Quantitative analysis of gene expression in preimplantation mouse embryos using green fluorescent protein reporter.
We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available.
Medvedev
Serguei Yuri
SY
Department of Developmental Biology, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan. sergyurimedvedev@netscape.net
Tokunaga
Tomoyuki
T
Schultz
Richard M
RM
Furukawa
Tsutomu
T
Nagai
Takashi
T
Yamaguchi
Manabu
M
Hosoe
Misa
M
Yakovlev
Alexander F
AF
Takahashi
Seiya
S
Izaike
Yoshiaki
Y
D000818:Animals; D001755:Blastocyst / Q000378:metabolism* / Q000528:radiation effects; D002138:Calibration; D004247:DNA / Q000096:biosynthesis / Q000302:isolation & purification; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology* / Q000528:radiation effects; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006207:Half-Life; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008845:Microinjections; D009924:Organ Culture Techniques; D010957:Plasmids / Q000235:genetics; D011247:Pregnancy; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014466:Ultraviolet Rays
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA