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

• Published in: PloS one 2020-03, Vol.15 (3), p.e0230126-e0230126
• Main Authors: Tan, Lei, Hu, Yi, Li, Yalan, Yang, Lingchen, Cai, Xiong, Liu, Wei, He, Jiayi, Wu, Yingxin, Liu, Tanbin, Wang, Naidong, Yang, Yi, Adelstein, Robert S, Wang, Aibing
• Format: Article
• Language: English
• Subjects: Analysis; Animal diseases; Animal genetic engineering; Animal models; Animal vaccines; Biology; Biology and Life Sciences; Biotechnology; Cardiology; Clinical trials; Cloning; CRISPR; Deoxyribonucleic acid; Disease prevention; DNA; Efficiency; Embryo cells; Embryonic stem cells; Gene targeting; Genes; Genetic engineering; Genetic modification; Genetically modified organisms; Genome editing; Genomes; Homologous recombination; Homology; House mouse; Insertion; Investigations; Laboratories; Medical research; Molecular biology; Muscle proteins; MYH9 gene; Myosin; Neomycin; Proteins; Proteomics; Research and Analysis Methods; Sequences; Stem cell research; Stem cell transplantation; Stem cells; Veterinary colleges; Veterinary medicine
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0230126

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