Comparison of two different mouse embryonic stem cell culture systems in generating chimeric embryos by blastocyst microinjection method

Safarpoor, D.; Heidari, B.; Naderi, M. M.; Sarvari, A.; Behzadi, B.; Borjian, S.; Akhondi, M. M.; Radmehr, B.; Shirazi, A.
June 2014
Iranian Journal of Reproductive Medicine;Jun2014 Supplement, Vol. 12, p90
Academic Journal
Introduction: The generation of chimeric embryos is an important step in knockout mouse production. As known the source of donor mouse embryonic stem cells (ESCs) is effective on production of chimeric mouse embryos and germline transmission. The present study was aimed to investigate the efficiency of two different mouse ESCs in generating chimeric embryos. Materials and Methods: The embryos were collected from the oviducts of 0.5-dpc C57BL/6 mice and after 3 days culture, at blastocyst stage, randomly divided into 3 groups. I) the blastocysts were microinjected by mouse ESCs co-cultured on mitomycinated MEF; II) the blastocysts were microinjected by mouse feeder free ESCs; III) the non-injected blastocysts were considered as a control. Approximately, 10-15 GFP positive ESCs were microinjected into blastocele cavity of groups I and II. The hatching rate and the presence of GFP positive cells in inner cell mass (ICM) were evaluated at Day 5 of culturing. Results: The results showed that the hatching rate was significantly higher in group 1 than control group (96.3±3.7 vs. 58.6±11.3) though, the presence of GFP positive ESCs was lower in group 1 than group 2 (57.2±9.2 vs. 86.3±5.9). Conclusion: The increased hatching rate in group 1 compared to control is probably due to zona pellucida drilling by laser. The presence of greater numbers of GFP positive ESCs in group II might be related to the higher purity and homogeneity of cultured ESCs in feeder free culture system compared to the co-cultured group.


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