Use of luciferase chimaera to monitor PLCzeta expression in mouse eggs.
The microinjection of cRNA encoding phospholipase Czeta (PLC zeta) causes Ca2+ oscillations and the activation of development in mouse eggs. The PLCzeta protein that is expressed in eggs after injection of cRNA is effective in causing Ca2+ oscillations at very low concentrations. In order to measure the amount and timecourse of protein expression we have tagged PLCzeta with firefly luciferase. The expression of the luciferase protein tag in eggs is then measured by incubation in luciferin combined with luminescence imaging, or by the lysis of eggs in the presence of Mg-ATP and luciferin in a luminometer. The use of luciferase to monitor protein expression after injection of cRNA is a sensitive and effective method that efficiently allows for sets of eggs to be used for PLCzeta quantitation, Ca2+ imaging, and studies of embryo development.
Swann
Karl
K
Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff, UK.
Campbell
Karen
K
Yu
Yuansong
Y
Saunders
Christopher
C
Lai
F Anthony
FA
D000818:Animals; D021621:Imaging, Three-Dimensional; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D008845:Microinjections / Q000379:methods*; D010063:Ovum / Q000201:enzymology*; D018075:RNA, Complementary / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism*
D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D010738:Type C Phospholipases