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

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.

What is claimed is: 
     
         1 . A composition useful for regulating eukaryotic promoter-driven gene expression in prokaryotes, comprising:
 a chemical agent, containing a structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or a mixture thereof.   
     
     
         2 . The composition as defined in  claim 1 , wherein said chemical agent is a transcription inducer for gene expression. 
     
     
         3 . The composition as defined in  claim 1 , wherein said composition further comprises a bacterial culturing medium. 
     
     
         4 . The composition as defined in  claim 3 , wherein said chemical agent has a v/v concentration of 0.01% to 1% in said bacterial culturing medium. 
     
     
         5 . The composition as defined in  claim 3 , wherein said bacterial culturing medium is Luria-Bertani (LB) broth. 
     
     
         6 . The composition as defined in  claim 1 , wherein said gene encodes at least a protein or non-coding RNA, or both, which is useful for pharmaceutical or therapeutic application. 
     
     
         7 . The composition as defined in  claim 6 , wherein said non-coding RNA is useful for pharmaceutical or therapeutic application. 
     
     
         8 . The composition as defined in  claim 6 , wherein said non-coding RNA is a miR-302 homologue. 
     
     
         9 . The composition as defined in  claim 6 , wherein said protein or peptide is useful for pharmaceutical or therapeutic application. 
     
     
         10 . The composition as defined in  claim 6 , wherein said pharmaceutical or therapeutic application is selected from generating pluripotent stem cells, stem cell research and therapy, cancer therapy and disease treatment, wound healing treatment, or generating high-yield food and drug supplies. 
     
     
         11 . A composition for regulating eukaryotic promoter-driven gene expression in prokaryotes, comprising:
 (a) at least a chemical agent capable of inducing or enhancing eukaryotic promoter-driven gene expression, wherein said chemical agent containing a structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or a mixture thereof; and   (b) a plurality of prokaryotic cells, said plurality of prokaryotic cells containing at least a gene mediated by a eukaryotic promoter-driven expression mechanism;   wherein (a) and (b) are mixed together under a condition to induce the eukaryotic promoter-driven gene expression of said gene in said prokaryotic cells.   
     
     
         12 . The composition as defined in  claim 11 , wherein each one of said prokaryotic cells has at least an inducible gene expression composition. 
     
     
         13 . The composition as defined in  claim 12 , wherein said inducible gene expression composition is a vector containing a eukaryotic promoter. 
     
     
         14 . The composition as defined in  claim 11 , wherein said gene encodes at least a non-coding RNA or protein-coding RNA, or both. 
     
     
         15 . The composition as defined in  claim 14 , wherein said non-coding RNA contains at least a sequence containing 30% to 100% homology to a microRNA. 
     
     
         16 . The composition as defined in  claim 14 , wherein said non-coding RNA is a small hairpin RNA. 
     
     
         17 . The composition as defined in  claim 14 , wherein said protein-coding RNA contains at least a sequence containing 30% to 100% homology to a microRNA. 
     
     
         18 . The composition as defined in  claim 14 , wherein said protein-coding RNA contains 30% to 100% homology to a cDNA sequence of a eukaryotic gene. 
     
     
         19 . The composition as defined in  claim 14 , wherein said non-coding RNA or protein-coding RNA is isolatable from said prokaryotes. 
     
     
         20 . The composition as defined in  claim 11 , wherein said gene encodes at least a protein or peptide, or both. 
     
     
         21 . The composition as defined in  claim 20 , wherein said protein contains at least a peptide sequence. 
     
     
         22 . The composition as defined in  claim 20 , wherein said protein or peptide is isolatable from said prokaryotes. 
     
     
         23 . The composition as defined in  claim 20 , wherein said protein is an enzyme or an antibody. 
     
     
         24 . The composition as defined in  claim 20 , wherein said protein contains a peptide sequence similar to insulin. 
     
     
         25 . The composition as defined in  claim 20 , wherein said protein contains a peptide sequence similar to a growth factor. 
     
     
         26 . The composition as defined in  claim 11 , wherein said prokaryotic cells are bacterial cells. 
     
     
         27 . The composition as defined in  claim 11 , wherein said prokaryotic cells are  Escherichia coli  ( E. coli ). 
     
     
         28 . The composition as defined in  claim 11 , wherein said chemical agent is a transcription inducer for gene expression. 
     
     
         29 . The composition as defined in  claim 11 , wherein said chemical agent contains a structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerin, or a mixture thereof. 
     
     
         30 . The composition as defined in  claim 11 , wherein said eukaryotic promoter is a type II RNA polymerase (pol-2) equivalent or a pol-2 compatible viral promoter. 
     
     
         31 . The composition as defined in  claim 30 , wherein said pol-2 compatible viral promoter is a cytomegaloviral (CMV) promoter or a retroviral long terminal repeat (LTR) promoter. 
     
     
         32 . The composition as defined in  claim 11 , wherein said eukaryotic promoter-driven expression is a cellular mechanism including RNA transcription and protein translation. 
     
     
         33 . The composition as defined in  claim 11 , wherein said condition is a bacterial culturing condition in Luria-Bertani (LB) broth at 37° C. 
     
     
         34 . The composition as defined in  claim 11 , wherein said chemical agent in said condition has a v/v concentration of 0.01% to 1%. 
     
     
         35 . The composition as defined in  claim 14 , wherein said non-coding RNA is useful for pharmaceutical or therapeutic application. 
     
     
         36 . The composition as defined in  claim 14 , wherein said protein-coding RNA is useful for pharmaceutical or therapeutic application. 
     
     
         37 . The composition as defined in  claim 14 , wherein said non-coding RNA is a miR-302 homologue. 
     
     
         38 . The composition as defined in  claim 15 , wherein said microRNA is a miR-302 homologue. 
     
     
         39 . The composition as defined in  claim 17 , wherein said microRNA is a miR-302 homologue. 
     
     
         40 . The composition as defined in  claim 20 , wherein said protein or peptide is useful for pharmaceutical or therapeutic application. 
     
     
         41 . The composition as defined in  claim 35 , wherein said pharmaceutical or therapeutic application is selected from generating pluripotent stem cells, stem cell research and therapy, cancer therapy and disease treatment, wound healing treatment, or generating high-yield food and drug supplies. 
     
     
         42 . The composition as defined in  claim 36 , wherein said pharmaceutical or therapeutic application is selected from generating pluripotent stem cells, stem cell research and therapy, cancer therapy and disease treatment, wound healing treatment, or generating high-yield food and drug supplies. 
     
     
         43 . The composition as defined in  claim 40 , wherein said pharmaceutical or therapeutic application is selected from generating pluripotent stem cells, stem cell research and therapy, cancer therapy and disease treatment, wound healing treatment, or generating high-yield food and drug supplies.