Gene Expression Cassette with Downstream Enhancer for Consistent Protein Production
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Solution Overview
Problem
Conventional gene expression promoters and enhancers, such as CMV and CAG promoters, often result in low or variable gene expression levels depending on cell type or gene type, posing challenges in biotechnology applications, particularly in medical diagnostics and treatments.
Innovation Solution
A gene expression cassette is developed with a DNA construct containing a gene to be expressed and a poly A addition sequence downstream of a 1st promoter, further enhanced by the inclusion of an enhancer or 2nd promoter, RU5′, UAS, or SV40-ori, which significantly increases gene expression efficiency across various cell types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional promoters (CMV, CAG) are used to increase gene expression, then gene expression efficiency is improved, but expression levels become variable and low depending on cell type or gene type
Solution Approach 1:
The patent combines multiple regulatory elements (promoter, enhancer, poly A addition sequence) into a single expression cassette system. This merging of components creates a unified system that works synergistically to achieve high and consistent gene expression across different cell types, resolving the contradiction between expression efficiency and reliability.
Solution Approach 2:
The expression cassette is designed with universal applicability across different cell types and gene types. The combination of promoter, enhancer, and poly A sequence creates a multi-functional system that can drive consistent gene expression regardless of the specific cell type or gene being expressed, addressing the variability problem of conventional promoters.
2Productivity
If conventional promoters are used, then gene expression can be achieved, but protein production levels remain extremely low in many cases
Solution Approach 1:
The patent incorporates a poly A addition sequence downstream of the gene in the expression cassette. This preliminary structural arrangement ensures proper mRNA processing and stability before translation occurs, thereby maximizing protein production levels from the outset rather than relying on post-transcriptional optimization.
Solution Approach 2:
The patent changes the structural parameters of the expression system by adding specific sequences (poly A addition sequence downstream, enhancer elements) to the conventional promoter structure. These parameter changes in the DNA construct directly increase the quantity of functional mRNA and subsequently the amount of expressed protein.
Data Source
AI summary
An object of the present invention is to provide a method for increasing the expression of foreign genes, in particular, using a promoter, an enhancer, and the like, and an expression cassette containing a promoter, an enhancer, and the like, by which gene expression can be increased. The purpose is achieved with the use of the gene expression cassette comprising a DNA construct containing a gene to be expressed and a poly A addition sequence that are located downstream of a 1st promoter, and further comprising an enhancer or a 2nd promoter ligated downstream of the DNA construct.