Generation Of EGFP Pigs At Rosa26 Site And Analysis EGFP Expression In Various Tissues

Swine is an important agricultural resource and animal model in biomedical research.CRISPR/Cas9 editing system is used to modify pig genomes efficiently and accurately,which greatly broadens the application of pig models in various research fields.Heterotopic expression of foreign genes is required to produce transgenic pigs with good phenotypes,but random integration of foreign genes usually leads to abnormal expression of foreign genes and unstable phenotypes.Thus,in order to avoid the side effects of random integration,the selection of Safe Harbor as a foreign gene insertion site is an effective way to ensure the stable expression of foreign genes.As one of the classic safe harbor,Rosa26 has been widely used in transgenic animal research,but the current research rarely systematically reveals the expression of Rosa26 in various tissues and organs of pigs.The aim of this paper was to obtain Rosa26 locus integrated eGFP transgenic pigs using homology-mediated End Join(HMEJ)and to detect the expression of eGFP in the tissues of transgenic pigs.Two eGFP/KI transgenic pigs were successfully obtained in this study.Then,we extracted the total RNA from each tissue and analyzed the m RNA expression level of eGFP in each tissue.The results showed that eGFP was expressed in different tissues of transgenic pigs,and the expression of the m RNA of eGFP/KI pig was high expression in Thyroid lymph node?kidney?fat?spleen and bladder.Finally,the frozen section results showed that eGFP was stably expressed in all tissues of pigs.Quantitative analysis of the average fluorescence intensity shows that eGFP is expressed in all tissues,and the expression level is different.The four tissues with the highest expression are ventricle,gastrocnemius,atrium,small intestine and atrium.In summary,this study used HMEJ-mediated gene knock-in strategy to obtain 2Rosa26 locus-integrated eGFP transgenic pigs.And in PBMCs,it is found that eGFP expression has cellular heterogeneity.This thesis systematically analyzed the expression of eGFP in various tissues and organs at the m RNA level and histological level,and it is of great research significance to optimize the gene editing program according to the experimental purpose.