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Inducible Gene Expression Composition for Using Eukaryotic Pol-2 Promoter-Driven Transcription in Prokaryotes and the Applications Thereof

a technology of eukaryotic pol2 and promoter, applied in the direction of drug composition, dermatological disorder, organic chemistry, etc., can solve the problems of inability or even impossible to produce eukaryotic rnas and peptides/proteins using eukaryotic rna promoters in prokaryotic cells, and the prior art was still limited

Inactive Publication Date: 2013-08-15
MELLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a way to change prokaryotic cells so they can use eukaryotic promoters to produce desired RNA and related proteins. This is done by adding some chemical agents that help the prokaryotic cells quickly adapt to use the eukaryotic promoters.

Problems solved by technology

These differences make a prokaryotic cell difficult or even impossible to produce eukaryotic RNAs and peptides / proteins using eukaryotic RNA promoters.
Nevertheless, these bacterial and bacteriophage promoters, such as Tac, Lac, T3, T7, and SP6 RNA promoters, are not pol-2 promoters and their transcription is an error-prone process that tends to cause mutations.
Due to lack of possible compatibility between eukaryotic and prokaryotic transcription systems, these prior arts were still limited by the use of prokaryotic RNA promoters in prokaryotes.
However, these Tet-On promoters are not eukaryotic pol-2 promoters and have never been tested in prokaryotes.

Method used

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  • Inducible Gene Expression Composition for Using Eukaryotic Pol-2 Promoter-Driven Transcription in Prokaryotes and the Applications Thereof
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  • Inducible Gene Expression Composition for Using Eukaryotic Pol-2 Promoter-Driven Transcription in Prokaryotes and the Applications Thereof

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1. Bacterial Cell Culture and Chemical Treatments

[0115]Competent E. coli DH5alpha cells are acquired from the z-competent E. coli transformation kit (Zymo Research, Irvine, Calif.) and transformed by mixing with 5 μg of a desired plasmid vector such as pLVX-Grn-miR302+367 or pLenti-EF1alpha-RGFP-miR302. Non-transformed bacterial cells are normally grown in Luria-Bertani (LB) broth supplemented with 10 mM MgSO4 and 0.2 mM glucose at 37° C. with frequent agitation at 170 rpm, whereas the transformed bacterial cells are cultivated in the above LB broth further supplemented with additional 100 μg / ml ampicillin. For chemical induction, 0.5 to 2 ml of MOPS, glycerin, and ethanol, respectively or in combination, is added into 1 litter LB broth supplemented with 10 mM MgSO4 and 0.2 mM glucose in the presence of 100 μg / ml ampicillin. For negative control, the transformed bacterial cells are cultivated in the above ampicillin-supplemented LB broth but without adding any chemical inducer.

2. Hu...

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Abstract

Eukaryotic protein-coding messenger RNAs and non-coding microRNAs are naturally transcribed by type II RNA polymerases (pol-2) but not prokaryotic RNA polymerases. As a result, current eukaryotic RNA and protein production is performed either using eukaryotic pol-2 promoters in hybridomas or mammalian cells or using prokaryotic promoters in bacterial cells. However, because prokaryotic RNA transcription tends to be error-prone, frequent mutation is a big problem. Also, growing hybridomas or mammalian cells is relatively laborious and costly. To overcome these problems, the present invention provides a novel inducible composition and method for producing eukaryotic RNAs and / or their related peptides / proteins directly using eukaryotic pol-2 promoter-driven gene expression in fast growing bacteria, without the need of changing to prokaryotic promoters or growing hybridomas / mammalian cells. The RNAs and peptides / proteins so obtained can be used to develop drugs, cure diseases, treat tumors / cancers, produce pluripotent stem (iPS) cells, enhance wound healing, and make foods.

Description

[0001]This application claims priority to the U.S. Provisional Application Ser. No. 61 / 522,843 filed on Aug. 12, 2011 which was entitled “An Inducible Gene Expression Composition for Using Eukaryotic Pol-2 Promoter-Driven Transcription in Prokaryotes and The Applications Thereof”.BACKGROUND OF THE INVENTION[0002]1. Field of Invention[0003]This invention generally relates to a composition and its application for producing ribonucleic acids (RNAs, i.e. messenger RNAs and microRNAs) and / or proteins / peptides (i.e. antibodies and enzymes) using eukaryotic RNA promoter-driven transcription in prokaryotes. Particularly, the present invention teaches a composition and its use for generating RNAs and / or proteins / peptides using eukaryotic type II RNA polymerase (pol-2) promoter-driven transcription in bacterial cells. Alternatively, the present invention is also an inducible gene expression composition using chemical agents rather than antibiotics to stimulate eukaryotic RNA promoter-driven t...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/70
CPCC12N15/70C12N1/38A61P17/02A61P35/00C12N15/635
Inventor LIN, SHI-LUNGCHANG, DONALD C.
Owner MELLO BIOTECH
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