Barcoded Transposon Vectors for Single-Cell TF Identification
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Solution Overview
Problem
Current methods for identifying transcription factor combinations in cellular reprogramming are inefficient due to the lack of effective methods for associating transcription factor combinations with specific cellular phenotypes, particularly in stem cells, where single cell RNA sequencing often fails to capture transcripts from exogenous nucleic acids with high sensitivity.
Innovation Solution
The use of barcoded transposon expression vectors, such as the piggyBAC™ expression vector, which includes a promoter operably linked to a transcription factor sequence and a barcode within 100 nucleotides of a terminator sequence, flanked by terminal repeats recognized by a transposase, enables high-resolution identification of transcription factor combinations driving specific cell states through droplet-based single cell RNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If droplet-based single cell RNA sequencing is used to detect transcription factor expression, then high resolution identification of transcription factor combinations is achieved, but sensitivity to capture transcripts from exogenous nucleic acids is insufficient
Solution Approach 1:
The patent introduces barcoded transposon expression vectors as an intermediary system between transcription factors and detection methods. The vectors contain unique barcodes that are transcribed along with transcription factor sequences, serving as detectable markers that amplify the signal from exogenous nucleic acids while maintaining the ability to resolve individual transcription factor combinations at high precision
Solution Approach 2:
The patent creates copies of transcription factor expression information by transcribing both the transcription factor sequence and its associated barcode into RNA. This copying mechanism allows the detection system to capture and identify transcription factor combinations through the barcode sequences, thereby improving detection sensitivity without compromising identification resolution
2Adaptability or versatility
If large-scale overexpression analyses are conducted to identify transcription factor combinations, then unbiased identification is achieved, but the association with specific cellular phenotypes becomes inefficient
Solution Approach 1:
The patent creates a universal barcoded transposon expression vector system that can identify transcription factor combinations and simultaneously associate them with cellular phenotypes through the same barcode sequences. The barcodes serve multiple functions: identifying the transcription factor combination, linking to cellular phenotype data, and enabling high-throughput analysis, thereby improving association efficiency without sacrificing unbiased identification
Solution Approach 2:
The patent merges the identification function and phenotype association function into a single integrated system using barcoded transposon expression vectors. The barcodes are incorporated into the same transcriptional unit as the transcription factors, allowing simultaneous detection of both the transcription factor combination and its relationship to cellular phenotypes in a unified high-throughput platform
Data Source
AI summary
Provided herein, in some embodiments, are methods and compositions for identifying combinations of transcription factors, for example, those involved in cell type conversion processes, such as cell differentiation.