Molecular Human Reproduction, Vol. 7, No. 11, 1039-1046,
November 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Expression of a green fluorescent protein variant in mouse oocytes by injection of RNA with an added long poly(A) tail
1 Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo,162-8666, 2 Department of Obstetrics and Gynecology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, 113-8241 and 3 Laboratory of Intracellular Metabolism, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan
Abstract
In oocytes, cytoplasmic 3' polyadenylation regulates translational activation of dormant mRNA during meiotic maturation. Thus exogenous proteins are hardly expressed after injection of conventional RNA. To circumvent this, we synthesized a long polyadenylated (~250 A) tail to encode RNA with an enhanced yellow fluorescent protein targeted to mitochondria (EYFP-mito), and injected it into mouse oocytes at the germinal vesicle (GV) stage. From this transcript, EYFP-mito was clearly expressed in ~80% of oocytes, while scarce expression from a transcript with only 30 A was observed. In strongly expressing oocytes, fluorescence was detected within 1–3 h after RNA injection, increased linearly up to 12 h, and reached a maximum at 12–15 h. The distribution of EYFP-mito matched the staining of mitochondria in these oocytes. About 80% of these oocytes underwent GV breakdown and 60% matured in vitro, comparable to non-expressing or non-RNA-injected oocytes. Some of the oocytes which strongly expressed EYFP-mito remained at the GV stage. Thus, the expression was not always accompanied by meiotic maturation, nor did it suppress the maturation process. Mature oocytes expressing EYFP-mito possessed normal fertilizability associated with intracellular Ca2+ oscillations, and developed into 2-cell embryos. Thus, polyadenylated RNA is a useful tool applicable to the expression of EYFP-fused functional proteins or of indicator protein probes for studies of mammalian fertilization.
gene expression/GFP variant/mouse oocyte/polyadenylated RNA/mitochondria-targeted protein
Notes
4 To whom correspondence must be addressed. shunm{at}research.twmu.ac.jp
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