Generation Of Double Gene Specific Knock-in Pigs At Rosa26 Locus

Many researchers have focused on knock-in pigs for site-specific integration,but little attention has been paid to genetically modified pigs with targeted integration of multi-gene recombination.However,a single recombinant gene transgenic animal is not enough to achieve the effect of improving two or more traits at the same time,which is a great waste of time and money for the time-consuming and laborious preparation of transgenic animals.Therefore,inserting multiple foreign genes at the same time to improve multiple traits of transgenic animals is a problem that needs to be solved urgently.The site-specific integration for multiple exogenous genes were achieved at Rosa26 locus of pig genome by CRISPR/Cas9 system in this reserch and an efficient and safe targeted integration technology of multiple genes was established.There are two modes of integration for exogenous genes: Fat-1 and IGF-1 were selected as exogenous genes for the first mode and regulated by self-cleaving 2A peptides(2A)and internal ribosome entry site(IRES).Fat-1 and the shRNA which against porcine epidemic diarrhea virus were selected as exogenous genes for the second mode and the Fat-1was driven by the promoter of EF1?.According to these two models,two targeting vectors were constructed and co-transfect into fetal fibroblasts respectively with Cas9/sg RNA vector by electrotransfection.A positive fibroblast clone was obtained by the limiting dilution and the integration efficiency was 5.2%.Then two genetically modified pigs were obtained with SCNT technology.The RT-PCR results of positive cell clones indicated that the exogenous gene was integrated into the porcine Rosa26 locus,and the endogenous promoter could drive the transcription of the exogenous gene;The integration of an exogenous promoter(EF1?)in the Rosa26 locus can initiate the transcription of exogenous genes;The integration of exogenous regulatory elements(such as 2A,IRES)at Rosa26 locus can regulate the expression of exogenous genes.The results of RT-q PCR and Western-Blotting in various tissues of F0 piglets further showed that multiple exogenous genes integrated at the Rosa26 locus of pigs can be safely and efficiently transcribed and expressed at the individual level.Exogenous genes can use Rose26 endogenous promoter to drive transcription,or Rose26 gene endogenous promoter to drive transcription and expression;The introduction of IRES and 2A before the exogenous gene can regulate the expression of the exogenous gene.The exogenous gene can be transcribed and expressed by the exogenous promoter EF1?.Importantly,gas chromatography analysis showed that the level of n-3PUFAs in these genetically modified pigs increased significantly,which led to a significant decrease in the ratio of n-6PUFAs/n-3PUFAs from 6.982 to 3.122(***P<0.001).The results of RT-q PCR and Western-blot in various tissues of F0 piglets further showed that multiple foreign genes integrated at the Rosa26 locus of pigs can be safely and efficiently transcribed and expressed at the individual level.In conclusion,the establishment of the editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.