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

doi:10.12300/j.issn.1674-5817.2022.154

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 5

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

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).

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.

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）.

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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