An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment modification.

Rats have long been an ideal model for disease research in the field of biomedicine, but the bottleneck of in vitro culture of rat embryonic stem (ES) cells hindered the wide application as genetic disease models. Here, we optimized a special medium which we named 5N-medium for rat embryonic stem cells, which improved the in vitro cells with better morphology and higher pluripotency. We then established a drug selection schedule harboring a prior selection of 12 h that achieved a higher positive selection ratio. These treatments induced at least 50% increase of homologous recombination efficiency compared with conventional 2i culture condition. Moreover, the ratio of euploid ES clones also increased by 50% with a higher germline transmission rate. Finally, we successfully knocked in a 175 kb human Bacterial Artificial Chromosome (BAC) fragment to rat ES genome through recombinase mediated cassette exchange (RMCE). Hence, we provide a promising system for generating sophisticated rat models which could be benefit for biomedical researches.

Production of p53 gene knockout rats by homologous recombination in embryonic stem cells.

The use of homologous recombination to modify genes in embryonic stem (ES) cells provides a powerful means to elucidate gene function and create disease models. Application of this technology to engineer genes in rats has not previously been possible because of the absence of germline-competent ES cells in this species. We have recently established authentic rat ES cells. Here we report the generation of gene knockout rats using the ES-cell-based gene targeting technology. We designed a targeting vector to disrupt the tumour suppressor gene p53 (also known as Tp53) in rat ES cells by means of homologous recombination. p53 gene-targeted rat ES cells can be routinely generated. Furthermore, the p53 gene-targeted mutation in the rat ES-cell genome can transmit through the germ line via ES-cell rat chimaeras to create p53 gene knockout rats. The rat is the most widely used animal model in biological research. The establishment of gene targeting technology in rat ES cells, in combination with advances in genomics and the vast amount of research data on physiology and pharmacology in this species, now provide a powerful new platform for the study of human disease.

Alterations in Brca1 expression in mouse ovarian granulosa cells have short-term and long-term consequences on estrogen-responsive organs.

Incessant menstrual cycle activity, uninterrupted by either pregnancy or oral contraceptive use, is the most important risk factor for sporadic ovarian cancer. Menstrual cycle progression is partly controlled by steroid hormones such as estrogens and others that are secreted by the ovarian granulosa cells. We showed earlier that mice carrying a homozygous granulosa cell-specific knockout of Brca1, the homolog of BRCA1 that is associated with familial ovarian cancer predisposition in humans, develop benign epithelial tumors in their reproductive tract. These tumors are driven, at least in part, by a prolongation of the proestrus phase of the estrus cycle (equivalent to the follicular phase of the menstrual cycle) in Brca1 mutant mice, resulting in prolonged unopposed estrogen stimulation. Mutant mice synchronized in proestrus also showed increased circulating estradiol levels, but the possibility that this change also has a role in tumor predisposition was not investigated. We sought to determine whether these changes in hormonal stimulation result in measurable changes in tissues targeted by estrogen outside the ovary. Here we show that mice carrying a Brca1 mutation in their ovarian granulosa cells show increased endometrial proliferation during proestrus, implying that the effects of Brca1 inactivation on estrogen stimulation have short-term consequences, at least on this target organ. We further show that mutant mice develop increased femoral trabecular thickness and femoral length, which are well-known consequences of chronic estrogen stimulation. Estrogen biosynthesis by granulosa cells was increased not only in mice carrying a homozygous Brca1 mutation, but also in heterozygous mutants mimicking the mutational status in granulosa cells of human BRCA1 mutation carriers. The results suggest that human germline BRCA1 mutations, although associated with increased cancer risk, may also have beneficial consequences, such as increased bone strength, that may have contributed to the maintenance of mutated BRCA1 alleles in the human gene pool.

Inactivation of Msx1 and Msx2 in neural crest reveals an unexpected role in suppressing heterotopic bone formation in the head.

In an effort to understand the morphogenetic forces that shape the bones of the skull, we inactivated Msx1 and Msx2 conditionally in neural crest. We show that Wnt1-Cre inactivation of up to three Msx1/2 alleles results in a progressively larger defect in the neural crest-derived frontal bone. Unexpectedly, in embryos lacking all four Msx1/2 alleles, the large defect is filled in with mispatterned bone consisting of ectopic islands of bone between the reduced frontal bones, just anterior to the parietal bones. The bone is derived from neural crest, not mesoderm, and, from DiI cell marking experiments, originates in a normally non-osteogenic layer of cells through which the rudiment elongates apically. Associated with the heterotopic osteogenesis is an upregulation of Bmp signaling in this cell layer. Prevention of this upregulation by implantation of noggin-soaked beads in head explants also prevented heterotopic bone formation. These results suggest that Msx genes have a dual role in calvarial development: They are required for the differentiation and proliferation of osteogenic cells within rudiments, and they are also required to suppress an osteogenic program in a cell layer within which the rudiments grow. We suggest that the inactivation of this repressive activity may be one cause of Wormian bones, ectopic bones that are a feature of a variety of pathological conditions in which calvarial bone development is compromised.

EphA4 as an effector of Twist1 in the guidance of osteogenic precursor cells during calvarial bone growth and in craniosynostosis.

Heterozygous loss of Twist1 function causes coronal synostosis in both mice and humans. We showed previously that in mice this phenotype is associated with a defect in the neural crest-mesoderm boundary within the coronal suture, as well as with a reduction in the expression of ephrin A2 (Efna2), ephrin A4 (Efna4) and EphA4 in the coronal suture. We also demonstrated that mutations in human EFNA4 are a cause of non-syndromic coronal synostosis. Here we investigate the cellular mechanisms by which Twist1, acting through Eph-ephrin signaling, regulates coronal suture development. We show that EphA4 mutant mice exhibit defects in the coronal suture and neural crest-mesoderm boundary that phenocopy those of Twist1(+/-) mice. Further, we demonstrate that Twist1 and EphA4 interact genetically: EphA4 expression in the coronal suture is reduced in Twist1 mutants, and compound Twist1-EphA4 heterozygotes have suture defects of greater severity than those of individual heterozygotes. Thus, EphA4 is a Twist1 effector in coronal suture development. Finally, by DiI labeling of migratory osteogenic precursor cells that contribute to the frontal and parietal bones, we show that Twist1 and EphA4 are required for the exclusion of such cells from the coronal suture. We suggest that the failure of this process in Twist1 and EphA4 mutants is the cause of craniosynostosis.