Screen And Application Of Friendly Transgene Integration Sites In Pigs

Foreign gene expression in recipient cells can be influenced by many factors.Among these factors,integration sites play a crucial role in determining the expression of the target gene.Random integration in host genome is subjected to position effects,which make the expression of foreign genes unreliable.Adding some specific DNA sequences or optimizing the design of expression cassettes to simulate the highly transcribed chromosome region could reduce the effects of integration sites.Alternatively,the integration sites on active transcribed region can be pre-selected,then the target gene will be inserted into those sites to achieve site-specific integration.Up to date,determining precisely where to integrate exogenous DNA sequences in pig genome to maximize transgene safety has received little attention.For high efficiency of foreign gene expression,discovering the friendly integration sites in pig genome becomes the most important task.To find candidate genomic loci,we firstly searched the pig genome for highly transcribed regions using gene expression data.These data were retrieved from a public pig gene expression altas set.We used a sliding window approach with the window size of 500 kb,and calculated the mean expression level of all 500 kb windows.Cancer-related genes might promote malignant cell transformation if their expression were disturbed.We excluded the windows containing cancer-related genes.In order to avoid disturbance to transcriptional units,we selected intergenic regions without non-coding RNA and large nucleosomal formation ability.We finally screened out two candidate integration sites,named Pifs501 and Pifs302,which located on chr15:137957000-137958000 and chr16:44288700-44289700.We performed site-specific integration by CRISPR/Cas9-mediated homologous recombination on Pifs501 and Pifs302 sites.CMV,PGK and EFla promoters driven EGFP stable expression were achieved on Pifs501 and Pifs302 in IBRS-2 cells and pig fibroblasts.EGFP integration on Pifs501 did not significantly interfere the expression of nearby genes 600 kb centering on insertion site.Pifs501 showed typical marks of actively transcribed chromatin with hypomethylation level of promoter and less repressive histone marks(H3K27me3).Pifs501 could support stable EGFP expression at different development stages,such as Pifs501-CMV-EGFP targeted cloned blastocysts,embryos and after birth.EGFP was ubiquitously expressed in various tissues of transgenic pigs,with higher expression in heart and pancreas.The widespread expression in these tissues did not interfere with the expression of neighboring genes adjacent to Pifs501.Since Pifs501 was suitable for the expression of exogenous gene,we established Master cell line with recognition site loxP of Cre recombinase on Pifs501 site.After electroporating a Cre expression plasmid and an exchange vector containing Follistatin into Pifs501 Master fetal fibroblasts,we obtained single cell clones with EGFP substituted by The establishment of the Master cell line on the Pifs501 site enable us to achieve site specific integration by recombination reaction of Cre/loxP system,which greatly improve the targeting efficiency of foreign gene into specific gene locus.In summary,using gene expression data,we proposed a de novo method for screening friendly transgene integration sites.We screened out a friencly integration site Pifs501.Furthermore,we established a Master cell line for site-specific integration on Pifs501.These works provide a good method for accurate editing of pig genome,and greatly improve the efficiency of integration and expression of exogenous genes in host cells.Our study has significant importance in the extensive application of transgenic technology in pig breeding and human disease model construction.