Targeted Integrated Cell Nucleic Acid Fragment Integration
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Traditional methods for developing host cells for recombinant protein production involve random integration of target genes, leading to high costs, unstable expression, unpredictable site effects, and resource wastage, with the process taking over six months and requiring extensive screening.
Innovation Solution
A nucleic acid fragment with defined integration sites, integrated using recombinase-mediated recombination, allows for precise targeting of exogenous nucleic acid fragments in CHO cells, simplifying the process and ensuring high stability and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If random integration method is used to construct cell lines, then cell line generation is achieved, but construction time exceeds 6 months and costs are extremely high
Solution Approach 1:
The patent pre-prepares a nucleic acid fragment with a specific sequence (SEQ ID No. 1) that contains defined integration sites before cell line construction. This preliminary preparation of the integration template enables direct targeted integration, eliminating the need for random integration followed by extensive screening, thereby reducing construction time from over 6 months to a significantly shorter period while improving integration precision.
Solution Approach 2:
The patent introduces a recombinase as an intermediary enzyme that mediates the specific recognition and integration of the exogenous nucleic acid fragment at the defined sites within the nucleic acid fragment. This recombinase-mediated recombination system acts as a bridge between the exogenous gene and the host genome, enabling precise targeted integration without random insertion, thus resolving the contradiction between integration precision and construction time.
2Manufacturing precision
If random integration method is used, then cell line generation is achieved, but extensive screening and repetitive work are required resulting in high costs
Solution Approach 1:
The patent pre-defines the integration sites within the nucleic acid fragment sequence (SEQ ID No. 1) before cell line construction. By having the integration targets predetermined in the nucleic acid fragment design, the method eliminates the need for post-integration screening to identify suitable clones, significantly reducing process complexity and costs while improving integration site control.
Solution Approach 2:
The recombinase serves as an intermediary that recognizes specific sequences and mediates precise integration at predetermined sites. This enzymatic mediator ensures that integration occurs only at the defined locations within the nucleic acid fragment, eliminating the need for extensive screening and repetitive process development for each cell strain, thereby simplifying the manufacturing process.
3Reliability
If random integration is used, then transgenic cells are obtained, but expression stability is difficult to predict and site effects cause significant decrease in target gene expression
Solution Approach 1:
The patent pre-identifies and pre-prepares a nucleic acid fragment with specific sequences (SEQ ID No. 1) that contain defined integration sites known to support stable expression. By selecting integration sites in advance based on their expression properties, the method ensures reliable and stable target gene expression from the beginning, avoiding the unpredictability of random integration while improving integration site precision.
4Productivity
If random integration method is used, then cell line generation is achieved, but resources are wasted due to tedious screening and process optimization for each strain
Solution Approach 1:
The patent pre-prepares the nucleic acid fragment with defined integration sites and pre-validates the integration target within the host cell genome. This preliminary preparation eliminates the need for tedious screening of multiple clones and repetitive process optimization for each cell strain, significantly improving cell line generation efficiency while reducing resource consumption in terms of time, materials, and labor.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method reduces construction time, lowers costs, and enhances controllability, resulting in stable and high-yielding expression of target genes, eliminating the need for rigorous culture condition optimization.
Implementation Method 1
integrated using recombinase-mediated recombination, allows for precise targeting of exogenous nucleic acid fragments in CHO cells
Data Source
Figure 1
Figure 2~3
Figure 4
AI summary
The present invention relates to the field of biotechnology, and in particular to a nucleic acid and a recombinant vector and targeted integrated cell comprising same, and a method for producing a target gene expression product and a use thereof. The nucleic acid comprises a nucleic acid fragment shown in SEQ ID No.1, and the nucleic acid fragment is used for integrating an exogenous nucleic acid fragment. Upon a large number of cell strain screenings in the early stage, it is found that for the nucleic acid fragment as shown in SEQID No.1 or a homologous fragment that maintains at least 90% identity with the nucleic acid fragment, an exogenous nucleotide sequence is integrated into the nucleic acid fragment or the homologous fragment thereof, and the correspondingly obtained targeted integrated cell has the characteristics of high identity, high stability, and high yield.