Transgene Vector Construction For The Tree Shrew Spermatagonial Stem Cell Line

Tree shrew is widespread in Southeast Asian countries and in Southwest China, which is between the mice and the monkey in genetics, and even once classificated in Lower primates.Tree shrew is very similar to human in mmunology, virology and oncology, it has several significant advantages, such as small size, short reproductive cycle, and low cost advantage. So it is an ideal experimental animal model owning to these advantages. But the tree shrew is not widely used, the main reason is the lack of genetic operations. Gene modification is the commonly and important method to model human diseases in modern medical biology research. The first to use is also more commonly used lentivirus infection method is with high efficiency of genetic operations. For the study of transgenic tree shrews, we will modify and select different lentiviral vectors, hoping to find a lentiviral vector with high infection efficiency and high expression of foreign genes.In this paper, a variety of lentiviral expression vectors were constructed by molecular cloning and many lentiviral were packaged. At last, we successfully infected the tree shrew spermatogonial stem cells through the screening of lentivirus, which has high infection efficiency and high expression efficiency. The main contents are as follows:1. Fragments of EF1a promoter and PGK promoter were obtained by high fidelity PCR reaction, and cloning vectors with different promoters were constructed by ligation reaction. After ligation, new lentiviral expression plasmids. pTomo-EFla, SIV-EFla and SIV-PGK, were obtained by double digestion and ligation.2. Lentiviral expression plasmids were amplified and lentivirus packaging, lentivirus packaging though different lentiviral expression plasmids with different transfection reagents. A variety of lentiviruses of different concentrations were obtained by collecting and enriching.3. Infection efficiency of SIV and SIV-EF1a virus was higher than others, which was detected by using different lentivirus to injecte tree shrew primary generation SSC.4. The tree shrew SSC were injected by using the selected lentiviruses.5. The transgenic tree shrew were obtained from transgenic tree shrew SSC, which proved that the high expression efficiency of lentiviral vector...