Fluorescent Lentiviral Vector via Bioorthogonal Click Chemistry
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Solution Overview
Problem
Current methods cannot continuously and dynamically observe the real-time infection behavior of lentiviruses in host cells, making it difficult to research the biological invasion process.
Innovation Solution
A fluorescently labeled lentiviral vector is prepared by azidating the cell membrane of 293T cells with azido sugars and co-transfecting with a plasmid packaging system, followed by mixing with azide dibenzocyclooctyne-cyanine 5 (DBCO-Cy5) to create a fluorescently labeled lentiviral vector, allowing for dynamic monitoring of infection behavior.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent protein labels are used to detect lentiviral transfection success, then detection capability is improved, but continuous and dynamic observation of real-time infection behavior is not possible
Solution Approach 1:
The patent incorporates azido groups into the lentivirus envelope structure during virus production, before the infection process begins. This preliminary incorporation of the labeling group enables subsequent real-time fluorescent tracking without requiring genetic modification of the target cell or virus at the time of infection, thus achieving both detection precision and real-time observation capability
2Ease of manufacture
If genetic modification is used to enable fluorescent labeling, then labeling capability is improved, but complexity of the method increases
Solution Approach 1:
The patent extracts the fluorescent labeling function from the genetic material and places it on the viral envelope instead. By using chemical labeling of the envelope protein with azido groups rather than genetic encoding of fluorescent proteins, the method simplifies the overall process while maintaining labeling capability, avoiding complex genetic modification procedures
Solution Approach 2:
The patent uses azido groups as an intermediary chemical handle on the viral envelope that can be subsequently labeled with fluorescent dyes. This intermediary approach allows flexible labeling without direct genetic modification, simplifying the methodology while enabling versatile fluorescent tagging for real-time observation
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 enables simple and cost-effective fluorescent labeling of lentiviral vectors without genetic modification, allowing for real-time observation of lentivirus infection dynamics and providing insights into the biological invasion mechanism.
Implementation Method 1
subjecting a 293T cell and an azido sugar to co-incubation, such that a cell membrane system is azidated through sugar metabolism
Implementation Method 2
mixing the N3-LVs with a sufficient amount of an azide dibenzocyclooctyne-cyanine 5 (DBCO-Cy5) to allow co-incubation
Data Source
AI summary
The present disclosure belongs to lentiviral labeling technology, and in particular relates to a fluorescently labeled lentiviral vector, and a preparation method and use thereof. The preparation method of a fluorescently labeled lentiviral vector includes: preparation of N3-LVs: subjecting a 293T cell and an azido sugar to co-incubation, such that a cell membrane system is azidated through sugar metabolism to obtain an N3-293T cell capable of expressing an azido group on an envelope surface, and subjecting the N3-293T cell and a plasmid packaging system to co-transfection to form a lentiviral vector N3-LVs; and preparation of Cy5-LVs: mixing the N3-LVs with a sufficient amount of an azide dibenzocyclooctyne-cyanine 5 (DBCO-Cy5) to allow co-incubation, removing excess DBCO-Cy5, and collecting a virus concentrate to obtain a fluorescently labeled lentiviral vector Cy5-LVs. In the present disclosure, a simple and easy lentiviral fluorescent labeling technology is provided by covalently modifying fluorescein on an envelope surface of a lentivirus based on bioorthogonal click chemistry with a low cost.