Expression of membrane targeted aequorin in Xenopus laevis oocytes.
We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.
Daguzan
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C
Centre de Biologie du Développpement, UMR 9925, Université Paul Sabatier, Toulouse, France.
Nicolas
M T
MT
Mazars
C
C
Leclerc
C
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Moreau
M
M
D000331:Aequorin / Q000096:biosynthesis*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000502:physiology; D018076:DNA, Complementary; D005260:Female; D015870:Gene Expression; D007150:Immunohistochemistry; D008163:Luminescent Measurements; D008845:Microinjections; D018613:Microscopy, Confocal; D009865:Oocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000008:administration & dosage; D011985:Receptors, Serotonin / Q000096:biosynthesis; D044263:Receptors, Serotonin, 5-HT1; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D037401:Scyphozoa; D014982:Xenopus laevis
D018076:DNA, Complementary; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011993:Recombinant Fusion Proteins; D000331:Aequorin