Fluorogenic Oligonucleotide Probes for Cell Engineering
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Solution Overview
Problem
Current cell engineering techniques, including CRISPR, face challenges in efficiently detecting and isolating cells expressing specific genetic sequences, particularly due to limitations in detecting and isolating cells expressing desired genetic sequences without cytotoxicity and with high specificity.
Innovation Solution
The development of expression vectors comprising a promoter, multiple cloning site, spacer, and detection tag, along with fluorogenic oligonucleotide signaling probes, allows for the detection and isolation of cells expressing specific sequences of interest using fluorescence-resonance energy transfer (FRET) and flow cytometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorogenic oligonucleotide signaling probes are used to detect cells expressing specific genetic sequences, then measurement precision is improved, but device complexity increases due to the need for specialized detection systems
Solution Approach 1:
The patent employs fluorogenic oligonucleotide signaling probes as intermediary molecules that specifically bind to target genetic sequences. These probes contain fluorophores and quenchers that act as mediators to convert molecular recognition events into detectable fluorescent signals, enabling high-specificity detection without complex instrumentation
Solution Approach 2:
The invention replaces complex mechanical or biochemical detection systems with a fluorescent optical detection system. By using fluorogenic probes that emit light upon binding to target sequences, the system substitutes elaborate detection mechanisms with simpler fluorescence-activated cell sorting (FACS) technology that is widely available and easier to operate
2Productivity
If traditional cell engineering methods are used, then ease of manufacture is maintained, but productivity decreases due to inefficient detection and isolation of expressing cells
Solution Approach 1:
The patent incorporates detection tags into the expression vectors before cell transfection. These pre-integrated tags enable direct detection of cells expressing the desired genetic sequences without requiring subsequent complex screening or selection processes, thereby improving productivity while maintaining ease of manufacture
Solution Approach 2:
The invention uses fluorogenic signaling probes that change their fluorescent properties (from quenched to fluorescent) when they bind to target sequences in expressing cells. This color/fluorescence change provides a visual and quantifiable indicator that enables rapid identification and isolation of positive cells through flow cytometry, significantly enhancing cell isolation efficiency
3Manufacturing precision
If detection tags are incorporated into expression vectors, then manufacturing precision is improved, but device complexity increases due to additional vector components
Solution Approach 1:
The patent merges the detection function with the expression vector by incorporating detection tags directly into the vector structure. This integration allows the same vector that delivers the genetic sequence to also provide the detection capability, eliminating the need for separate detection systems and reducing overall complexity despite adding vector components
Solution Approach 2:
The detection tags incorporated into the expression vectors serve multiple functions: they enable detection of sequence expression, facilitate cell isolation, and can be used for quantitative analysis. This multi-functionality improves manufacturing precision by providing built-in detection capabilities without requiring additional separate systems
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 the efficient isolation and characterization of cells expressing specific genes, such as ion channels and sensory receptors, with high specificity and stability, facilitating drug discovery and biotechnology applications.
Implementation Method 1
each terminus of the signaling probe is covalently linked to a fluorophore or quencher paired to absorb its emission, for instance via fluorescence-resonance energy transfer (FRET)
Data Source
AI summary
The present disclosure relates to novel expression vectors, host cells and methods useful to produce cells comprising one or more sequences of interest.