Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells

Abstract

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.

Investigation of the molecular biology underlying the pronounced high gene targeting frequency at the Myh9 gene locus in mouse embryonic stem cells

“…As demonstrated in previous studies, gene targeting at the Myh9 gene locus, in particular the exon2 site, had a pronounced high HR efficiency (>90%) in mESCs [27,[29][30][31]. Although GT efficiency in mESCs is considered 10-100 fold higher than that in mammalian somatic cells [34].…”

“…The constructs targeting to the Myh9 gene exon2, intron2 and exon3 sites were separately generated by using the same strategy as described previously [27,31]. Briefly, DNA fragments for the 5' and 3' homologous arms were amplified by PCR using the primer pairs listed in S1 Table and the template of 129/Sv genomic BAC clone DNA, and then cloned into the vector mpNTKV-LoxP described previously [27].…”

“…HR events occurring at the Myh9 gene exon2 (including those truncated targeting constructs), intron2 and exon3 sites were separately identified by PCR with the primer pair (the forward primer located at the neomycin, while the reverse primer resided immediately outside the 3' short arm) listed in S1 Table . The preparation of genomic DNA, the components of PCR system and the PCR reaction conditions completely followed the previous description [31]. The PCR products were analyzed by electrophoresis on 1.0% agarose gel, the wild-type allele produced no band while the targeted allele yielded an expected �2.1, 2.2, 2.3 kb band, respectively, as indicated in S1 Table . At least five randomly selected PCR-amplified products were excised, mixed, extracted and cloned into the T-easy vector for sequencing (Promega).…”

“…This unique property has been applied to the production of a series of nonmuscle myosin II (NM II) related mutant mouse lines (�7) [27][28][29][30]. Moreover, this finding has also been extended to the site-specific insertion of other exogenous genes in mouse ES cells [31]. In the present study, we intended to investigate the molecular biology underlying this distinctive HR efficiency from multiple aspects.…”

Replacing nonmuscle myosin 2A with myosin 2C1 permits gastrulation but not placenta vascular development in mice

“…A floxed Neo r cassette, which aided in selection of targeted clones, was removed by crossing the mutant mice with CMV-Cre mice. Homologous recombination was made easier by the presence of a “safe harbor” site in Myh9 ( Liu et al. , 2018 ).…”