Fluorescent Protein Markers for Gene Expression Correlation

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Solution Overview

Problem

The transformation of living cells with genetic constructs often results in heterogeneous populations due to varying interactions and integration locations within the chromosome, leading to differences in gene expression levels, making it challenging to identify and separate cells with desired expression levels of specific genes.

Innovation Solution

Incorporating internal ribosome entry sites (IRES) and cis-acting hydrolase elements (CHYSEL) within genetic constructs to correlate the expression levels of proteins, allowing for the use of selectable proteins to identify and select cells based on their expression levels, and employing linker sequences to form fusion proteins for enhanced expression assessment.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If genetic constructs are transformed into living cells, then gene expression occurs, but heterogeneous populations result due to varying integration locations and interactions

Engineering Contradiction:

Improvegene expression consistencyVSAvoidcell population homogeneity

Core Design Contradiction:

ReliabilityVSEase of operation

Solution Approach 1:

The patent employs fluorescent proteins that emit different colors or fluorescence intensities to visually indicate the expression level of genes in transformed cells. This allows researchers to quickly identify and sort cells with desired expression levels without complex analysis, directly addressing the heterogeneity problem by providing an intuitive visual marker for expression variability.

Inventive Principle:

Principle #32Color changes

2Measurement precision

If expression levels of multiple genes are measured individually, then accurate quantification is achieved, but extensive analysis time and resources are required

Engineering Contradiction:

Improvegene expression quantification accuracyVSAvoidanalysis time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent combines multiple gene expression measurements into a single visual assessment by using fluorescent proteins whose expression correlates with the expression levels of multiple genes of interest. This merging approach allows researchers to evaluate multiple genes simultaneously through a single fluorescence readout, dramatically reducing analysis time while maintaining measurement precision.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The fluorescent protein markers serve multiple functions: they indicate successful transformation, report gene expression levels, and enable cell sorting. This multi-functionality eliminates the need for separate analysis procedures for each gene, reducing both time and resource requirements while maintaining accurate expression quantification.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Measurement precision

If traditional methods like Western blot are used to measure protein expression, then detailed expression analysis is obtained, but the process becomes laborious and time-consuming

Engineering Contradiction:

Improveprotein expression measurement accuracyVSAvoidcell screening efficiency

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent replaces laborious mechanical procedures like Western blotting with a fluorescence-based optical system. The fluorescent proteins provide direct visual indication of expression levels that can be assessed rapidly using microscopy or flow cytometry, maintaining measurement accuracy while dramatically improving screening efficiency and productivity.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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 approach enables efficient identification and selection of cells with desired gene expression levels by correlating the expression of one protein with another, simplifying the process and reducing the need for extensive analysis, such as Western blots, thereby streamlining the identification of cells expressing genes at desired levels.

Implementation Method 1

The fluorescent and colored proteins absorb light energy to achieve their characteristic fluorescence or color properties for detection

Methodology Applied

Scientific EffectAbsorption (EM radiation): Absorption (EM radiation)

Implementation Method 2

The fluorescent and colored proteins emit light at specific wavelengths to enable detection and visualization of gene expression levels

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS8975042B2Fluorescent and colored proteins and methods for using them

Publication Date: 2015.03.10 DNA TWOPOINTO INC

AI summary

The field of this invention relates to methods for combining genetic elements such that the activity of one of the elements provides a means for identifying, enriching, selecting for, or enhancing the activity of a second element. The invention also includes specific elements and combinations of elements.