Coupled cytoplasmic transcription-and-translation--a method of choice for heterologous gene expression in Xenopus oocytes.
We demonstrate here that the intracellular environment of Xenopus oocytes is quite compatible with the requirements of T7 RNA polymerase (T7 RNAP)-mediated transcription. This reaction runs robustly in the oocyte cytoplasm for many hours. The coinjection of a T7 promoter-driven luciferase-encoding plasmid DNA and purified T7 RNAP into oocytes results in the prolonged production of luciferase protein. Thus, the efficient coupling of T7 RNAP-mediated transcription with the intrinsic oocyte translation machinery occurs in the oocyte cytoplasm. The coupled protein synthesis generates high expression yield, displays little variation in the expression level between individual oocytes, requires very limited amounts of DNA template and T7 RNAP, and does not affect the oocyte viability and functional status. Our detailed, quantitative comparison of the existing expression methods in Xenopus oocytes highlights the advantages of the technique based on the cytoplasmic coinjection of T7 RNAP and T7 promoter-driven plasmid DNA and demonstrates that it is greatly superior to the alternative methods of heterologous gene expression.
Tokmakov
Alexander A
AA
Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi-ku, Kanagawa, Japan. tokmak@gsc.riken.go.jp
Matsumoto
Eiko
E
Shirouzu
Mikako
M
Yokoyama
Shigeyuki
S
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D009865:Oocytes / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015533:Transcriptional Activation / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics*; D014982:Xenopus laevis
D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases