Genetic Constructs for Tracking and Ablating Quiescent Cells

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Solution Overview

Problem

Current methods for analyzing and monitoring cellular cycles, particularly for quiescent cells, lack real-time observation capabilities and do not allow for effective tracking and ablation of cells based on their proliferation/quiescence state.

Innovation Solution

The development of genetic constructs such as CreERT2-p27K− and DTA-p27K−, which are specifically active in quiescent cells, enabling real-time tracking and ablation of quiescent cellular populations, including stem cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If conventional methods (e.g., BrdU label and anti-BrdU antibody) are used to analyze cellular cycle phases, then cellular cycle analysis can be performed, but real-time observation capability is lost

Engineering Contradiction:

Improvecellular cycle phase analysis capabilityVSAvoidreal-time observation capability

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent replaces conventional mechanical/chemical labeling methods (BrdU incorporation and antibody staining) with a genetic construct-based system. The FusionProtein expresses a fluorescent marker under the control of a phase-specific promoter, allowing direct optical observation of cellular cycle phases in real-time without mechanical intervention or complex staining procedures.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the parameter of observation from indirect biochemical detection (antibody-antigen binding) to direct optical detection (fluorescence emission). By using a fluorescent protein marker driven by phase-specific gene expression, the system transforms the measurement parameter from chemical composition to optical signal, enabling real-time monitoring.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If no specific tracking method is used for quiescent cells, then general cellular analysis can be performed, but tracking and ablation of quiescent cells based on proliferation/quiescence state cannot be achieved

Engineering Contradiction:

Improvegeneral cellular analysis capabilityVSAvoidquiescent cell tracking precision

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent applies local quality by designing the genetic construct to be specifically active only in quiescent cells (G0 phase). The FusionProtein expression is controlled by a promoter that is selectively activated in quiescent cells, providing localized functional identification without affecting other cellular states. This allows precise tracking of quiescent cells while maintaining general applicability to different cell types.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20250043305A1Genetic construct for tracking and/or ablating quiescent cells

Publication Date: 2025.02.06 UNIVERSITA DEGL STUDI DI TRENTO

AI summary

A genetic construct is described having a nucleotide sequence A (Cre+ERT2) with a nucleotide sequence SEQ ID NO. 1 coding for an enzyme recombinase Cre and a sequence SEQ ID NO. 2 coding for a mutated receptor for estrogen ERT2; or a nucleotide sequence A′ with a nucleotide sequence SEQ ID NO. 3 coding for the fragment A of the diphtheria toxin (DTA); and a nucleotide sequence B with a nucleotide sequence SEQ ID NO. 4 coding for the inhibitor of a mutant cyclin dependent kinase (CDK) p27K−.