Establishing transgene mouse models: construction and identification of human leucocyte antigen-A*0206 gene lentiviral vector

doi:10.3969/j.issn.2095-4344.2015.24.009

Abstract

Abstract:

BACKGROUND: Studies have shown that human leukocyte antigen (HLA)-A*0206 subtype is related to the abscess of nasopharyngeal carcinoma, but there is no corresponding transgenic animal models that could used to judge the relationship between HLA-A*0206 and nasopharyngeal carcinoma on the overall level and further research of immunotherapy and gene therapy.
OBJECTIVE: To construct lentiviral vectors carrying pLVX-CMV-HLA-A*0206-HA-mCMV-ZsGreen and establish HLA-A*0206 transgenic mice.
METHODS: The HLA-A*0206 sequence was synthesized. EcoRI recognition site was introduced in the 5’ end by polymerase chain reaction, and influenza virus hemagglutinin labels and BamHI recognition site were introduced in the 3’ end. Eco RI and Bam HI double enzyme digestion target fragments and the pLVX-CMV-mCMV-ZsGreen plasmids were connected to the digested productions and transfected JM109 competent cells. The positive clones were selected and identified by double enzyme digestion and sequencing. The positive plasmid and packaging plasmids were transfected into 293T cells, which were human renal epithelial cell line that can express SV40 large T antigen. The lentivirus containing target sequence was produced.
RESULTS AND CONCLUSION: Gel electrophoresis and sequencing results showed that, HLA-A*0206 was successfully inserted into pLVX-CMV-mCMV-ZsGreen frame plasmids. Transfection efficiency was 92% after 48 hours of transfecting 293T cells. The viral suspension titer was 5 × 108 measured by fluorescence method. Experimental findings indicate that, the lentivirus containing cytomegalovirus promoter, HLA-A*0206, influenza virus hemagglutinin label and Zsgreen report gene was successfully constructed.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lentivirus, Nasopharyngeal Carcinoma, Transgenic Mice, Green Fluorescent Proteins

CLC Number:

R318

Zhang Xiu-yan, Zhan Chun-lie, Zhang Xiao-yu. Establishing transgene mouse models: construction and identification of human leucocyte antigen-A*0206 gene lentiviral vector[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(24): 3813-3817.

Figures/Tables 1

References

[1] Chang ET, Adami HO. The enigmatic epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 2006;15(10):1765-1777.

[2] Jia WH, Huang QH, Liao J, et al. Trends in incidence and mortality of nasopharyngeal carcinoma over a 20-25 year period (1978/2002) in sihui and Cangwu counties in southern China. BMC Cancer. 2006;6:178.

[3] Lin C, Meng X, Hazawa M, et al. The genomic landscape of nasopharyngeal carcinoma. Nat Genet. 2014;46(8):866-871.

[4] Guo X, Winkler CA, Li J, et al. Evaluation and integration of genetic signature for prediction risk of nasopharyngeal carcinoma in Southern China. Biomed Res Int. 2014. doi:10.1155/2014/434072.

[5] Chien YC, Chen JY, Liu MY, et al. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men. New Engl J Med. 2001;345(26):1877-1882.

[6] Zhang YX, Kang SY, Chen G, et al. ABO blood group, Epstein-Barr virus infection and prognosis of patients with non-metastatic nasopharyngeal carcinoma. Asian Pac J cancer Prev. 2014;15(17):7459-7465.

[7] Hutajulu SH, Kurnianda J, Tan IB, et al. Therapeutic implications of Epstein-Barr virus infection for the treatment of nasopharyngeal carcinoma. Ther Clin Risk Manag. 2014;10: 721-736.

[8] Xie SH, Yu IT, Tse LA, et al. Domestic incense burning and nasopharyngeal carcinoma: a case-control study in Hong Kong Chinese. Environ MoI Mutagen. 2014. doi:10.1002/em.21894.

[9] Simons MJ, Day NE, Wee GB, et al. Nasopharyngeal carcinoma V: immol/Lunogenetic studies of Southeast Asian ethnic groups with high and low risk for the tumor. Cancer Res. 1974;34(5):1192-1195.

[10] Tang M, Zeng Y, Poisson A, et al. Haplotype-dependent HLA susceptibility to nasopharyngeal carcinoma in a Southern Chinese population. Genes Immol. 2010;11(4):334-342.

[11] 蓝轲,乔贵林,沈新民,等.鼻咽癌来源LMP1转基因小鼠的制备[J].动物医学进展,2002,23(1):69-71.

[12] 肖志强,姚开泰.携带EB病毒潜伏膜蛋白基因转基因小鼠的制备[J].生物化学与生物物理进展,1995,22(2):149-153.

[13] 何迎春,田道法,卢芳国,等.人突变型p53和EB病毒LMP1转基因小鼠的建立[J].第四军医大学学报,2006,27(1):6-9.

[14] Sakuma T, Barry MA, Ikeda Y. Lentiviral vectors:basic to translational. Biochem J. 2012;443(3):603-618.

[15] Coghill AE, Hildesheim A. Epstein-Barr Virus Antibodies and the risk of associated malignancies: review of the literature. Am J Epidemiol. 2014;180(7):687-695.

[16] Chin YM, Mushiroda T, Takahashi A, et al. HLA-A SNPs and amino acid variants are associated with nasopharyngeal carcinoma in Malaysian Chinese. Int J Cancer. 2015;136(3): 678-687.

[17] Goldsmith DB, West TM, Morton R. HLA associations with nasopharygeal carcinoma in Southern Chinese: a meta-analysis. Clin Otolaryngol Allied Sci. 2002;27(1):61-67.

[18] Demayo JL, Wang J, Liang D, et al. Genetically engineered mice by pronuclear DNA microinjection. Curr Protoc Mouse Biol. 2012;2:245-262.

[19] Liao X, Cui H, Wang F. Establishment of a transgenic mouse model of corneal dystrophy overexpressing human BIGH3. Int J Mol Med. 2013;32(5):1110-1114.

[20] Meng Q, Xiao Z, Yuan H, et al. Transgenic mice with overexpression of mutated human optineurin (E50K) in the retina. MoI Biol Rep. 2012;39(2):1119-1124.

[21] Chandrashekran A, Sarkar R, Thrasher A, et al. Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa. FASEB J. 2014;28(2):569-576.

[22] Moreira PN, Montoliu L. Intracytoplasmic sperm injection (IC SI)-mediated transgenesis in mice. Methods MoI Biol. 2014; 1194:141-156.

[23] García-Vázquez FA, Ruiz S, Matás C, et al. Production of transgenic piglets using IC SI-sperm-mediated gene transfer in combination with recombinase RecA Reproduction. 2010; 140(2):259-272.

[24] Nickerson HD, Colledge WH. A LacZ-based transgenic mouse for detection of somatic gene repair events in vivo. Gene Ther. 2004;11(17):1351-1357.

[25] Delzor A, Escartin C, Déglon N. Lentiviral vectors: a powerful tool to target astrocytes in vivo. Curr Drug Targets. 2013; 14(11):1336-1346.

[26] Monzani PS, Sangalli JR, De Bem TH, et al. Breeding of transgenic cattle for human coagulation factor Ⅸ by a combination of lentiviral system and cloning. Genet MoI Res. 2013;12(3):3675-3688.

[27] Liu C, Wang L, Li W, et al. Highly efficient generation of transgenic sheep by lentivirus accompanying the alteration of methylation status. PLoS One. 2013;8(1):e54614.

[28] Baxa M, Hruska-Plochan M, Juhas S, et al. A transgenic minipig model of Huntington’s Disease. J Huntingtons Dis. 2013;2(1):47-68.

[29] Matsuzaki Y, Oue M, Hirai H. Generation of a neurodegenerative disease mouse model using lentiviral vectors carrying an enhanced synapsin 1 promoter. J Neurosci Methods. 2014;223:133-143.

[30] Tomkowiak M, Ghittoni R, Teixeira M, et al. Generation of transgenic mice expressing EGFP protein fused to NP68 MHC classⅠepitope using lentivirus vectors. Genesis. 2013;51(3):193-200.