Methological Studies On Lentiviral Transgenesis In Mice

1. BackgroundTransgenic technology is an important technique plateforme for in vivo investigation of gene functions. Currently, the applicable transgene techniques include micro-injection, ES cell-mediated transgenesis, sperm-mediated gene transfer and nuclear transfer. In all the techniques mentioned above, micro-injiection is most widely used and applicable to most animal species. However, this technique is inefficient, costly and requires sophisticated equipments and skills, especially when it comes to large animal species. Lentiviral vector provides an alternative method to produce transgenic animals. Lentiviral vector is a retroviral vecor. After infection of cells, the RNA genome of the viral vector can be reverse transcribed into DNA, and the DNA can be efficiently integrated into the genome of host cells. Besides, in contrast to the congene retroviral vectors, lentivral vector is capable of infecting both deviding and quiescient cells. On the basis of this point, Lois(2002) et al successfully produced transgenic mice with lentiviral vector. In this work, the transgene delivered by lentiviral vector expressed efficiently and was not subject to silence during germline transmission.Detrentic cell (DC) is the main professional antigen-presentation cells (APC) in human and mammal bodies. In addition to stimulating immune response, DC is also capable of inducing peripheral immune tolerance. Therefore, to understand the precise mechanism of antigene presentation, DC is the suitable target cells. The process of antigen presentation in DC is mediated by vestricle translocation, which is a highly organized process regulated by Rab gene farmily. On the basis of this point, to understand the detailed mechanism of antigen presentation, it is necessary to investigate the roles of Rab genes in the process of antigene presentation. In this work, we established a DC-specific Rab32 knock-down mouse model by lentiviral transgenesis which had significantly reduced Rab32 expression specificanlly in DCs, and laid down the foundation to further investigate the roles of Rab32 protein in antigen presentation.2. ResultsIn the first part, methological researches on lentiviral transgenesis was performed. The mouse eggs was collected and subjected to perivitelline space injection (PSI) with concentrated lentiviral vectors. The effects of PSI on egg development were investigated. The results were the following:1) Through the three-plasmd packgeing system, concentrated lentivial suspension of which the TU was >1×109 was prepared; after PSI of the vector, 2-cell cleavage rate of the eggs injected with lentivral vector was 81.8%(1189/1453), with no significant difference from the untreated eggs, indicating that PSI with lentiviral vector had no impactt on the development of the injected eggs; the GFP positive rate (based on counting under microscope) was more than 90%(1178/1189), indicating that the lentiviral vector was properly packaged and capable of transducing mouse eggs efficiently; 2) the 2-cell eggs which had been subjected to PSI were transferred into pseudo-pregnant female mice, and the pregnance rate of the recipients was 42.8%(12/28), which was also comparable to the untreated egg group, suggesting that in vivo development capacity of the injected eggs was not impacted by PSI process either; 3) the transgenic rate of the founder mice was 60.8%(73/120), and the overall transgenic mouse production efficiency (transgenic mice/injected eggs) was 5.0%(73/1453), further indicating that lentiviral vector-mediated transgenesis is an efficient and practicable method for transgenic mouse production; 4) based on the eGFP transgenic founder mice, a transgenic line was established by mating the transgenic founder mice with wild-type mice of the same strain. Results indicated that transgene expression remained stable at least over six generations, suggesting that the expression of transgenes delivered by lentiviral vector can be passed on through germline transmission.In the second part, based on RNA interference (RNAi) strategy, we produced transgenic knock-down mice targeting Rab32 gene in DCs by lentiviral transgenesis. The vector for expressing siRNA molecules was constructed based on the FUGW vector which was used in the first part. To express the siRNA molecules specifically in DCs, the siRNA coding sequence was cloned downstream the CD11c promoter. To trace the siRNA expression visibly, the eGFP coding sequence was placed downstream the same promoter in the same orientation. The results are the following: 1) the siRNA expression cassette was detected in the genomic DNAs of both the founder mice and their F1 offspring by PCR. Besides, eGFP expression was observed in the DCs located in the skin of ear concha under fluorescent microscope. These results indicated that transgenic mice haboring the siRNA expression cassette was produced. 2) By flow cytometry analysis of Bone Marrow Detrentic Cells (BMDC), it was shown that eGFP was specifically expressed in DCs, indicating that the used CD11c promoter drive siRNA expression specifically in DCs. 3) By Fluorenscent Quantitative PCR ( FQ-PCR) analysis, the Rab32 expression in DCs was shown to be reduced by 78.3%, indicating that the siRNA molecule delivered by lentiviral vector was expressed and knocked down the target gene expression significantly. Conclusions1. An efficient and praticable lentiviral transgenesis system, which consists of lentiviral vector package, perivitllent space injection (PSI) and mouse egg manipulation, was successfully established.2. Using the established lentiviral transgenesis system, transgenic knock-down mice targeting Rab32 specifically in DCs was produced, further indicating that the established lentiviral transgenesis can be applied to expressing both structural genes and siRNA molecules.