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.

journal_name

Mol Hum Reprod

authors

Aida T,Oda S,Awaji T,Yoshida K,Miyazaki S

doi

10.1093/molehr/7.11.1039

subject

Has Abstract

pub_date

2001-11-01 00:00:00

pages

1039-46

issue

11

eissn

1360-9947

issn

1460-2407

journal_volume

7

pub_type

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