Multiplexed Cell Lineage Quantification via Barcoding
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Solution Overview
Problem
Current methods for analyzing gene function in cancer are limited by the inability to precisely quantify clonal population sizes and the impact of genetic alterations in vivo, leading to incomplete understanding of tumor growth and response to therapies.
Innovation Solution
Combining cell barcoding with high-throughput sequencing and genetically engineered mouse models, using multiplexed CRISPR/Cas9-mediated genome editing and somatic homology directed repair to quantify tumor growth and identify functional tumor suppressors with unprecedented resolution.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cell barcoding with high-throughput sequencing and multiplexed CRISPR/Cas9-mediated genome editing is used, then measurement precision of clonal population sizes is improved, but device complexity increases
Solution Approach 1:
The patent segments the analysis by assigning unique barcodes to individual cells or cell clusters, allowing parallel tracking of multiple clonal populations. This segmentation enables precise measurement of each clone's population size while maintaining overall system manageability through modular barcode design and sequential processing steps.
Solution Approach 2:
The patent introduces barcodes as intermediary markers that mediate between the genetic alterations and the measurement system. These barcodes serve as detectable proxies for clonal populations, enabling indirect but precise quantification without directly observing the genetic alterations themselves, thus improving measurement precision while managing complexity.
2Ease of operation
If in vivo systems with crude measurements of tumor growth are used, then ease of operation is improved, but measurement precision deteriorates
Solution Approach 1:
The patent replaces crude mechanical or visual measurement systems with molecular-based detection using barcodes and sequencing. This substitution maintains the in vivo operational simplicity while dramatically improving measurement precision by enabling quantitative analysis of clonal population sizes through high-throughput sequencing of barcode sequences.
3Ease of manufacture
If cell lines in culture are used for gene function analysis, then ease of manufacture is improved, but reliability of in vivo phenotype insight deteriorates
Solution Approach 1:
The patent performs preliminary barcoding and genetic alteration introduction in easily manufactured cell cultures, then transplants these pre-prepared cells into in vivo systems. This preliminary action in culture allows controlled manipulation and initial tracking setup, while the subsequent in vivo environment provides reliable physiological context for phenotype assessment, combining the advantages of both approaches.
Data Source
AI summary
Compositions and methods are provided for measuring population size for a plurality of clonal cell populations in the same individual, e.g., for measuring tumor size for a plurality of clonally independent tumors within the same individual. A subject method can include: (a) contacting an individual with a plurality of cell markers that are heritable and distinguishable from one another, to generate a plurality of distinguishable lineages of heritably marked cells; (b) after sufficient time has passed for the heritably marked cells to undergo at least one round of division, detecting and measuring quantities of at least two of the plurality of cell markers present in the contacted tissue, thereby generating a set of measured values; and (c) using the set of measured values to calculate the number of heritably marked cells that are present (e.g., for at least two of the distinguishable lineages of heritably marked cells).