title: Expression of a green fluorescent protein variant in mouse oocytes by injection of RNA with an added long poly(A) tail.
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 (approximately 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 approximately 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 Ca(2+) 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.
authors.affiliation: Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666.
authors.forename: T
authors.initials: T
authors.lastname: Aida
authors.forename: S
authors.initials: S
authors.lastname: Oda
authors.forename: T
authors.initials: T
authors.lastname: Awaji
authors.forename: K
authors.initials: K
authors.lastname: Yoshida
authors.forename: S
authors.initials: S
authors.lastname: Miyazaki
mesh_terms: D000818:Animals; D001483:Base Sequence; D020013:Calcium Signaling; D005260:Female; D005307:Fertilization in Vitro; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology*; D011061:Poly A / Q000235:genetics* / Q000378:metabolism; D026723:Polyadenylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011061:Poly A