Reporter Construct for Enriching Nuclease-Modified Cells
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Solution Overview
Problem
Current methods for using synthetic nucleases in gene therapy and research face challenges in distinguishing and isolating gene-modified cells from wild-type cells, as existing systems lack efficient methods for enriching or selecting cells with specific endogenous nucleotide sequence modifications.
Innovation Solution
A reporter construct system is developed, comprising a target sequence recognized by a specific nuclease and reporter genes, allowing for the identification and enrichment of cells by expressing fluorescent proteins upon nuclease activity, using flow cytometry or magnetic-activated cell sorting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If synthetic nucleases are used to induce targeted gene disruption, then gene modification is achieved, but the ability to distinguish and isolate gene-modified cells from wild-type cells deteriorates
Solution Approach 1:
A reporter construct is introduced as an intermediary element that links nuclease activity to detectable signals. The construct contains a reporter gene whose expression is controlled by a promoter that is inactivated by the nuclease when it binds to the target sequence. This intermediary system allows indirect detection of nuclease activity and gene modification through reporter gene expression levels.
Solution Approach 2:
The reporter construct utilizes fluorescent proteins (such as GFP, YFP, or RFP) as the reporter gene, which produce detectable color/fluorescence signals. When the nuclease binds to the target sequence in the reporter construct, the promoter is inactivated, leading to loss of fluorescent signal. This color/fluorescence change provides a direct visual and measurable indicator of successful nuclease binding and gene modification.
2Reliability
If laborious screening of numerous clones is performed to obtain gene-disrupted cells, then gene-modified cells can be isolated, but the time required for the process increases
Solution Approach 1:
The manual, laborious process of screening numerous clones through mechanical inspection and manual sorting is replaced by an automated flow cytometry-based sorting system. The fluorescent signal from the reporter construct allows automated detection and sorting of gene-modified cells based on their fluorescence intensity, dramatically reducing the time and labor required for cell isolation.
Solution Approach 2:
The reporter construct provides self-service by automatically marking gene-modified cells with fluorescent signals that make them visible to flow cytometry. The system self-indicates which cells have been successfully modified by the nuclease, eliminating the need for complex manual screening procedures and enabling rapid, automated selection of the desired cell population.
3Ease of operation
If a reporter construct with target sequence and reporter gene is prepared and introduced into cells, then cell sorting based on reporter expression is enabled, but the complexity of the system increases
Solution Approach 1:
The reporter construct merges multiple functional elements into a single integrated plasmid: the promoter region that responds to nuclease binding, the target sequence that the nuclease recognizes and binds to, the reporter gene that produces the detectable signal, and the necessary regulatory elements. This merging of components into one construct simplifies the overall system by eliminating the need for separate plasmids and reducing the number of separate steps required.
Data Source
AI summary
The present invention relates to a reporter construct and method for identifying or enriching the cells, wherein a specific endogenous nucleotide sequence is cleaved by a specific nuclease or modified by such cleavage; a host cell comprising the reporter construct; and a system for monitoring a nuclease activity. The reporter system of the present invention is simple and non-invasive, and allows for an efficient enrichment of the gene-modified cells. Therefore, the present invention will promote the application of a nuclease in the field of gene therapy and genetic engineering as well as basic research.