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Inducible Rbpms-CreERT2 mouse line for functional studies of genes in retinal ganglion cell physiology and disease

ABSTRACT: Retinal ganglion cells (RGCs) are the sole output neurons conveying visual stimuli from the retina to the brain and the dysfunction or loss of RGCs is the primary determinant of visual loss in traumatic and degenerative ocular conditions. Currently, there is a lack of RGC-specific Cre mouse lines that serve as invaluable tools for manipulating genes in RGCs and studying the genetic basis of RGC diseases. The RNA-binding protein with multiple splicing (RBPMS) is identified as the specific marker of all RGCs. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused in-frame to the C-terminus of endogenous RBPMS, allowing the co-expression of RBPMS and CreERT2. The inducible Rbpms-CreERT2 mice exhibited a high recombination efficiency in activating the expression of tdTomato reporter gene in nearly all adult RGCs as well as in differentiated RGCs starting at E13.5. Additionally, heterozygous Rbpms-CreERT2 knock-in mice showed no detectable defect in the retinal structure, visual function and transcriptome. Altogether, our results demonstrated that the Rbpms-CreERT2/+ knock-in mouse can serve as a powerful and highly desired genetic tool for lineage tracing, genetic manipulation, retinal physiology study and ocular disease modeling in RGCs.

ORGANISM(S): Mus musculus

PROVIDER: GSE232726 | GEO | 2023/08/21

REPOSITORIES: GEO

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Project description:To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested at 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (p<0.05, FDR<1%) were quantified by SWATH-MS. Bioinformatics data analysis showed that there were 35 up-regulated and 24 down-regulated proteins in the temporal quadrant while 19 and 5 proteins were up-regulated and down-regulated, respectively, in the nasal quadrant respectively (n=4, p<0.05; fold change ≥1.4 fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants including CLU, GFAP, GNG5, IRF2BPL, L1CAM and CPLX1. Linear regression analysis indicated a strong association between the data obtained by SWATH-MS and MRM-MS (temporal, R2=0.97; nasal, R2=0.96). Gene ontology analysis reveals statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

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Inducible Rbpms-CreERT2 mouse line for functional studies of genes in retinal ganglion cell physiology and disease

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