hKDR+/+ 人源化及Rag1-/-基因缺陷新型双靶点遗传修饰荷瘤小鼠模型的建立

doi:10.12300/j.issn.1674-5817.2022.154

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (2): 103-111.DOI: 10.12300/j.issn.1674-5817.2022.154

• 人类疾病动物模型 • 下一篇

hKDR^+/+ 人源化及Rag1^-/-基因缺陷新型双靶点遗传修饰荷瘤小鼠模型的建立

刘甦苏, 吴勇, 曹愿, 赵皓阳, 翟世杰, 孙晓炜, 李琳丽, 范昌发()()

中国食品药品检定研究院实验动物资源研究所, 国家啮齿类实验动物资源库, 北京 102629

收稿日期:2022-10-09 修回日期:2022-11-27 出版日期:2023-04-25 发布日期:2023-05-16
通讯作者: 范昌发（1970—），男，研究员，主要从事模式动物研究。E-mail： fancf@nifdc.org.cn。ORCID：0000-0001-5556-2025
作者简介:刘甦苏（1981—），女，硕士，副主任技师，主要从事模式动物研究。E-mail： liususu@nifdc.org.cn
基金资助:
国家科技重大专项课题“创新生物技术药评价及标准化关键技术研究”(2018ZX09101-001)

Establishment of hKDR^+/+ Humanized and Rag1^-/- Gene Knockout Double Genetically Modified Mouse Model

Susu LIU, Yong WU, Yuan CAO, Haoyang ZHAO, Shijie ZHAI, Xiaowei SUN, Linli LI, Changfa FAN()()

Institute for Laboratory Animal Resources, National Institutes for Food and Drug Control; National Rodent Laboratory Animal Resources Center, Beijing 102629, China

Received:2022-10-09 Revised:2022-11-27 Published:2023-04-25 Online:2023-05-16
Contact: FAN Changfa (ORCID: 0000-0001-5556-2025), E-mail: fancf@nifdc.org.cn

摘要/Abstract

摘要：

目的通过增强血管内皮生长因子受体（vascular endothelial growth factor receptor，VEGFR）人源化小鼠（hKDR^+/+）接纳不同来源的小鼠肿瘤细胞系的潜力，建立能支持多种肿瘤细胞系成瘤的、可评价针对VEGFR靶点药物的新型荷瘤小鼠模型。方法首先建立基于hKDR^+/+人源化小鼠模型评价针对VEGFR靶点抗体体内活性的方法，同时采用CRISPR/Cas9技术构建重组激活基因1（recombination activating gene 1，Rag1）敲除的C57BL/6N小鼠（命名为Rag1^-/-小鼠）。然后将Rag1^-/-小鼠与hKDR^+/+小鼠交配，筛选获得双基因纯合、双靶点基因修饰的hKDR^+/+/Rag1^-/-小鼠。最后在hKDR^+/+、Rag1^-/-、hKDR^+/+/Rag1^-/-和C57BL/6N小鼠上测试不同小鼠品系来源肿瘤细胞系的体内肿瘤生长差异。结果针对VEGFR靶点设计的抗体在hKDR^+/+小鼠体内表现出明显的抗肿瘤活性，与PBS组相比，肿瘤体积明显减小（P<0.01），肿瘤质量明显减轻（P<0.05）。Rag1^-/-小鼠、hKDR^+/+/Rag1^-/-小鼠的外周血B细胞和T细胞数量均明显减少（P<0.05，P<0.001）。C57BL/6小鼠来源的MC38细胞接种7 d后，在hKDR^+/+/Rag1^-/-、Rag1^-/-、C57BL/6N和hKDR^+/+四组小鼠体内均可见肿瘤生长。BALB/c小鼠来源的CT26细胞接种10 d后，仅在hKDR^+/+/Rag1^-/-和Rag1^-/-小鼠体内见到肿瘤生长，在C57BL/6N及hKDR^+/+小鼠中均未见肿瘤生长；接种后3周，hKDR^+/+/Rag1^-/-小鼠的成瘤体积明显大于Rag1^-/-小鼠（P<0.01）。结论获得了Rag1基因敲除小鼠，并进一步筛选获得新型hKDR^+/+/Rag1^-/-双靶点基因修饰小鼠模型；Rag1基因缺失时，不同品系小鼠来源的肿瘤细胞系更易成瘤。这提示可以通过降低hKDR^+/+人源化小鼠的免疫反应能力，增强或扩大该模型小鼠接纳肿瘤细胞系的范围，构建多种可用于评价VEGFR靶点药物的荷瘤小鼠模型。

关键词: hKDR^+/+人源化小鼠, Rag1^-/-基因敲除小鼠, VEGFR靶点, 抗体, 肿瘤

Abstract:

Objective Through improving the potential of vascular endothelial growth factor receptor (VEGFR)-humanized mouse model (hKDR^+/+) with C57BL/6N background to allow the growth of different mouse tumor cell lines, to establish novel tumor-bearing mouse models which can support in vivo tumorigenesis of different mouse tumor cell lines and be used to evaluate drugs targeting VEGFR. Methods Firstly, a method to evaluate the in vivo activity of antibody targeting VEGFR based on the hKDR^+/+ humanized mouse model was established. Recombinant activating gene 1 (Rag1) knockout mice (Rag1^-/-) were established using CRISPR/Cas9 technology. Then these Rag1^-/- mice were crossed with hKDR^+/+ mice to get a double gene modified homozygous hKDR^+/+/Rag1^-/- mouse model by screening. Finally, tumor cell lines derived from different mouse strains were tested on the double gene-modified mouse model to compare the differences in tumor growth. Results Antibodies designed for VEGFR showed significant anti-tumor activity in hKDR^+/+ mice, which significantly reduced tumor volume and weight compared with the PBS group (P<0.01, P<0.05). The number of B cells and T cells in the peripheral blood of Rag1^-/- mice and hKDR^+/+/Rag1^-/- mice decreased (P<0.05, P<0.001). Tumors were observed in hKDR^+/+/Rag1^-/-, Rag1^-/-, wild-type, and hKDR^+/+ mice after 7 d of inoculation of MC38 cells derived from C57BL/6 mice. Tumors were only observed in groups of hKDR^+/+/Rag1^-/- and Rag1^-/- mice, but not in the wild-type and hKDR^+/+ mice after 10 d of inoculation with CT26 cells derived from BALB/c mice. After 3 weeks of inoculation, the tumor volume of hKDR^+/+/Rag1^-/- mice was significantly larger than that of Rag1^-/- mice (P<0.01). Conclusion Rag1 knockout mice were obtained and a novel hKDR^+/+/Rag1^-/- double genes modified mouse model was further screened. The tumor cell lines from different mouse strain origins were more prone to growth in mice with Rag1 gene deficiency. The results suggest that the reduced immune response of hKDR^+/+ humanized mice will improve the capacity of supporting the growth of mouse tumor lines in the model. As a result, more tumor-bearing mouse models may be constructed for the evaluation of drugs targeting VEGFR in this way.

Key words: hKDR^+/+ humanized mice, Rag1^-/- knockout mice, VEGFR target, Antibodies, Tumor

中图分类号:

Q95-33

刘甦苏, 吴勇, 曹愿, 赵皓阳, 翟世杰, 孙晓炜, 李琳丽, 范昌发. hKDR^+/+ 人源化及Rag1^-/-基因缺陷新型双靶点遗传修饰荷瘤小鼠模型的建立[J]. 实验动物与比较医学, 2023, 43(2): 103-111.

Susu LIU, Yong WU, Yuan CAO, Haoyang ZHAO, Shijie ZHAI, Xiaowei SUN, Linli LI, Changfa FAN. Establishment of hKDR^+/+ Humanized and Rag1^-/- Gene Knockout Double Genetically Modified Mouse Model[J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 103-111.

图/表 5

表1 模型建立所需引物信息

Table 1 Primers for model development

引物 Primer	序列 (5’→3’) Sequence (5’→3’)
Rag1-T7 sgRNA-F	TAGGTGAAACGATTCCCACAGATG
Rag1-T7 sgRNA-R	AAACCATCTGTGGGAATCGTTTCA
Rag1-F	GGGGAAACCTTACCTAGAACAG
Rag1-R	AGAATTCCGTCGGGTGGAT
KDR-F	TTTATGTTTCAGGTTCAGGGGGAG
KDR-R	CCAGCACTGTCTAAATTCAACGGC
KDR-WF	GTATGTGTTTCTCTGCCCTCCTCG

图1 针对VEGFR2靶点的抗体能抑制hKDR+/+小鼠的肿瘤生长注：A为用人源化抗体或PBS治疗MC38荷瘤hKDR+/+小鼠的实验方案示意图（D0、D10、D13、D16、D20、D24和D25分别表示注射肿瘤细胞当天及之后的第10、13、16、20、24、25天）；B为在治疗结束时，对每只小鼠的肿瘤进行解剖，并确定肿瘤大小；C为Ramucirumab和VEGFR-HK19抗体能显著降低肿瘤体积（每组4只小鼠，**P<0.01）；D为治疗结束时，各组肿瘤质量的比较（每组4只小鼠，*P<0.05）。

Figure 1 Antibody targeting VEGFR2 targets can inhibit tumor growth in hKDR+/+ mouseNote: A shows the experimental flow diagram of treating MC38 tumor bearing hKDR+/+ mice with humanized antibody or PBS （D0, D10, D13, D16, D20, D24和D25 show the day of injection and the 10th, 13th, 16th, 20th, 24th和25th day after the injection of tumor cells, respectively); B shows the tumors were dissected from each mouse in different groups at the end of the treatment period; C shows Ramucirumab and VEGFR-HK19 can significantly reduce tumor volume (n=4,**P<0.01); D shows the tumor weight in different groups at the end of the treatment (n=4, *P<0.05).

图2 Rag1-/-基因敲除小鼠的构建及验证注：A为模型的构建设计图示；B为Rag1基因敲除（Rag1-/-）小鼠的基因型鉴定图（hom，基因纯合小鼠；het，基因杂合小鼠；WT，野生型小鼠）；C为Rag1-/- 基因小鼠和C57BL/6N野生型小鼠外周血T、B淋巴细胞百分含量散点图；D为2组小鼠T、B淋巴细胞百分含量分析柱状图（每组3只小鼠，*P<0.05，***P<0.001）；E为Rag1基因目的片段测序及序列对比，其中DEL表示敲除的片段。

Figure 2 Design strategy and validation of Rag1-/- gene knockout miceNote：A shows model construction design; B shows genotype identification map of Rag1-/- mice (hom shows homozygous mice; het shows heterozygous mice; WT shows wild-type mice). C shows scatter diagrams of the percentage of T and B lymphocytes in peripheral blood of Rag1-/- mice and C57BL/6N wild-type mice. D shows the bar chart of percentage analysis of T and B lymphocytes in the two groups of mice (n=3, *P<0.05, ***P<0.001). E shows the sequence comparison of target fragments of the Rag1 gene. DEL shows the knockout segment.

图3 hKDR+/+/Rag1 -/-双靶点基因修饰小鼠的获得及验证注：A为hKDR+/+/Rag1-/-人源双靶点基因修饰小鼠繁育流程；B为hKDR+/+/Rag1-/-人源双靶点基因小鼠基因型鉴定（1、2、3为双基因纯合小鼠，hom指基因纯合小鼠，WT指野生型小鼠）；C为hKDR+/+/Rag1 -/- 人源双靶点基因修饰小鼠的外周血T、B淋巴细胞百分含量散点图；D为2组小鼠T、B淋巴细胞百分含量分析柱状图（每组3只小鼠，*P<0.05，***P<0.001）。

Figure 3 Design strategy and validation of hKDR+/+ humanized and Rag1-/- deficient double genetically modified mouse modelNote：A shows breeding process of hKDR+/+/Rag1-/- humanized double target gene-modified mice; B shows genotype identification of hKDR+/+/Rag1-/- humanized double target gene mice (1, 2, 3 show homozygous mice with two genes，hom shows homozygous mice, WT shows wild-type mice); C shows scatter diagrams of percentage of T and B lymphocytes in the peripheral blood of hKDR+/+/Rag1-/- humanized double target gene-modified mice; D shows the bar chart of percentage analysis of T and B lymphocytes in the two groups of mice（n = 3, *P<0.05，***P<0.001）.

图4 MC38和CT26肿瘤细胞在Rag1-/-基因缺失背景的小鼠模型中正常生长注：A为MC38鼠源肿瘤细胞移植后的4种小鼠肿瘤体积变化比较；B为CT26鼠源肿瘤细胞移植后的4种小鼠生长情况；C为实验末期接种CT26细胞（左，红框所示）和MC38细胞（右）的成瘤小鼠外观；D为实验末期接种CT26细胞hKDR+/+/Rag1-/-和Rag1-/-小鼠的肿瘤取材。每组5只小鼠，**P<0.01。

Figure 4 Tumor growth of MC38 and CT26 cells in mice with Rag1-/- gene deficiencyNote： A shows comparison of tumor volume changes in the four types of mice after transplantation of MC38 tumor cells; B shows comparison of tumor volume changes in the four types of mice after transplantation of CT26 tumor cells; C shows the appearances of tumor forming mice inoculated with CT26 cells (left, showed in the red boxes) and MC38 cells (right) at the end of the treatment period; D shows tumor samples of hKDR+/+/Rag1-/- and Rag1-/- mice inoculated with CT26 cells at the end of the experiment. n=5, **P<0.01.

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hKDR^+/+ 人源化及Rag1^-/-基因缺陷新型双靶点遗传修饰荷瘤小鼠模型的建立

Establishment of hKDR^+/+ Humanized and Rag1^-/- Gene Knockout Double Genetically Modified Mouse Model

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