Construction of sporadic colorectal cancer mouse model expressed simultaneously KrasLSL-G12D/- and Smad4loxp/loxp genes _CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

YANG Chao ,  ZHENG Yongbin , SONG Dan , XIAO Kuang , TONG Shilun

Department of Gastrointestinal Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, P. R. China;

Corresponding author：

ZHENG Yongbin, Email: wqandzyb@163.com

Keywords：

genetically engineered mice; sporadic colorectal cancer; Kras gene; Smad4 gene

DOI：

10.7507/1007-9424.201801070

Video：

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Objective To construct and verify a genetically engineered mouse model which is similar to clinical sporadic colorectal cancer and simultaneously expresses Kras^LSL-G12D/- and Smad4^loxp/loxp genes. Methods The Kras^tm4Tyj/J mouse and Smad4^tm2.1Cxd/J mouse were transformed into the genetic background, and the genotypes of the offspring mice were identified by the PCR to obtain the mice expressed simultaneously Kras^LSL-G12D/- and Smad4^loxp/loxp genes. The Lentivirus^{Cre-IRES-Luciferase} was injected into the submucosa of the model mice and the tumorigenicity was observed under the IVIS system. The tumor tissues of the model mice were sampled and the HE staining was used to verify the tumorigenicity of the model mice. Results The genetically engineered mouse model which could simultaneously express Kras^LSL-G12D/- and Smad4^loxp/loxp genes was obtained by the breeding and selection. The mouse intestinal epithelial cell carcinogenesis was successfully induced by the viral vector containing Cre recombinase. Conclusion Mouse model expressed simultaneously Kras^LSL-G12D/- and Smad4^loxp/loxp genes is capable of sporadic tumorigenicity by Cre recombinase and could simulate pathological process of human sporadic colorectal cancer.

Citation： YANG Chao, ZHENG Yongbin, SONG Dan, XIAO Kuang, TONG Shilun. Construction of sporadic colorectal cancer mouse model expressed simultaneously Kras^LSL-G12D/- and Smad4^loxp/loxp genes . CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2018, 25(8): 917-922. doi: 10.7507/1007-9424.201801070 Copy

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2.	Inamoto S, Itatani Y, Yamamoto T, et al. Loss of SMAD4 promotes colorectal cancer progression by accumulation of myeloid-derived suppressor cells through the CCL15-CCR1 chemokine axis. Clin Cancer Res, 2016, 22(2): 492-501.
3.	Wosiak A, Wodziński D, Kolasa M, et al. SMAD-4 gene expression in human colorectal cancer: Comparison with some clinical and pathological parameters. Pathol Res Pract, 2017, 213(1): 45-49.
4.	Cheng D, Zhao S, Tang H, et al. MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4. Oncotarget, 2016, 7(29): 45199-45213.
5.	Belmont PJ, Budinska E, Jiang P, et al. Cross-species analysis of genetically engineered mouse models of MAPK-driven colorectal cancer identifies hallmarks of the human disease. Dis Model Mech, 2014, 7(6): 613-623.
6.	Pino MS, Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology, 2010, 138(6): 2059-2072.
7.	Gelsomino F, Barbolini M, Spallanzani A, et al. The evolving role of microsatellite instability in colorectal cancer: A review. Cancer Treat Rev, 2016, 51: 19-26.
8.	Fleet JC. Animal models of gastrointestinal and liver diseases. New mouse models for studying dietary prevention of colorectal cancer. Am J Physiol Gastrointest Liver Physiol, 2014, 307(3): G249-G259.
9.	Hung KE, Maricevich MA, Richard LG, et al. Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment. Proc Natl Acad Sci U S A, 2010, 107(4): 1565-1570.
10.	Roper J, Martin ES, Hung KE. Overview of genetically engineered mouse models of colorectal carcinoma to enable translational biology and drug development. Curr Protoc Pharmacol, 2014, 65: 14.
11.	Abi-Ghanem J, Samsonov SA, Pisabarro MT. Insights into the preferential order of strand exchange in the Cre/loxP recombinase system: impact of the DNA spacer flanking sequence and flexibility. J Comput Aided Mol Des, 2015, 29(3): 271-282.
12.	Puppa MJ, White JP, Sato S, et al. Gut barrier dysfunction in the Apc (Min/+) mouse model of colon cancer cachexia. Biochim Biophys Acta, 2011, 1812(12): 1601-1606.
13.	Marecki JC, Parajuli N, Crow JP, et al. The use of the Cre/loxP system to study oxidative stress in tissue-specific manganese superoxide dismutase knockout models. Antioxid Redox Signal, 2014, 20(10): 1655-1670.
14.	Amankwatia EB, Chakravarty P, Carey FA, et al. MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms. Br J Cancer, 2015, 112(9): 1480-1490.
15.	Saud SM, Li W, Morris NL, et al. Resveratrol prevents tumorigenesis in mouse model of Kras activated sporadic colorectal cancer by suppressing oncogenic Kras expression. Carcinogenesis, 2014, 35(12): 2778-2786.
16.	Voorneveld PW, Kodach LL, Jacobs RJ, et al. The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC. Br J Cancer, 2015, 112(1): 122-130.
17.	Luo F, Brooks DG, Ye H, et al. Mutated K-ras (Asp12) promotes tumourigenesis in Apc (Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways. Int J Exp Pathol, 2009, 90(5): 558-574.
18.	Panza P, Maier J, Schmees C, et al. Live imaging of endogenous protein dynamics in zebrafish using chromobodies. Development, 2015, 142(10): 1879-1884.
19.	Kocher B, Piwnica-Worms D. Illuminating cancer systems with genetically engineered mouse models and coupled luciferase reporters in vivo. Cancer Discov, 2013, 3(6): 616-629.
20.	Koba W, Jelicks LA, Fine EJ. MicroPET/SPECT/CT imaging of small animal models of disease. Am J Pathol, 2013, 182(2): 319-324.
21.	Qin X, Hu X, Wu C, et al. Hepatocellular carcinoma cells carrying a multimodality reporter gene for fluorescence, bioluminescence, and magnetic resonance imaging in vitro and in vivo. Acad Radiol, 2016, 23(11): 1422-1430.
22.	Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet, 2014, 383(9927): 1490-1502.
23.	Chen J, Chen Z. The effect of immune microenvironment on the progression and prognosis of colorectal cancer. Med Oncol, 2014, 31(8): 82.
24.	Patel SA, Gooderham NJ. IL6 mediates immune and colorectal cancer cell cross-talk via miR-21 and miR-29b. Mol Cancer Res, 2015, 13(11): 1502-1508.
25.	Maglietta A, Maglietta R, Staiano T, et al. The immune landscapes of polypoid and nonpolypoid precancerous colorectal lesions. PLoS One, 2016, 11(7): e0159373.

1. Rui Y, Wang C, Zhou Z, et al. K-Ras mutation and prognosis of colorectal cancer: a meta-analysis. Hepatogastroenterology, 2015, 62(137): 19-24.
2. Inamoto S, Itatani Y, Yamamoto T, et al. Loss of SMAD4 promotes colorectal cancer progression by accumulation of myeloid-derived suppressor cells through the CCL15-CCR1 chemokine axis. Clin Cancer Res, 2016, 22(2): 492-501.
3. Wosiak A, Wodziński D, Kolasa M, et al. SMAD-4 gene expression in human colorectal cancer: Comparison with some clinical and pathological parameters. Pathol Res Pract, 2017, 213(1): 45-49.
4. Cheng D, Zhao S, Tang H, et al. MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4. Oncotarget, 2016, 7(29): 45199-45213.
5. Belmont PJ, Budinska E, Jiang P, et al. Cross-species analysis of genetically engineered mouse models of MAPK-driven colorectal cancer identifies hallmarks of the human disease. Dis Model Mech, 2014, 7(6): 613-623.
6. Pino MS, Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology, 2010, 138(6): 2059-2072.
7. Gelsomino F, Barbolini M, Spallanzani A, et al. The evolving role of microsatellite instability in colorectal cancer: A review. Cancer Treat Rev, 2016, 51: 19-26.
8. Fleet JC. Animal models of gastrointestinal and liver diseases. New mouse models for studying dietary prevention of colorectal cancer. Am J Physiol Gastrointest Liver Physiol, 2014, 307(3): G249-G259.
9. Hung KE, Maricevich MA, Richard LG, et al. Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment. Proc Natl Acad Sci U S A, 2010, 107(4): 1565-1570.
10. Roper J, Martin ES, Hung KE. Overview of genetically engineered mouse models of colorectal carcinoma to enable translational biology and drug development. Curr Protoc Pharmacol, 2014, 65: 14.
11. Abi-Ghanem J, Samsonov SA, Pisabarro MT. Insights into the preferential order of strand exchange in the Cre/loxP recombinase system: impact of the DNA spacer flanking sequence and flexibility. J Comput Aided Mol Des, 2015, 29(3): 271-282.
12. Puppa MJ, White JP, Sato S, et al. Gut barrier dysfunction in the Apc (Min/+) mouse model of colon cancer cachexia. Biochim Biophys Acta, 2011, 1812(12): 1601-1606.
13. Marecki JC, Parajuli N, Crow JP, et al. The use of the Cre/loxP system to study oxidative stress in tissue-specific manganese superoxide dismutase knockout models. Antioxid Redox Signal, 2014, 20(10): 1655-1670.
14. Amankwatia EB, Chakravarty P, Carey FA, et al. MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms. Br J Cancer, 2015, 112(9): 1480-1490.
15. Saud SM, Li W, Morris NL, et al. Resveratrol prevents tumorigenesis in mouse model of Kras activated sporadic colorectal cancer by suppressing oncogenic Kras expression. Carcinogenesis, 2014, 35(12): 2778-2786.
16. Voorneveld PW, Kodach LL, Jacobs RJ, et al. The BMP pathway either enhances or inhibits the Wnt pathway depending on the SMAD4 and p53 status in CRC. Br J Cancer, 2015, 112(1): 122-130.
17. Luo F, Brooks DG, Ye H, et al. Mutated K-ras (Asp12) promotes tumourigenesis in Apc (Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways. Int J Exp Pathol, 2009, 90(5): 558-574.
18. Panza P, Maier J, Schmees C, et al. Live imaging of endogenous protein dynamics in zebrafish using chromobodies. Development, 2015, 142(10): 1879-1884.
19. Kocher B, Piwnica-Worms D. Illuminating cancer systems with genetically engineered mouse models and coupled luciferase reporters in vivo. Cancer Discov, 2013, 3(6): 616-629.
20. Koba W, Jelicks LA, Fine EJ. MicroPET/SPECT/CT imaging of small animal models of disease. Am J Pathol, 2013, 182(2): 319-324.
21. Qin X, Hu X, Wu C, et al. Hepatocellular carcinoma cells carrying a multimodality reporter gene for fluorescence, bioluminescence, and magnetic resonance imaging in vitro and in vivo. Acad Radiol, 2016, 23(11): 1422-1430.
22. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet, 2014, 383(9927): 1490-1502.
23. Chen J, Chen Z. The effect of immune microenvironment on the progression and prognosis of colorectal cancer. Med Oncol, 2014, 31(8): 82.
24. Patel SA, Gooderham NJ. IL6 mediates immune and colorectal cancer cell cross-talk via miR-21 and miR-29b. Mol Cancer Res, 2015, 13(11): 1502-1508.
25. Maglietta A, Maglietta R, Staiano T, et al. The immune landscapes of polypoid and nonpolypoid precancerous colorectal lesions. PLoS One, 2016, 11(7): e0159373.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction of sporadic colorectal cancer mouse model expressed simultaneously Kras^LSL-G12D/- and Smad4^loxp/loxp genes

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