Establishment Of HPV16E_6/7 Transgenic Mice

Human papillomaviruses(HPVs) are double-stranded DNA tumor viruses that induce hyperproliferative lesions in cutaneous and mucosal epithelia, which belongs to Papoviruidae. Various HPV types have been implicated as causative agents in the genesis of epithelial diseases in laryngeal, oral, epidermal and cervical epithelia. Infection with a human papillomaviruses, particularly a high risk virus, can account for the subsequent development of cervical and cutaneous cancers. HPVs encode two proteins, E6 and E7, that are important for cell immortalization. E6 and E7 interact with the tumor suppressor proteins p53 and pRb, respectively. Eg facilitates degradation of p53 through association with an accessory protein, E6-AP. E7 binds to pRb, leading to functional inactivation of these proliferation regulators and uncontrolled cell proliferation and tumor formation. P_inv is a promotor which promotes its downstream gene expressed in specific cells and tissues, has been successfully applied to study gene expression of transgenic mice.We constructed the P_inv recombinant plasmid that included gene E₆/7 in its MCS, gene E6/7 was acquired by PCR from fresh cervial cancer tissue of Jiangsu district and had been sequenced. the results show the E6E7 gene are highly homogeneous with German standard strand, the sequences of E6 is as same as German standard strand,there is only one mutation in 109^th bp(T→C) of E7 and the mutation doesn'tcause any change of amino acid. After that, we verified the tissue speciality of P_INV by transfection to several kinds of cells. To establish the pINV-E6/7 transgenic mice ,we micro injected the Pinv-E6/7 DNA fragment into male nuclei. In this research, total 129 mice were born. By PCR, five transgenic mice were detected positive for PINV-E6/7.T0 confirm the positive transgenic mice, southern blot was used to detect these positive transgenic mice. The result showed four transgenic mice were positive for pINV-E6/7. After propagating, the Fl positive rate is 50.3%(19/36), the F2 positive rate is 53%(62/117) by PCR detection, which suggested that this was conformed to the laws of Mondel inheritance and transgene could be passaged to its descendants.To veritify the tissue-specificity of Pinv in our positive pINV-E6/7 transgenic mice, we test the mRNA of E6/7 and observe the pathological changes of various tissues, the results show our experiments are successful.These suggested that the gene construct contained Pinv and gene E6/7 is successful, the transgenic mice model will become strongly useful tool for exploring the related vaccine and studing the relationship of Rb, p53 genes and E6/7 gene.