Nuclear-Anchored Cell Labeling for Specific Nuclei Isolation
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Solution Overview
Problem
Current genomic assays on bulk tissue are difficult to interpret due to the heterogeneity of cell types, necessitating improved techniques for specific cell type labeling and isolation to understand cell type-specific mechanisms.
Innovation Solution
Development of nucleic acid constructs and viral delivery systems, such as SNAIL and cSNAIL, that utilize a truncated Sun1-GFP fusion protein and cell type-specific promoters to label and isolate specific cell types, enabling efficient and cost-effective isolation using affinity purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bulk tissue genomic assays are used, then the assay can be performed on entire tissue samples, but the results are difficult to interpret and disentangle for cell type-specific mechanisms
Solution Approach 1:
The patent segments the tissue sample into individual nuclei through mechanical disruption and filtration, then further segments by cell type through magnetic bead affinity purification. This segmentation allows genomic assays to be performed on specific cell types rather than bulk tissue, resolving the contradiction between performing assays on entire tissues and obtaining cell type-specific information.
Solution Approach 2:
The patent uses magnetic beads coated with antibodies against cell type-specific markers as intermediaries to selectively bind and isolate desired cell nuclei from the mixed population. This intermediary mechanism enables precise separation of cell types without requiring complex sorting procedures, thereby maintaining cell type information while enabling targeted genomic analysis.
2Productivity
If traditional nuclear isolation methods are used, then nuclei can be isolated from tissue, but the methods are complex, time-consuming, and resource-intensive
Solution Approach 1:
The patent extracts only the essential components needed for nuclear isolation: mechanical disruption to release nuclei, filtration to remove debris, and magnetic bead affinity purification to separate cell types. By taking out and eliminating unnecessary complex procedures from traditional isolation methods, the patent achieves high productivity while reducing overall complexity.
Solution Approach 2:
The patent employs nuclei themselves as the target for purification, using their inherent nuclear envelope structures and associated proteins to bind magnetic beads. The nuclei essentially serve their own isolation function through their intrinsic properties, eliminating the need for complex external sorting mechanisms and thereby simplifying the overall process while maintaining high efficiency.
3Adaptability or versatility
If existing cell type-specific labeling methods are used, then specific cell types can be identified, but the methods are not compatible with multiplexing and transgenic models
Solution Approach 1:
The patent creates a universal labeling system using magnetic beads that can be conjugated to multiple different antibodies targeting various cell type-specific markers. This allows the same basic isolation platform to be applied across multiple cell types and experimental models simultaneously, enabling multiplexing and compatibility with transgenic models while maintaining reliable cell type-specific labeling through the specificity of the antibody-bead conjugates.
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
The methods provide precise labeling and isolation of cell types, allowing for cell type-specific genomics and are compatible with multiplexing and various species, including humans, enhancing the practicality of genomic studies.
Implementation Method 1
The Sun1 portion enables the fusion polypeptide to localize to the nuclear membrane
Implementation Method 2
allowing for efficient and cost-effective cell type-specific genomics across various species, including humans, by utilizing a tagged Sun1 fusion polypeptide and Cre-dependent mechanisms
Implementation Method 3
The tag polypeptide can be a fluorescent polypeptide (e.g., a green fluorescent protein (GFP), such as a GFP with an amino acid sequence that at least 95% identical to the superfolder GFP sequence set forth in SEQ ID NO:2)
Implementation Method 4
a promoter sequence specific for a selected cell type, where the promoter sequence is operably linked to the sequence encoding the tagged Sun1 fusion polypeptide, and is effective to drive expression of the sequence encoding the tagged Sun1 fusion polypeptide in the selected cell type
Implementation Method 5
a first lox sequence flanking the 5′ end of the sequence encoding the tagged Sun1 fusion polypeptide and a second lox sequence flanking the 3′ end of the sequence encoding the tagged Sun1 fusion polypeptide, and (c) a promoter sequence downstream of the lox sequence flanking the 3′ end of the sequence encoding the tagged Sun1 fusion polypeptide, such that the sequence encoding the tagged Sun1 fusion polypeptide is in reverse orientation with respect to the promoter
Data Source
AI summary
Materials and methods for labeling and isolating particular cell types from mixed cell populations are provided herein. Also provided herein are methods for generating data representing a synthetic genetic sequence configured for labeling at least one cell type by causing expression of a marker in the at least one cell type.