Construction and Identification of Transgenic Mice Carrying Human Cytomegalovirus IE2 Gene

Author(s): Wenhua Zhu, Mengyuan Wang, Zhifei Wang, Ming Hu, Bin Wang, Dongmeng Qian

The infection of human cytomegalovirus causes several diseases of congenitally infected neonates and immunocompromised populations. The immediate–early 2 gene of human cytomegalovirus plays an essential role in viral replication and the pathogenic process. Due to the strict species-specific of HCMV infection, any animals besides human beings cannot be infected with HCMV. For this reason, few animal model which is used to explore the effect of the IE2 gene on the mRNA or protein level has been constructed to. In this study, the cDNA of IE2 mRNA (1,743 bp) was cloned into the mammalian expression vector pAV.Des1d then identified by polymerase chain reaction and sequencing analysis. Next, the expressed vector was transferred into mouse fertilized eggs from C57BL/6 mice by pronuclear microinjection to obtain the first generation of transgenic mice. In the 59 F0 generation mice, there are only 3 offspring mice were identified as IE2 positive mice. Furthermore, the positive ratios detected by PCR from F0 to F3 were 53.8%, 43.1%, 62.1%, respectively. The expression of exogenous IE2 mRNA was detected in kidney, heart, muscle, brain, spleen, and adipose tissue of F1 transgenic mice. These results suggest that we have successfully constructed a stable transgenic mouse line that can be used as a tool to explore the specific effects of IE2 gene on the growth and development of animals as well as mechanisms of its influence.

Human Cytomegalovirus, Immediate Early 2 Gene, Transgenic Mice

1. Biron, K. K. (2006). Antiviral drugs for cytomegalovirus diseases. Antiviral Res, 71(2-3), 154-163. doi:10.1016/j.antiviral.2006.05.002
2. Brenin, D. R., Talamonti, M. S., & Iannaccone, P. M. (1997). Transgenic technology: an overview of approaches useful in surgical research. Surg Oncol, 6(2), 99-110.
3. Davison, A. J., Dolan, A., Akter, P., Addison, C., Dargan, D. J., Alcendor, D. J., . . . Hayward, G. S. (2003). The human cytomegalovirus genome revisited: comparison with the chimpanzee cytomegalovirus genome. J Gen Virol, 84(Pt 1), 17-28. doi:10.1099/vir.0.18606-0
4. Gealy, C., Humphreys, C., Dickinson, V., Stinski, M., & Caswell, R. (2007). An activation-defective mutant of the human cytomegalovirus IE2p86 protein inhibits NF-kappaB-mediated stimulation of the human interleukin-6 promoter. J Gen Virol, 88(Pt 9), 2435-2440. doi:10.1099/vir.0.82925-0
5. Geist, L. J., & Dai, L. Y. (2000). Immediate early gene 2 of human cytomegalovirus increases interleukin 2 receptor-alpha gene expression. J Investig Med, 48(1), 60-65.
6. Geist, L. J., Monick, M. M., Stinski, M. F., & Hunninghake, G. W. (1991). The immediate early genes of human cytomegalovirus upregulate expression of the interleukin-2 and interleukin-2 receptor genes. Am J Respir Cell Mol Biol, 5(3), 292-296. doi:10.1165/ajrcmb/5.3.292
7. Geist, L. J., Monick, M. M., Stinski, M. F., & Hunninghake, G. W. (1992). Cytomegalovirus immediate early genes prevent the inhibitory effect of cyclosporin A on interleukin 2 gene transcription. J Clin Invest, 90(5), 2136-2140. doi:10.1172/jci116099
8. Griffiths, P. D. (2012). Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation. Lancet Infect Dis, 12(10), 790-798. doi:10.1016/s1473-3099(12)70197-4
9. Harris, S. M., Bullock, B., Westgard, E., Zhu, H., Stenberg, R. M., & Kerry, J. A. (2010). Functional properties of the human cytomegalovirus IE86 protein required for transcriptional regulation and virus replication. J Virol, 84(17), 8839-8848. doi:10.1128/jvi.00327-10
10. Huang, R., Qian, D., Hu, M., Zhang, X., Song, J., Li, L., . . . Wang, B. (2015). Association between human cytomegalovirus infection and histone acetylation level in various histological types of glioma. Oncol Lett, 10(5), 2812-2820. doi:10.3892/ol.2015.3638
11. Khan, Z., Yaiw, K. C., Wilhelmi, V., Lam, H., Rahbar, A., Stragliotto, G., & Soderberg-Naucler, C. (2014). Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis. Carcinogenesis, 35(1), 145-154. doi:10.1093/carcin/bgt292
12. Kim, K. S., & Carp, R. I. (1972). Abortive infection of human diploid cells by murine cytomegalovirus. Infect Immun, 6(5), 793-797.
13. Lafemina, R. L., & Hayward, G. S. (1988). Differences in cell-type-specific blocks to immediate early gene expression and DNA replication of human, simian and murine cytomegalovirus. J Gen Virol, 69 ( Pt 2), 355-374. doi:10.1099/0022-1317-69-2-355
14. Listman, J. A., Race, J. E., Walker-Kopp, N., Unlu, S., & Auron, P. E. (2008). Inhibition of IL-1beta transcription by peptides derived from the hCMV IE2 transactivator. Mol Immunol, 45(9), 2667-2677. doi:10.1016/j.molimm.2007.12.024
15. Malouli, D., Hansen, S. G., Nakayasu, E. S., Marshall, E. E., Hughes, C. M., Ventura, A. B., . . . Fruh, K. (2014). Cytomegalovirus pp65 limits dissemination but is dispensable for persistence. J Clin Invest, 124(5), 1928-1944. doi:10.1172/jci67420
16. Manicklal, S., Emery, V. C., Lazzarotto, T., Boppana, S. B., & Gupta, R. K. (2013). The "silent" global burden of congenital cytomegalovirus. Clin Microbiol Rev, 26(1), 86-102. doi:10.1128/cmr.00062-12
17. Marchini, A., Liu, H., & Zhu, H. (2001). Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes. J Virol, 75(4), 1870-1878. doi:10.1128/jvi.75.4.1870-1878.2001
18. Murphy, E., Rigoutsos, I., Shibuya, T., & Shenk, T. E. (2003). Reevaluation of human cytomegalovirus coding potential. Proc Natl Acad Sci U S A, 100(23), 13585-13590. doi:10.1073/pnas.1735466100
19. Reddehase, M. J., Podlech, J., & Grzimek, N. K. (2002). Mouse models of cytomegalovirus latency: overview. J Clin Virol, 25 Suppl 2, S23-36.
20. Reeves, M., Murphy, J., Greaves, R., Fairley, J., Brehm, A., & Sinclair, J. (2006). Autorepression of the human cytomegalovirus major immediate-early promoter/enhancer at late times of infection is mediated by the recruitment of chromatin remodeling enzymes by IE86. J Virol, 80(20), 9998-10009. doi:10.1128/jvi.01297-06
21. Ross, S. A., & Boppana, S. B. (2005). Congenital cytomegalovirus infection: outcome and diagnosis. Semin Pediatr Infect Dis, 16(1), 44-49. doi:10.1053/j.spid.2004.09.011
22. Soroceanu, L., Matlaf, L., Khan, S., Akhavan, A., Singer, E., Bezrookove, V., . . . Cobbs, C. S. (2015). Cytomegalovirus Immediate-Early Proteins Promote Stemness Properties in Glioblastoma. Cancer Res, 75(15), 3065-3076. doi:10.1158/0008-5472.can-14-3307
23. Stenberg, R. M. (1996). The human cytomegalovirus major immediate-early gene. Intervirology, 39(5-6), 343-349.
24. Stenberg, R. M., Thomsen, D. R., & Stinski, M. F. (1984). Structural analysis of the major immediate early gene of human cytomegalovirus. J Virol, 49(1), 190-199.
25. Stenberg, R. M., Witte, P. R., & Stinski, M. F. (1985). Multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region 2 and evidence for a common initiation site within immediate-early region 1. J Virol, 56(3), 665-675.
26. Stinski, M. F., & Petrik, D. T. (2008). Functional roles of the human cytomegalovirus essential IE86 protein. Curr Top Microbiol Immunol, 325, 133-152.
27. Tang, Q., & Maul, G. G. (2006). Mouse cytomegalovirus crosses the species barrier with help from a few human cytomegalovirus proteins. J Virol, 80(15), 7510-7521. doi:10.1128/jvi.00684-06
28. Taylor, R. T., & Bresnahan, W. A. (2005). Human cytomegalovirus immediate-early 2 gene expression blocks virus-induced beta interferon production. J Virol, 79(6), 3873-3877. doi:10.1128/jvi.79.6.3873-3877.2005
29. Taylor, R. T., & Bresnahan, W. A. (2006a). Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor alpha-induced NFkappaB-dependent gene expression. J Virol, 80(21), 10763-10771. doi:10.1128/jvi.01195-06
30. Taylor, R. T., & Bresnahan, W. A. (2006b). Human cytomegalovirus immediate-early 2 protein IE86 blocks virus-induced chemokine expression. J Virol, 80(2), 920-928. doi:10.1128/jvi.80.2.920-928.2006
31. Wang, D., & Fu, T. M. (2014). Progress on human cytomegalovirus vaccines for prevention of congenital infection and disease. Curr Opin Virol, 6, 13-23. doi:10.1016/j.coviro.2014.02.004
32. Wang, X., Hu, M., Xing, F., Wang, M., Wang, B., & Qian, D. (2017). Human cytomegalovirus infection promotes the stemness of U251 glioma cells. J Med Virol, 89(5), 878-886. doi:10.1002/jmv.24708
33. Yue, Y., & Barry, P. A. (2008). Rhesus cytomegalovirus a nonhuman primate model for the study of human cytomegalovirus. Adv Virus Res, 72, 207-226. doi:10.1016/s0065-3527(08)00405-3