Construction and Phenotypic Analysis of Gene p53 Knockout Rat Model

doi:10.13560/j.cnki.biotech.bull.1985.2018-0086

Abstract

Abstract: The objectives of this work are to construct rat model with gene p53 knockout using TALEN,to establish colony,and to analyze the phenotypes of the rats. TALEN protein specifically recognizing exon 2 of gene p53 was designed and the corresponding vectors were constructed. In vitro transcribed TALEN mRNA was injected into fertilized eggs of SD rats. New born rats with right splicing at the target site were determined by sequencing. Together 11new-born rats were obtained after injection and 10 of them were having right splicing at target site by sequencing. Four rats were selected as founder to establish the colony. The homozygotes with gene p53 knockout presented tumor susceptibility and the main type of spontaneous tumor was malignant fibrous histiocytoma. In addition,the homozygous rats with gene p53 knockout also had eye developmental defects,such as retina degeneration and disorganized fiber of lens. In conclusion,rat model with p53knockout is efficiently constructed by TALEN,the model exhibits tumor susceptibility and eye developmental defects,indicating that the model can be used for studying the mechanisms of tumorigenesis and the function of gene p53 in eye development.

Key words: TALEN, gene p53 knockout, SD rat, tumor, eye developmental defect

HUO Gui-tao, YANG Yan-wei, WU Xi, LIU Su-su, LI Qian-qian, ZHOU Shu-ya, LIU Quan-ming, WANG San-long, SHEN Yue-lei, LÜ Jian-jun, FAN Chang-fa. Construction and Phenotypic Analysis of Gene p53 Knockout Rat Model[J]. Biotechnology Bulletin, 2018, 34(8): 170-174.

References

[1] Chang F, Syrijänen S, Tervahauta A, et al.Tumourigenesis associated with the p53 tumour suppressor gene[J]. British Journal of Cancer, 1993, 68(4):653-661.
[2] Hollstein M, Sidransky D, Vogelstein B, et al.p53 mutations in human cancers[J]. Science, 1991, 253(5015):49-53.
[3] Donehower LA and Lozano G. 20 years studying p53 functions in genetically engineered mice[J]. Nature Reviews Cancer, 2009, 9(11):831-841.
[4] Jacks T, Remington L, Williams BO, et al.Tumor spectrum analysis in p53-mutant mice[J]. Curr Biol, 1994, 4(1):1-7.
[5] Purdie CA, Harrison DJ, Peter A, et al.Tumour incidence, spectrum and ploidy in mice with a large deletion in the p53 gene[J]. Oncogene, 1994, 9(2):603-609.
[6] Malkin D, Li FP, Strong LC, et al.Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms[J]. Science, 1990, 250(4985):1233-1238.
[7] Jacob HJ.Functional genomics and rat models[J]. Genome Research, 1999, 9(11):1013-1016.
[8] Geurts AM, Cost GJ, Freyvert Y, et al.Knockout rats via embryo microinjection of zinc-finger nucleases[J]. Science, 2009, 325(5939):433-433.
[9] Tong C, Li P, Wu NL, et al.Production of p53 gene knockout rats by homologous recombination in embryonic stem cells[J]. Nature, 2010, 467(7312):211-213.
[10] van Boxtel R, Kuiper RV, Toonen PW, et al. Homozygous and heterozygous p53 knockout rats develop metastasizing sarcomas with high frequency[J]. The American Journal of Pathology, 2011, 179(4):1616-1622.
[11] Amos-Landgraf JM, Kwong LN, Kendziorski CM, et al.A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer[J]. Proc Natl Acad Sci, 2007, 104(10):4036-4041.
[12] Cotroneo M, Haag JD, Zan Y, et al.Characterizing a rat Brca2 knockout model[J]. Oncogene, 2007, 26(11):1626-1635.
[13] van Boxtel R, Toonen PW, van Roekel HS, et al. Lack of DNA mismatch repair protein MSH6 in the rat results in hereditary non-polyposis colorectal cancer-like tumorigenesis[J]. Carcinogenesis, 2008, 29(6):1290-1297.
[14] Ikeda S, Hawes NL, Chang B, et al.Severe ocular abnormalities in C57BL/6 but not in 129/Sv p53-deficient mice[J]. Investigative Ophthalmology & Visual Science. 1999, 40(8):1874-1878.
[15] Tendler Y, Pokroy R, Panshin A, et al.p53 protein subcellular localization and apoptosis in rodent corneal epithelium cell culture following μLtraviolet irradiation[J]. Int J Mol Med, 2013, 31(3):540-546.