Dataset Information

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.

SUBMITTER: Liu T

PROVIDER: S-EPMC5811019 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Liu Tanbin T Hu Yi Y Guo Shiyin S Tan Lei L Zhan Yang Y Yang Lingchen L Liu Wei W Wang Naidong N Li Yalan Y Zhang Yingfan Y Liu Chengyu C Yang Yi Y Adelstein Robert S RS Wang Aibing A

PloS one 20180213 2

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, ...[more]

PMID: 29438440

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Project description:The generation of genetically modified mouse models derived from gene targeting (GT) in mouse embryonic stem (ES) cells (mESCs) has greatly advanced both basic and clinical research. Our previous finding that gene targeting at the Myh9 exon2 site in mESCs has a pronounced high homologous recombination (HR) efficiency (>90%) has facilitated the generation of a series of nonmuscle myosin II (NM II) related mouse models. Furthermore, the Myh9 gene locus has been well demonstrated to be a new safe harbor for site-specific insertion of other exogenous genes. In the current study, we intend to investigate the molecular biology underlying for this high HR efficiency from other aspects. Our results confirmed some previously characterized properties and revealed some unreported observations: 1) The comparison and analysis of the targeting events occurring at the Myh9 and several widely used loci for targeting transgenesis, including ColA1, HPRT, ROSA26, and the sequences utilized for generating these targeting constructs, indicated that a total length about 6 kb with approximate 50% GC-content of the 5' and 3' homologous arms, may facilitate a better performance in terms of GT efficiency. 2) Despite increasing the length of the homologous arms, shifting the targeting site from the Myh9 exon2, to intron2, or exon3 led to a gradually reduced GT frequency (91.7, 71.8 and 50.0%, respectively). This finding provides the first evidence that the HR frequency may also be associated with the targeting site even in the same locus. Meanwhile, the decreased trend of the GT efficiency at these targeting sites was consistent with the reduced percentage of simple sequence repeat (SSR) and short interspersed nuclear elements (SINEs) in the sequences for generating the targeting constructs, suggesting the potential effects of these DNA elements on GT efficiency; 3) Our series of targeting experiments and analyses with truncated 5' and 3' arms at the Myh9 exon2 site demonstrated that GT efficiency positively correlates with the total length of the homologous arms (R = 0.7256, p<0.01), confirmed that a 2:1 ratio of the length, a 50% GC-content and the higher amount of SINEs for the 5' and 3' arms may benefit for appreciable GT frequency. Though more investigations are required, the Myh9 gene locus appears to be an ideal location for identifying HR-related cis and trans factors, which in turn provide mechanistic insights and also facilitate the practical application of gene editing.

Dataset Information

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

Publications

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

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