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Targeted integration of exogenous genes into so-called safe harbors/friend sites, offers the advantages of expressing normal levels of target genes and preventing potentially adverse effects on endogenous genes. However, the ideal genomic loci for this purpose remain limited. Additionally, due to the inherent and unresolved issues with the current genome editing tools, traditional embryonic stem (ES) cell-based targeted transgenesis technology is still preferred in practical applications. Here, we report that a high and repeatable homologous recombination (HR) frequency (>95%) is achieved when an approximate 6kb DNA sequence flanking the MYH9 gene exon 2 site is used to create the homology arms for the knockout/knock-in of diverse nonmuscle myosin II (NM II) isoforms in mouse ES cells. The easily obtained ES clones greatly facilitated the generation of multiple NM II genetic replacement mouse models, as characterized previously. Further investigation demonstrated that though the targeted integration site for exogenous genes is shifted to MYH9 intron 2 (about 500bp downstream exon 2), the high HR efficiency and the endogenous MYH9 gene integrity are not only preserved, but the expected expression of the inserted gene(s) is observed in a pre-designed set of experiments conducted in mouse ES cells. Importantly, we confirmed that the expression and normal function of the endogenous MYH9 gene is not affected by the insertion of the exogenous gene in these cases. Therefore, these findings suggest that like the commonly used ROSA26 site, the MYH9 gene locus may be considered a new safe harbor for high-efficiency targeted transgenesis and for biomedical applications.

Porcine circovirus type 2 (PCV2) is the causative pathogen of porcine circovirus-associated diseases (PCVAD). This virus evolves mostly through point mutations and genome recombination between different PCV2 genotypes (e.g. PCV2a and PCV2b), as has been confirmed in swine herds. In the current work, the complete PCV2 genome sequences of 69 clones derived from various tissues (lymph node, spleen and lung,) of an infected individual, were subjected to phylogenetic and alignment analyses. The results not only demonstrate co-infection with distinct PCV2b subtypes (e.g. 1B and 1C) in the same animal, but also highlight another mechanism of evolution - diverse point mutations acquired during immune evasion by this virus.

Background Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G 710 to A 710 ) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively. Results Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID 50 ), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal. Conclusions These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

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Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells. PLoS ONE 15(3): e0230126.