Optimization Of Mouse Zygotes Microinjection Technique And Construction Of Trex1 Knockout Mice

Microinjection technique is one of the major tools used to construct transgenic animal model, the success rate of using this method is affected by many factors.Trexl is the major 3' end DNA exonuclease in mammalian cells. There is a large number of cells in the body that damaged or apoptosis, which can release the DNA even in normal conditions, if human Trex1 gene mutations, DNA release by those cells will can not be removed efficiently and result in massive accumulation, which can Induce innate immune system to recognize and clear the DNA from the creature itself, and likely to produce autoimmune diseases, such as Aicardi-Gouti Res syndrome or systemic lupus erythematosus (SLE). Using gene editing technology to knock out the Trexl gene, we may construct an animal model that may present autoimmune disease features to study the changes and their underlying mechanisms, therefore to provide a basis for understanding the pathogenesis of these diseases and exploring the novel targets used for the diagnosis and treatment of autoimmune diseases.In this study, we successfully constructed three targeting Trexl gene knockout plasmids, and we verified the effectiveness of these plasmids in L929 cells first. To further introduce these plasmids to mouse fertilized eggs, we optimize the conditions used for microinjection including four factors:the age of donor mice, the site of the injection on the fertilized eggs, and the contents and concentration of the injection materials. The microinjection efficiency was tested by using the px459-sg1-Trex1 knockout plasmid and the transcription product of Trex1-sg1RNA, the cas9mRNA and cas9 protein. We show that the fertilized eggs derived from 4.5 week old donor mice are best to be used for the microinjection, and the optimal Cas9mRNA/sg1mRNA concentration is 50/25 (ng/?l), and the best site for injection is the nucleus, but not cytoplasm.We obtained 140 newborn mice, including 1 homozygous Trexl knockout mice,38 heterozygous mice and 101 wild type mice. After the analysis of the physiological and biochemical indexes of these mice, we found that the spleen size of Trex1-/- mice was larger than that of Trexl+/- and Trex1+/- mice. And WB results showed that Trex1 was expressed in Trex1+/+ and Trex1+/- mice while Trex1-/- mice was not, which means that it may have been produced autoimmune disease due to the absence of Trexl. In conclusion, we successfully construct a Trexl knockout mouse model by using the zygotes microinjection method and CRISPR/Cas9 technology, provide a solid base for further study the mechanisms underlying Trex1-regulations related to autoimmune disorders.