研究目的
To investigate the role of soluble adenylyl cyclase (sAC) in neuronal differentiation during retinal development, specifically in retinal ganglion cells, amacrine cells, and photoreceptors.
研究成果
sAC is crucial for the differentiation and development of retinal ganglion cells, amacrine cells, and photoreceptors during early retinal development. The effects are more pronounced with earlier deletion (Chx10cre) and persist into adulthood for certain cell types, highlighting sAC's role in cell fate specification and maturation.
研究不足
The Cre-mediated excision may not be 100% penetrant, leading to residual sAC expression. Differences in retinal sectioning angles could introduce small variations. The study does not identify specific progenitor subsets or cell-cell interactions dependent on sAC, which are areas for future research.
1：Experimental Design and Method Selection:
The study used conditional gene deletion via Chx10 or Math5 promoter-driven Cre-Lox recombination to eliminate sAC from retinal progenitor cells at different developmental stages. Immunofluorescence staining and Western blot analysis were employed to assess protein expression, cell markers, and retinal morphology.
2：Sample Selection and Data Sources:
Mice with conditional sAC knockout (cKO) were generated by crossing sAC floxed mice with Chx10cre or Math5cre transgenic mice. Age-matched littermates without Cre alleles served as controls. Retinal tissues from adult and newborn (postnatal day 1) mice were used, with six eyes from three mice per group for analysis.
3：List of Experimental Equipment and Materials:
Equipment included Western blot apparatus (e.g., gels, membranes), fluorescence microscopes, and software for image analysis (ImageJ, SPSS). Materials included antibodies (e.g., anti-sAC, anti-Brn3a), lysis buffers, and reagents for immunofluorescence.
4：Experimental Procedures and Operational Workflow:
Mice were euthanized, retinas dissected and processed for Western blot or fixed for immunofluorescence. Sections were stained with specific antibodies, imaged, and analyzed for cell counts and layer thickness. Statistical analysis was performed using Student's t-tests and ANOVA.
5：Data Analysis Methods:
Data were analyzed using SPSS software, with means and standard deviations calculated. Statistical significance was set at P < 0.05, using t-tests for comparisons and ANOVA for multiple groups.
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lysis buffer
Cell Signaling Technology
Used for lysing retinal tissues to extract proteins for Western blot analysis.
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phenylmethanesulfonyl fluoride
Sigma-Aldrich Corp.
Added to lysis buffer to inhibit proteases during protein extraction.
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bicinchoninic acid assay kit
Thermo Fisher Scientific
Used to determine protein concentration in retinal lysates.
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Tris-glycine precast gels
4% to 20% gradient
Invitrogen, Life Technologies
Used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis to fractionate proteins.
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polyvinylidene difluoride membrane
Millipore
Used for transferring proteins from gels in Western blotting.
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primary antibody against sAC
Ab82854
Abcam
Used to detect sAC protein in Western blot and immunofluorescence staining.
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secondary antibody
horseradish peroxidase-conjugated anti-goat or rabbit
Santa Cruz Biotechnology
Used in Western blotting to visualize labeled proteins.
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chemiluminescence substrate
Thermo Fisher Scientific
Used for developing horseradish peroxidase in Western blots.
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image capture system
GE imageQuant
GE Healthcare Biosciences
Used to capture images of Western blots.
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ImageJ software
National Institutes of Health
Used for analyzing relative band intensities in Western blots.
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optimal cutting temperature compound
Sakura Finetek
Used for mounting retinal tissues for frozen sectioning.
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primary antibodies for immunofluorescence
e.g., anti-ADCY10 (ab203204), anti-Brn3a (AB5945), anti-recoverin (AB5585), anti-rhodopsin (MAB5356), anti-β-tubulin (MMS-410P), Pax6 (sc-7750), PKC-α (sc-208), anti-glutamine synthetase (G2781)
Various (Abcam, EMD Millipore, Covance, Santa Cruz Biotech, Sigma Corporation)
Used to stain specific cell markers in retinal sections for immunofluorescence analysis.
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secondary antibodies
Alexa fluorophore-conjugated
Invitrogen
Used in immunofluorescence to detect primary antibodies.
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DAPI
Used for nuclear staining in immunofluorescence.
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SPSS software
20.0
IBM Corp.
Used for statistical analysis of experimental data.
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