Mammalian Expression Vector SV40 Enhancer Hbb Intron
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Solution Overview
Problem
Current mammalian expression vectors require multiple selection steps and multiple copies of gene integration to achieve high expression levels, making cell line development time-consuming and inefficient, especially for predicting stable expression of secreted proteins.
Innovation Solution
The use of mammalian expression vectors combining the simian virus 40 enhancer and early promoter region with the human Hbb intronII, allowing for single-step selection with G418 or MTX, which enables high and reproducible expression of genes of interest without the need for multiple gene copies or selection steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple selection steps and multiple copies of gene integration are used to achieve high expression levels, then expression level is improved, but cell line development time and workload increase
Solution Approach 1:
The patent changes the parameters of the expression vector by incorporating specific regulatory elements (SV40 enhancer and early promoter region, Hbb intronII) to achieve high expression levels through qualitative improvement rather than quantitative multiplication. This allows single-copy integration with single-step selection, resolving the contradiction between expression level and development time.
2Quantity of substance
If multiple selection steps are performed to generate high producing cell pool, then expression level is improved, but productivity decreases
Solution Approach 1:
The patent modifies the vector parameters by incorporating specific regulatory elements that enable high expression from single-copy integration. This allows progression from multiple selection steps to single-step selection, thereby improving productivity while maintaining high expression levels.
3Ease of operation
If transient expression levels are determined using intracellular reporter proteins, then measurement is simplified, but prediction accuracy of stable expression levels decreases
Solution Approach 1:
The patent changes the regulatory element parameters to create a system where transient and stable expression are better correlated. The specific combination of SV40 enhancer/promoter and Hbb intronII regulates both intracellular and secreted protein expression, improving the predictive value of transient expression assays for stable production levels.
Data Source
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AI summary
The present invention describes new mammalian expression vectors comprising a novel combination of regulatory elements and one or more selection marker gene(s). The vector allows for incorporation of at least one, preferably two or more genes of interest, its/their subsequent expression, and for selection of transfected cells using, e.g., G418 and/or MTX. The pDGP?GOI vector as an example for a mammalian expression vector according to the present invention exhibits a 9555 bp sequence, one strand of which is represented by SEQ ID NO:2.