Related Experiment Video
Updated: Oct 27, 2025
CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus AAV and 2-Cell Embryo Electroporation
Published on: March 15, 2024
An optimized proliferation system of embryonic stem cells for generating the rat model with large fragment
Chang Liu1, Zhonglin Cui2, Youzhen Yan3
1Key Laboratory of Arrhythmias, Ministry of Education of China, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Research Center for Translational Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200120, China; Department of Medical Genetics, Tongji University School of Medicine, Shanghai, 200092, China.
Abstract:
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.
