after the addition of 2-mercaptobenzothiazole; and the expression of luciferase was observed.
  • the concentration of 2-mercaptobenzothiazole has a dose relationship. With the increase of 2-mercaptobenzothiazole concentration, the expression of luciferase is up to 30 times in the detection concentration range (4 ⁇ M ⁇ 1000 ⁇ M).
  • Tecan 200Pro multi-function microplate reader select luminescence, integration 0.5s reading.
  • Induction multiple (L sample - L blank) / (L solvent - L blank);

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Abstract

一种化合物皮肤致敏体外评估细胞模型及其构建方法。该细胞模型的构建方法包括如下步骤：利用CRISPR/Cas9设计并构建sgRNA表达载体；设计并构建能将连接自裂解肽序列的报告基因定点敲入HMOX1基因的表达框内的同源重组载体；将同源重组载体，与hCas9质粒、sgRNA表达载体共转染细胞，进行单克隆化扩大培养，即得细胞模型。通过CRISPR/Cas9结合细胞单克隆技术，获得HMOX1基因终止密码子前敲入荧光素酶基因的HaCaT细胞模型。该细胞模型实现荧光素酶基因与HMOX1基因的同步表达，从而有效分辨致敏化合物和非致敏化合物。

Description

一种化合物皮肤致敏体外评估细胞模型及其构建方法 技术领域
本发明涉及基因工程领域，具体涉及一种化合物皮肤致敏体外评估细胞模型及其构建方法，特别涉及一种CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型及其方法。
背景技术
皮肤致敏是一种由皮肤接触诱发物导致的迟发性超敏反应，致敏化合物与内源蛋白结合形成半抗原，引起皮肤树突细胞激活及角质细胞系列反应，进而激活T细胞，导致淋巴结增生及机体皮肤炎症反应，这一过程也称皮肤致敏有害结局路径AOP。
皮肤致敏的动物实验主要有豚鼠皮肤实验和小鼠局部淋巴结试验(local lymph node assay，LLNA)，其中示踪剂改良版的LLNA:BrdU ELISA成为应用最为广泛的全球标准方法，也是目前我国皮肤致敏评价的主要方法。但是随着国际社会对实验动物福利伦理及3R原则的不断推行，且动物皮肤反应与人亦存在差异，替代动物的体外皮肤致敏评价方法研发迫在眉睫。由于皮肤致敏有害结局路径AOP已经比较清晰，因此基于AOP的关键步骤，开发模拟体内致敏过程的系统模型，是目前国内外体外替代方法研发的基本策略。其中模拟致敏过程细胞反应的关键事件，建立荧光素酶报告细胞模型是致敏评估准确高效性及实现高通量的重要手段。
目前国内外较为认可的报告细胞模型多集中在角质细胞及树突状细胞激活路径两个水平，包括针对Keap1-Nrf2-ARE通路的KeratinoSensTM、LuSens、
Figure PCTCN2018112411-appb-000001
ARE-bla HepG2、HMOX1-Luciferase等模型及评估IL-8表达的THP-G8细胞，其中KeratinoSens TM已成为OECD指导原则，LuSens和THP-G8也已得到欧洲ECVAM认可。以上报告细胞模型均是通过将靶基因调控元件与荧光素酶基因组合，通过荧光素酶表达间接反应靶基因表达。KeratinoSens TM细胞的调控元件为SV40与人AKR1C2基因的ARE元件组合，同样LuSens细胞的ARE调控元件来自大鼠NQO1基因，两个细胞模型的评估准确率分别可高达75～96％和71～85％。类似方法跟踪IL-8细胞表达的THP-G8细胞准确率高达 82％。部分细胞模型的检测准确率甚至高于小鼠局部淋巴结实验LLNA，表明基于AOP的报告细胞是LLNA的体外有效替代方法。
但是这些报告细胞模型仅仅间接反应靶基因表达，而非真正意义的实时报告基因表达。模型实质为调控元件-荧光素酶随机插入的转基因细胞，虽然细胞在一定程度上可通过荧光素酶的表达间接评估靶基因的表达，但是并非真正意义的同步追踪内源基因表达的报告细胞，导致模型仅可评估化合物是否致敏，难以定量评估致敏强弱。
发明内容
为了克服现有技术的缺点与不足，本发明的首要目的在于提供一种CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法。
本发明的另一目的在于提供上述CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型。
本发明的再一目的在于提供上述CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的应用。
本发明的目的通过下述技术方案实现：
一种CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，包括如下步骤：
(1)利用CRISPR/Cas9设计对HMOX1基因靶点特异性的sgRNA，并构建sgRNA表达载体；
(2)设计并构建能将连接自裂解肽序列的报告基因定点敲入HMOX1基因的表达框内的同源重组载体；
(3)将构建的同源重组载体，与hCas9质粒、sgRNA表达载体共转染哺乳动物的角质细胞、树突状细胞或单核细胞，进行单克隆化扩大培养，即得HMOX1基因靶向性敲入报告基因的细胞模型。
优选的，步骤(1)中所述的sgRNA的序列为SEQ ID NO：1或SEQ ID NO：3所示。
优选的，步骤(1)中，以U6启动子表达sgRNA，将设计的sgRNA序列合成Oligo，构建sgRNA表达载体；
优选的，步骤(2)中所述的报告基因为荧光素酶基因、氯霉素乙酰转移酶基因(cat)、β-半乳糖苷酶基因(LacZ)或二氢叶酸还原酶基因等；
优选的，步骤(2)中所述的自裂解肽为T2A肽、E2A肽、F2A肽或P2A 肽等。
优选的，步骤(2)中所述的定点位于HMOX1基因(NG_023030)的17529位与17530位碱基之间，但不限于该位置。
所述的HMOX1基因均指NCBI登录号为NG_023030，含有非编码区的HMOX1基因。
优选的，步骤(2)中所述的同源重组载体的序列如SEQ ID NO：4所示。
优选的，步骤(3)中所述的哺乳动物的角质细胞为HaCaT细胞，但不限于此。
优选的，步骤(3)中所述的哺乳动物的树突状细胞为CD34来源的树突状细胞，但不限于此。
优选的，步骤(3)中所述的哺乳动物的单核细胞为THP-1细胞，但不限于此。
一种CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型，通过上述构建方法构建得到。
所述的细胞模型在分辨化合物致敏性中的应用。
本发明的机理是：
基于文献报道的皮肤致敏细胞转录组数据及获国际认可几种皮肤致敏报告细胞模型，针对AOP路径的角质细胞水平反应选择HMOX1基因作为靶基因。随着近年来CRISPR/Cas9基因组编辑技术的发展，基因组定点编辑的效率显著提高，因此借助该技术可将荧光素酶报告基因定点插入到内源靶基因表达框内，荧光素酶基因伴随着靶基因表达而表达，可达到真正实时报告内源靶基因的表达，从而更加准确科学的评估皮肤致敏反应。
本发明相对于现有技术具有如下的优点及效果：
本发明通过CRISPR/CAS9结合细胞单克隆技术，获得HMOX1基因终止密码子前敲入荧光素酶基因的HaCaT细胞模型。该细胞模型实现荧光素酶基因与HMOX1基因的同步表达，从而有效分辨致敏化合物和非致敏化合物，为化合物致敏性研究提供更特异，更灵敏的细胞模型。
附图说明
图1是sgRNA靶点切割的T7E1酶切鉴定结果图。
图2是HaCaT细胞单克隆(6号)的示意图。
图3是HaCaT细胞单克隆(8号)的示意图。
图4是HaCaT细胞单克隆(10号)的示意图。
图5是HaCaT细胞单克隆(38号)的示意图。
图6是HaCaT细胞单克隆(43号)的示意图。
图7是6、10号单克隆荧光素酶基因敲入PCR鉴定结果图。
图8是8号单克隆荧光素酶基因敲入PCR鉴定结果图。
图9是38号单克隆荧光素酶基因敲入PCR鉴定结果图。
图10是43号单克隆荧光素酶基因敲入PCR鉴定结果图。
图11是38号单克隆荧光素酶基因敲入测序鉴定结果图。
图12是38号单克隆检测肉桂醇、2-巯基苯并噻唑、磺胺致敏性的结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。
实施例1
本发明实施例中，CRISPR/CAS9介导HMOX1基因靶向性敲入荧光素酶基因的HaCaT细胞模型的构建方法，该方法用于非诊断或治疗目的，包括如下步骤：
1.针对HMOX1基因设计靶点特异性的sgRNA，构建其表达载体，并进行靶点切割有效性检测。
针对HMOX1(NCBI登录号：NG_023030HMOX1)的基因终止密码子附近，设计特异性sgRNA，并进行脱靶分析，筛选三条特异性好，脱靶低的sgRNA，设计结果如表1所示。
表1 特异性sgRNA序列
名称 sgRNA序列(5'-3') 基因组位置
sgRNA-1 TTAACAGGTGGGCGTGCATCAGG Exon 5
sgRNA-10 GGTCCTTACACTCAGCTTTCTGG Exon 5
sgRNA-13 GCTTTATGCCATGTGAATGCAGG Exon 5
以U6启动子表达sgRNA，将设计的sgRNA序列合成Oligo，构建sgRNA 表达载体pU6-sgRNA。经测序分析均构建成功。具体方法如下：
U6-sgRNA质粒构建过程：
(1)设计的sgRNA合成Oligo，正义链(即与靶位点相同的序列)：5'-CACC-GN19-3'，反义链：5'-AAAC-19NC-3'(反义链N19为正义链N19的反向互补序列)；
(2)Oligo退火；U6进行BbsI酶切线性化，37℃反应2h，切胶回收线性化片段；
(3)退火的Oligo与线性化U6酶切产物连接过夜；连接产物转化大肠杆菌DH5α感受态细胞，涂布于含卡那霉素的LB平板生长，挑取单菌落于1mL LB液体培养基中(含卡那霉素)，37℃培养2～3h，采用sp6引物和正义链进行菌落PCR；菌落PCR为阳性的单克隆菌进行测序鉴定。序列正确者进行扩大培养和质粒制备。
sp6引物的序列为5'-GATTTAGGTGACACTATAG-3'(SEQ ID NO：5)。
选择sgRNA-1，sgRNA-10，sgRNA-13质粒与hCas9质粒分别共转染293T细胞，72h后提取基因组以表2的引物进行靶位点扩增，PCR产物进行T7EI酶切鉴定。酶切产物电泳显示，sgRNA-1有四条带，较浅；sgRNA-10有两条带，较清晰；sgRNA-13有四条带，较浅，如图1所示，其中sg1，sg10，sg13为sgRNA切割组，WT为未切割的野生型，M为DL2000marker。图1表明设计构建的sgRNA-1、sgRNA-13均能有效切割靶点。
Figure PCTCN2018112411-appb-000002
转染293T细胞过程
sgRNA-1，sgRNA-10，sgRNA-13质粒分别与H-Cas9质粒共转染293T细胞(采用脂质体转染方法)，转染步骤：
(1)弃去旧培养基，加入2mL新鲜培养基进行孵育；
(2)取0.5μg sgRNA重组质粒和0.5μg hCas9质粒溶于100μL DMEM(H)中，取3μL脂质体转染试剂稀释至100μL，将稀释的脂质体转染试剂加入到质粒稀释液中，轻轻吹均匀，配成转染复合物，室温反应15min；
(3)将转染复合物滴入6well中，37℃反应5h，弃去反应液，加入新鲜培养基培养。
Figure PCTCN2018112411-appb-000003
基因组提取过程
(1)转染72h后，60mm dish 293T细胞全部0.25％胰酶消化，1000rpm离心3min，弃上清；
(2)加入1mL PBS重悬，转移到1.5mL离心管中，1000rpm离心3min弃 上清，加入200μL PBS悬浮细胞；
(3)基因组提取试剂盒裂解法提取基因组DNA，最后用40μL DDH 2O洗脱；
(4)DNA浓度测定；
(5)基因组DNA 1％琼脂糖凝胶电泳。
Figure PCTCN2018112411-appb-000004
靶点扩增
以转染后提前的基因组为模板，对sgRNA-1、sgRNA-10、sgRNA-13进行PCR扩增；以野生型(WT)为模板，进行PCR扩增，扩增引物SEQ ID NO：6～7如表2。
表2 PCR扩增引物
Figure PCTCN2018112411-appb-000005
PCR扩增反应程序：95℃3min；95℃45s，61℃45s，72℃30s，30个循环；72℃5min；4℃保存。
Figure PCTCN2018112411-appb-000006
T7EI酶切检测过程：
(1)使用PCR产物，取200ng统一稀释到20μL进行变性、退火，程序如：95℃，5min；95–85℃at-2℃/s；85–25℃at-0.1℃/s；hold at 4℃。
(2)在20μL体系中加入T7EI 0.3μL，37℃酶切30分钟后，加入2μL 10×Loading Buffer(上样缓冲液)，用2％的琼脂糖胶电泳检测。
2.以pcDNA3.1(-)(Invitrogen)为骨架，同源左臂(500bp，SEQ ID NO：4中自5'端第934位～1433位碱基)和同源右臂(800bp，SEQ ID NO：4中自5'端第3138位～3937位碱基)间(其中，同源左臂为NG_023030第17529位碱基(包含)上游500bp的序列，同源右臂为NG_023030第17530位碱基(包含)下游800bp的序列)插入连接T2A肽序列的荧光素酶基因，构建同源重组载体，序列如SEQ ID NO：4所示。具体方法如下：
(1)以野生型基因组为模板，表3引物SEQ ID NO：8～9扩增含有同源臂左臂与同源臂右臂的片段；
(2)将pGL4.10(Promega)上的荧光素酶基因(荧光素酶基因序列为SEQ ID NO：4中自5'端第1488位～3137位碱基)连上T2A肽序列(SEQ ID NO：4中自5'端第1434位～1487位碱基)后同源重组至同源臂左臂与同源臂右臂间；
(3)同源臂左臂-T2A-荧光素酶基因-同源臂右臂片段加入NotI/BamHI酶 切位点后，连接于NotI/BamHI酶切线性化的pcDNA3.1(-)载体上，连接产物经转化、质粒提取，测序验证正确。
表3 扩增含有同源臂左臂与同源臂右臂的片段的引物
Figure PCTCN2018112411-appb-000007
3.HMOX1基因靶向性敲入荧光素酶基因HaCaT单克隆细胞建立及鉴定
将构建的sgRNA-13质粒、荧光素酶基因同源重组载体与hCas9质粒共转染HaCaT细胞，72h后加800μg/mL G418(遗传霉素)筛选，每2天换药一次，7天后消化消化细胞，有限稀释法稀释细胞，以100个细胞每孔的密度铺5个10cm平皿，15天左右待单克隆细胞长至0.5cm左右，可进行挑取。挑取45个细胞单克隆至24孔板培养，长满后取1/10细胞鉴定基因型，9/10的细胞传至12孔板，进行单克隆细胞扩大培养，长满后冻存。经测序鉴定，获得一系列单克隆敲入细胞，分别为6号，8号，10号，38号，43号克隆，(见图2、图3、图4、图5、图6)。
单克隆细胞扩大培养后，以表4的引物SEQ ID NO：10～11分别进行PCR扩增及测序鉴定。鉴定结果如图7～图10所示，电泳显示，6号、8号、10号、38号、43号单克隆，有目的条带，证明荧光素酶基因插入至基因组中；图11为38号单克隆基因组PCR产物测序图鉴定图，测序验证正确。
表4 PCR扩增引物
Figure PCTCN2018112411-appb-000008
4.基因敲入后化合物致敏性测定分析
采用38号克隆进行化合物致敏性的检测。
(1)选取致敏化合物肉桂醇进行实验。荧光素酶表达检测及MTT活性检测结果如图12所示，构建的细胞模型，与阴性对照组相比，加入肉桂醇后荧光素酶的表达增加；且荧光素酶的表达与肉桂醇浓度具有剂量关系，随着肉桂醇浓度的提高，荧光素酶的表达在检测浓度范围(4μM～1000μM)内最高激活21倍。
(2)选取致敏化合物2-巯基苯并噻唑进行实验。荧光素酶表达检测及MTT活性检测结果如图12所示，构建的细胞模型，与阴性对照组相比，加入2-巯基 苯并噻唑后荧光素酶的表达增加；且荧光素酶的表达与2-巯基苯并噻唑浓度具有剂量关系，随着2-巯基苯并噻唑浓度的提高，荧光素酶的表达在检测浓度范围(4μM～1000μM)内最高激活30倍。
(3)选取非致敏化合物磺胺进行实验。荧光素酶表达检测及MTT活性检测结果如图12所示，构建的细胞模型，与阴性对照组相比，加入磺胺后荧光素酶的表达无显著变化。
Figure PCTCN2018112411-appb-000009
给药过程：
将预先培养的细胞离心和去上清，用新鲜完全培养基重悬细胞。稀释10倍用细胞计数器计算细胞浓度，最终将浓度调整为50000cell/well，每孔添加200μL于白色不透明96孔板，确保每孔细胞数量基本一致。置于37℃、5％CO 2培养箱孵育24h。24h后去除培养基，用1％FBS的培养基配制药物工作液，药物浓度从1000μM以连续两倍稀释得到9个浓度，每孔加入200μL，设置阴性对照。工作液DMSO浓度均为0.5％。每孔滴加1～2滴石蜡油封板，孵育48h。48h后，检测荧光素酶表达情况，使用MTT检测细胞活性。
Figure PCTCN2018112411-appb-000010
荧光素酶检测过程：
(1)加药48h后，弃掉含有药物的培养基，PBS洗2遍；
(2)每孔加入100μL PBS，再加入等体积的Luciferase复合物；
(3)200rpm振荡2min，使细胞充分裂解；
(4)使用Tecan
Figure PCTCN2018112411-appb-000011
200Pro多功能酶标仪酶标仪，选择luminescence，integration 0.5s读数。
Figure PCTCN2018112411-appb-000012
MTT检测细胞活性过程：
(1)加药48h后，弃掉含有药物的培养基，PBS洗2遍，更换200μL新鲜的1％FBS培养基；
(2)每孔加入27μL MTT溶液，放入培养箱中继续孵育4h；
(3)孵育结束后，用1mL注射器小心吸走药物工作液，注意不要吸到孔底的紫色结晶，以免影响实验结果。加入200μL DMSO，250rpm振荡5min，使细胞裂解，紫色结晶充分溶解。
(4)在Tecan
Figure PCTCN2018112411-appb-000013
200Pro多功能酶标仪酶标仪上测定OD 570nm的吸光值。
Figure PCTCN2018112411-appb-000014
结果结算方法：
A.诱导倍数计算(fold induction)
诱导倍数＝(L样品-L空白)/(L溶剂-L空白)；
式中：
L样品：受试物荧光读数；
L空白：空白荧光(不含细胞不含受试物)读数；
L溶剂：溶剂对照(阴性对照)荧光读数均值。
B.细胞活性计算(viability)
细胞活性＝[(V样品-V空白)/(V溶剂-V空白)]*100；
式中：
V样品：受试物的MTT吸收值；
V空白：空白(不含细胞不加受试物)的MTT吸收值；
V溶剂：溶剂对照(阴性对照)的MTT吸收值。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于包括如下步骤：
    (1)利用CRISPR/Cas9设计对HMOX1基因靶点特异性的sgRNA，并构建sgRNA表达载体；
    (2)设计并构建能将连接自裂解肽序列的报告基因定点敲入HMOX1基因的表达框内的同源重组载体；
    (3)将构建的同源重组载体，与hCas9质粒、sgRNA表达载体共转染哺乳动物的角质细胞、树突状细胞或单核细胞，进行单克隆化扩大培养，即得HMOX1基因靶向性敲入报告基因的细胞模型。
  2. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(1)中所述的sgRNA的序列为SEQ ID NO：1或SEQ ID NO：3所示。
  3. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(1)中，以U6启动子表达sgRNA，将设计的sgRNA序列合成Oligo，构建sgRNA表达载体。
  4. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(2)中所述的报告基因为荧光素酶基因、氯霉素乙酰转移酶基因、β-半乳糖苷酶基因或二氢叶酸还原酶基因。
  5. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(2)中所述的自裂解肽为T2A肽、E2A肽、F2A肽或P2A肽。
  6. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(2)中所述的定点位于HMOX1基因的17529位与17530位碱基之间。
  7. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(2)中所述的同源重组载体的序列如SEQ ID NO：4所示。
  8. 根据权利要求1所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型的构建方法，其特征在于：
    步骤(3)中所述的哺乳动物的角质细胞为HaCaT细胞；
    步骤(3)中所述的哺乳动物的树突状细胞为CD34来源的树突状细胞；
    步骤(3)中所述的哺乳动物的单核细胞为THP-1细胞。
  9. 一种CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型，其特征在于通过权利要求1～8任一项所述的构建方法构建得到。
  10. 权利要求9所述的CRISPR/CAS9介导HMOX1基因靶向性敲入报告基因的细胞模型在分辨化合物致敏性中的应用。
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