Expression of exogenous proteins in porcine maturing oocytes after mRNA injection: Kinetic analysis and oocyte selection using EGFP mRNA
2001
Ohashi, S. (Tokyo Univ. (Japan)) | Naito, K. | Liu, J. | Sheng, Y. | Yamanouchi, K. | Tojo, H.
We investigated translation kinetics after enhanced green fluorescent protein (EGFP) mRNA injection into maturing porcine oocytes and then established a method for selecting the oocytes that were translating the injected mRNA without fixing and staining. The EGFP signal was observed within 3h of the mRNA injection, and the EGFP expression rate reached a maximum (about 30%) at 6h after injection. The high EGFP expression rate increased gradually until 18h after injection. In the EGFP expressing group, degenerated oocytes were absent and the maturation rate was comparable to that of non-injected oocytes, while the maturation rate was low and some oocytes were degenerated in the non-translating group. There were a significant number of morphologically normal oocytes without EGFP expression, therefore, we tried to select the oocytes translating the injected mRNA by co-injecting objective mRNA with EGFP mRNA as a marker. We found that the oocytes expressing EGFP also highly translated the co-injected mRNAs. These results suggest that translation of injected mRNA starts within 3h and continues for 18h after injection in maturing porcine oocytes, and that co-injection of objective mRNA with EGFP mRNA is a useful method for finding the oocytes that are translating objective mRNAs.
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