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Coupled cytoplasmic transcription-and-translation—a method of choice for heterologous gene expression in Xenopus oocytes

Paper ID Volume ID Publish Year Pages File Format Full-Text
25573 43582 2006 11 PDF Available
Title
Coupled cytoplasmic transcription-and-translation—a method of choice for heterologous gene expression in Xenopus oocytes
Abstract

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.

Keywords
Expression technique; Xenopus oocytes; T7 RNA polymerase; Luciferase
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 122, Issue 1, 9 March 2006, Pages 5–15
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us