Conditional Transgenic Surface Marker for Cell Sorting
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Solution Overview
Problem
Current methods for isolating specific cell types from a population are limited, as they often require surface markers that are not always available or are not suitable for all cell types, and existing transgenic surface markers can trigger immune responses and are not resistant to proteolytic digestion, making them unsuitable for certain applications.
Innovation Solution
A conditional transgenic surface marker gene is introduced, comprising a promoter, a STOP DNA sequence that prevents transcription, and a surface marker sequence that is conditionally removable, allowing the marker to be expressed on the cell surface without membrane rupture. The marker is designed to be resistant to proteolytic digestion and immune responses, enabling sorting through magnetic or flow cytometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional surface markers are used for cell sorting, then cells can be separated using flow cytometry or magnetic sorting, but the markers may trigger immune responses and are not resistant to proteolytic digestion
Solution Approach 1:
The patent modifies the chemical and structural parameters of the surface marker protein to achieve proteolytic resistance and immune evasion. The marker protein is designed with specific amino acid sequences and structural features that prevent proteolytic degradation and reduce immunogenicity, while maintaining its functionality for cell sorting applications.
Solution Approach 2:
The invention creates a composite marker system combining multiple functional elements: the marker protein itself, associated structural domains, and regulatory elements. This composite design allows the marker to simultaneously achieve proteolytic resistance, immune response evasion, and effective cell sorting capability.
2Adaptability or versatility
If intracellular markers are used to characterize cell types, then more cell types become accessible for sorting, but the cell membrane must be ruptured or destroyed to access these markers
Solution Approach 1:
The patent employs an intermediary mechanism where a conditional transgenic surface marker acts as a bridge between intracellular markers and the cell surface. This surface marker is conditionally expressed and can be removed without disrupting cell membrane integrity, allowing access to intracellular markers while maintaining cell viability.
Solution Approach 2:
The invention segments the marker system into separate functional components: the intracellular marker, the conditional transgenic surface marker, and the removal mechanism. This segmentation allows the surface marker to be removed selectively without affecting the cell membrane or other cellular structures.
3Ease of operation
If transgenic surface markers are introduced to enable sorting of living cells, then cell sorting becomes possible, but the markers are not resistant to proteolytic digestion and can trigger immune responses
Solution Approach 1:
The patent fundamentally changes the biochemical parameters of the transgenic surface marker to achieve proteolytic resistance and immune response evasion while maintaining its sorting capability. The marker protein is engineered with specific structural and sequence characteristics that provide stability in physiological conditions.
4Ease of operation
If conditional transgenic surface markers are expressed on the cell surface, then cells can be sorted without membrane rupture, but the markers must be conditionally removable to avoid immune rejection
Solution Approach 1:
The invention introduces dynamic control over marker expression through conditional transgenic elements. The surface marker is conditionally expressed and can be removed at specific times or under specific conditions, allowing temporary presence for sorting purposes while avoiding permanent expression that would trigger immune rejection.
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 allows for the isolation of cells characterized by intracellular markers or spatio-temporal properties, avoiding immune rejection and ensuring the marker's stability during sorting and subsequent grafting, thus expanding the range of accessible cell types and applications.
Implementation Method 1
a recombinantly expressed green fluorescent protein (GFP) could be located to the cytosolic face of the plasma membrane of mouse embryonic stem cells
Implementation Method 2
magnetic cell sorting (e.g. using magnetic bead-conjugated antibodies, e.g. MACS, Miltenyi Biotec)
Data Source
AI summary
The invention relates to a cell containing a gene encoding a conditional transgenic surface marker that is detectable upon expression on the surface of the cell, wherein the gene encoding a conditional transgenic surface marker comprises: (i) a promoter, operably linked to (ii) a first transcription sequence, and (iii) a second transcription sequence encoding the surface marker, whereby the first transcription sequence prevents the transcription of the second transcription sequence, whereby the first transcription sequence is conditionally removable such that the second transcription sequence is transcribable, and whereby the surface marker renders the cell sortable through the detection of the conditional transgenic surface marker. Furthermore, the invention relates to a construct for generating such a cell, and to a method for separating such a cell from a population of cells.