| Rats are important and earliest used mammlian animal models for biological researches and human diseases.Compared to mice,the physiological composition,cellular regulatory mechanisms,and intelligence levels are more similar to those of humans.The laboratory rat(Rattus norvegicus)has been served for varieties of researching fields including physiology,pathology and pharmacology in the past 150 years.Given its physiological similarities to human,rat is currently the most commonly used animal model for many pre-clinical tests,especially those related to cardiovascular disease,breast cancer,diabetes,chronic inflammatory diseases and age-related diseases.In 1981 the first mouse ES cell line was successfully established with the capacities of self-renewal and differentiation to all three germ layers cells.However,relative studies on rat models have been lagged far behind mouse due to the lack of authentic rat embryonic stem cell.As the first clone mouse was generated by wakayma in 1998 and in 2003 Qi Zhou successfully botain the first cloned rat,nevertheless the application of rat model was still limited.In 2008,???et al successfully derived germline competent rat ES cell lines which could be maintained in undifferentiated self-renewing status in long-term in vitro cultures based on a chemically-defined basal culture system that contains N2B27 medium supplemented by two small molecules(2i:CHIR99021 and PD0325901).The development of rat ES cell-based gene targeting technologies brings a bright future for genetic manipulations in rat models.Yet it is more time-consuming and costly to generate transgenic or knockout rats than mice,which has severely limited the application of rat models in biomedical studiesThe recently developed mouse haploid embryonic stem cells(ESCs)have proven the feasibility of producing viable transgenic mice via oocyte injection.Thus,haploid ESCs can serve as an efficient way to generate transgenic animals with heritable genetic modifications.We derived rat haploid ES cells from pre-implantation haploid blastocysts of Dark Agouti(DA)rats by using N2B27-2i culture system.The rat ES cells we derived in such conditions displayed compacted colonly morphologies similar with rat ES cells.Further experiments comfirmed the expressions of pluripotent markers including Oct4,Sox2,Nanog and Rexl in rat haploid ES cells.The RahESCs express high levels of pluripotent marker genes and can be maintained in an undifferentiated status for more than 30 passages.They possess the ability to differentiate into various cell types of all three germ layers andcan contribute to the germline of chimeric rats.RahESCs can be used to establish genome-wide mutant libraries by piggyBac(PB)transposons.Homozygous gene knockout can be easily achieved in RahESCs by either homologous recombination or the CRISPR-Cas system.In addition,RahESCs can fertilize oocytes and generate fertile offspring and can therefore serve as a powerful strategy for the generation of transgenic rats.In summary,it is the first case of successful establishment of rat haploid ES cell lines.A series of identification in molecular,cellular and developmental level demonstrated our rat haploid ES cells are classic pluripotent stem cells,which could contribute to germline.It therefore can serve as a powerful strategy for the generation of transgenic rats.Overall rat haploid ESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat. |
PDF Full Text Request