pOEV: a Xenopus oocyte protein expression vector.
We have constructed a Xenopus laevis oocyte expression vector, pOEV, which allows cloned DNA to be transcribed and translated directly in the oocyte. Since proteins translated in oocytes are post-translationally modified according to conserved eukaryotic signals, these cells offer a convenient system for performing structural and functional analyses of cloned genes. pOEV can be used for direct analysis of proteins encoded by cloned cDNAs without preparing mRNA in vitro, simplifying existing protocols for translating proteins in oocytes with a very high translational yield. Transcription of the vector in oocytes is driven by the promoter for the TFIIIA gene, which can generate 1-2 ng (per oocyte within 2 days) of stable mRNA template for translation. The vector also contains SP6 and T7 promoters for in vitro transcription to make mRNA and hybridization probes. DNA clones encoding chloramphenicol acetyltransferase (CAT) were injected into oocyte germinal vesicles and CAT protein accumulated in the cell over a 2- to 4-day period. We found that the concentration of DNA injected affected protein yields; surprisingly relatively low concentrations in the range 25-50 pg DNA per oocyte gave maximum yields of CAT protein. When as little as 5 pg of pOEV DNA is injected we typically expressed 40 fmol of CAT protein per oocyte, after 4-day incubations. In addition, we have shown that this system is amenable to the expression of nuclear and membrane proteins.
Pfaff
S L
SL
Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232.
Tamkun
M M
MM
Taylor
W L
WL
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic; D014982:Xenopus laevis / Q000235:genetics*
D011506:Proteins; D013462:Sulfur Radioisotopes; D012313:RNA; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase