研究目的
Investigating the regulation of Frmpd1 expression in rod photoreceptors through an alternative promoter and its implications for cell-type-specific gene silencing.
研究成果
The study demonstrates that the Frmpd1 gene is regulated by an alternative promoter in rod photoreceptors, which is activated by NRL and CRX. CRISPR/Cas9-mediated deletion of this promoter region effectively silences Frmpd1 in rods without affecting its expression in other tissues, providing a strategy for cell-type-specific gene editing. This approach could be applied to other genes to study their function in specific cell types.
研究不足
The study focuses on the retina-specific expression of Frmpd1 and its regulation by NRL and CRX. The applicability of the findings to other tissues or genes requires further investigation. The CRISPR/Cas9 approach, while effective, may have off-target effects that were not fully explored.
1：Experimental Design and Method Selection:
The study involved in vivo and in vitro experiments to identify and characterize the Frmpd1 alternative promoter in the retina. Techniques included electroporation, CRISPR/Cas9-mediated genome editing, and electrophoretic mobility shift assays (EMSA).
2：Sample Selection and Data Sources:
Mouse retina and other tissues were used to analyze Frmpd1 expression. RNA-seq data from human and mouse tissues were also utilized.
3：List of Experimental Equipment and Materials:
Equipment included confocal microscopes, PCR machines, and electroporation devices. Materials included plasmids, CRISPR/Cas9 components, and antibodies.
4：Experimental Procedures and Operational Workflow:
The workflow involved cloning promoter regions, performing in vivo electroporation, conducting EMSA, and analyzing gene expression through qPCR and in situ hybridization.
5：Data Analysis Methods:
Data were analyzed using bioinformatics tools for RNA-seq data, statistical analysis for qPCR results, and image analysis for in situ hybridization.
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Confocal microscope
LSM 700
Zeiss
Used for imaging electroporated retinas.
ZEISS LSM 990 Spectral Multiplex
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CRISPR/Cas9
Genome editing tool used to delete the genomic region including NRL and CRX binding sites in vivo.
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Electroporation device
Used for in vivo electroporation of Frmpd1 promoter region into mouse retina.
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PCR machine
Used for genotyping and quantitative PCR.
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Dual-Luciferase Reporter Assay System
Promega
Used for measuring luminescence of firefly and renilla luciferases in luciferase assays.
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SMARTer RACE 5’/3’ kit
Clontech
Used for 5’-RACE amplification.
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MEGAshortscript T7 Transcription Kit
Thermo Fisher Scientific
Used for in vitro transcription of crRNA-tracrRNA.
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Q5 Site-Directed Mutagenesis Kit
New England Biolabs
Used for introducing mutations into Frmpd1 promoter constructs.
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Lipofectamine 2000
Invitrogen
Used for transfection in luciferase assays.
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Fluoromount-G
Southern Biotech
Used for mounting retinas for imaging.
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