Major
Biology
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Abstract
Synaptic strength at glutamatergic synapses is regulated in part through activity-dependent degradation of PSD-95, a postsynaptic scaffolding protein whose presence at the synapse determines the number of AMPA receptors available for glutamatergic transmission. Murine double minute 2 (MDM2), as an E3 ubiquitin ligase, ubiquinates PSD-95 as a result of NMDA receptor activation, driving proteasomal removal and enabling synaptic weakening. Despite this significance, a deeper investigation is needed to directly identify its contribution to synaptic remodeling. Here we describe the construction of a two-vector plasmid, pMDM2-IRES2-EGFP, designed to enable the simultaneous expression of MDM2 alongside fluorescent EGFP from a single transcript. [LK1] The MDM2 sequence was PCR amplified from a pcDNA3-MDM2 donor plasmid using primers containing NheI and SacII restriction sites, selected because each enzyme cuts only once within the pIRES-EGFP cloning site without disrupting the MDM2 insert, enforcing directional, single-site integration. Following restriction digestion, gel purification, and ligation, the product was transformed into competent E. coli, and positive clones were identified through miniprep and Sanger sequencing. Resulting plasmid couples EGFP fluorescence directly with MDM2 expression via an internal ribosomal entry site (IRES), providing a reliable method to identify successfully transfected cells. This strategy provides a validated overexpression tool for investigating MDM2-mediated regulation of PSD-95 and its downstream effects on AMPA receptor regulation and synaptic plasticity.
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Construction and Validation of pIRES-EGFP Vector for Co-expression of MDM2 and Enhanced Green Fluorescent Protein
Synaptic strength at glutamatergic synapses is regulated in part through activity-dependent degradation of PSD-95, a postsynaptic scaffolding protein whose presence at the synapse determines the number of AMPA receptors available for glutamatergic transmission. Murine double minute 2 (MDM2), as an E3 ubiquitin ligase, ubiquinates PSD-95 as a result of NMDA receptor activation, driving proteasomal removal and enabling synaptic weakening. Despite this significance, a deeper investigation is needed to directly identify its contribution to synaptic remodeling. Here we describe the construction of a two-vector plasmid, pMDM2-IRES2-EGFP, designed to enable the simultaneous expression of MDM2 alongside fluorescent EGFP from a single transcript. [LK1] The MDM2 sequence was PCR amplified from a pcDNA3-MDM2 donor plasmid using primers containing NheI and SacII restriction sites, selected because each enzyme cuts only once within the pIRES-EGFP cloning site without disrupting the MDM2 insert, enforcing directional, single-site integration. Following restriction digestion, gel purification, and ligation, the product was transformed into competent E. coli, and positive clones were identified through miniprep and Sanger sequencing. Resulting plasmid couples EGFP fluorescence directly with MDM2 expression via an internal ribosomal entry site (IRES), providing a reliable method to identify successfully transfected cells. This strategy provides a validated overexpression tool for investigating MDM2-mediated regulation of PSD-95 and its downstream effects on AMPA receptor regulation and synaptic plasticity.