研究目的
To introduce a pooled approach for optical genetic screens in mammalian cells that combines high-content imaging with in situ sequencing to identify genes affecting spatially and temporally defined phenotypes.
研究成果
The study establishes a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries, identifying known and novel regulators of NF-kB signaling. The method enables systematic analysis of genetic components underpinning a wide range of cellular phenotypes.
研究不足
The approach is limited by the complexity of in situ sequencing and the need for high-quality imaging data. Additionally, the scalability to genome-scale libraries and the ability to detect multiple perturbations within the same cell are areas for further optimization.
1：Experimental Design and Method Selection:
The approach uses targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping.
2：Sample Selection and Data Sources:
Screened a set of 952 genes across millions of cells for involvement in nuclear factor kB (NF-kB) signaling by imaging the translocation of RelA (p65) to the nucleus.
3：List of Experimental Equipment and Materials:
Utilized lentiviral vectors for genetic perturbations, high-content imaging for phenotype assessment, and in situ sequencing for perturbation identification.
4：Experimental Procedures and Operational Workflow:
Cells were stimulated with cytokines, imaged for phenotype assessment, and then processed for in situ sequencing to link phenotypes to genetic perturbations.
5：Data Analysis Methods:
Translocation scores were calculated based on the correlation between nuclear stain and p65-mNeonGreen signal, and statistical analysis was performed to identify significant perturbations.
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