| Epstein-Barr virus (EBV) is associated with a number of human malignancies, especlialy with nasopharyngeal carcinoma (NPC). because of deficiency of animal model. However, due to lacking of evidence of animal models, the role of EB virus in NPC occurrence and development remains in researching hostspot .The reason for this may be that the regulators could not direct the transgene to be efficiently expressed in mouse nasopharynx. In view of this, the feasible strategy to get EBV -related transgenic model of NPC might be to select some appropriate regulators, which can lead target gene to be expressed efficiently and specifically in nasopharynx. In order to resolve this problem We took the following studies:1. Expression and evaluation of recombinant LMP1 lentiviral vector. ED-L2 promoter was amplified by polymerase chain reaction (PCR) from pED-L2-N-LMPl vector template. PacI enzymed cite was inserted into 5 ' of forward primer, and BamHI enzymed cite was inserted into 5 downstream primer. Then production of PCR was ligated with pUC118 vector. Using the DNA recombinant techniques, two eukaryotic expression vectors N-LMP1 lentiviral vector and B-LMP1 lentiviral vector were constructed respectively, both target genes N-LMP1 and B-LMP1 inserted in sense orientation were confirmed and by restriction endonuclease digestion analysis.2. Gene transfection in vitro and analysis of transgenic vectors .the N-LMP1 lentiviral vector and B-LMP1 lentiviral vector were transferred into CNE1, 5-8F, 6-10B and 293T cells. All of cell lines with N-LMP1 lentiviral-4-vector and B-LMP1 lentiviral vector transient transfection was investigated under fluorescence microscope, and the expression of enhanced green fluorescent protein could be observed. Reverse transcriptase PCR was used to detect the expression of LMP1 gene at the level of messenger RNA (mRNA) and so did immunohistochemical method to test the expression of LMP1 gene. Results showed the target gene could express correctly in host cells. These two identified vectors were also transferred into mouse fibroblasts NIH3T3 cells. The target gene couldn't express in NIH3T3 cells and blank control.3. Establishment of transgenic mouse model. Using ED-L2-N-LMP1-EGFP lentiviral vector; 8.9; VSVG (vesicular stomatitis virus glycoprotein, VSVG) -pseudotyped were transferred together into 293T cells, the viral supernatant is ready for collection 60 hours after transfection. Adjust centrifuge to spin for 90 minutes at 4 25000 rpm, When spin is finished, remove all traces of supernatant, then add 100 l of cold PBS, keep tubes at 4 癈 for at least 12 hours to dissolve the pellet. The virus was injected into the perivitelline space of each embryo. Then zygotes were transplanted into the oviduct of pseudo-pregnant female mice, thus the founder transgenic mice were obtained and their biological characteristic were observed by PCR and Southern blot analysis.hi this study, we construct the vector using specific nasopharynx tissue promoter ED-L2 carrying the enhanced green fluorescent protein (EGFP) gene driven by a specific nasopharynx tissue expressing promoter, To construct ED-L2-EGFP lentiviral expression vector, ED-L2-N-LMP1-EGFP lentiviral expression vector and ED-L2-B-LMP1-EGFP lentiviral expression vector. Produce transgenic mouse model. The transgenic mice were built by microinjection. The DNA was injected into the perivitelline space of each embryo. Then zygotes were transplanted into the oviducts of pseudo-pregnant recipient female mice, we obtained 3 founder mice of the ED-L2-N-LMP1-EGFP lentiviral vector group, and these mice were examined by PCR and Southern blot analysis. All of them were positive for gene integration.-5-...
|
PDF Full Text Request