To construct pMcp-1.BDNF and pMcp-1.NRF2 vectors, a fragment of the murine Mcp-1 promoter (560 bp immediately upstream of the initiation codon ATG; accession number U12470 ) and BDNF cDNA (KIBB9810; Promega) or NRF2 cDNA (KIBB7828; Promega) were inserted into a pAAV-MCS Promoterless Expression Vector (Cell Biolabs). To construct pMcp-1.NRF2-2A-EGFP, the same fragment of the Mcp-1 promoter, NRF2 cDNA, linker peptide 2A cDNA, and EGFP cDNA (Clontech) were inserted into the pAAV-MCS Promoterless Expression Vector using the Gibson assembly system (New England Biolabs). To construct pCMVNRF2-2A-EGFP, Nrf2 cDNA, 2A peptide, and egfp cDNA (Clontech) were inserted into a pAAV-CMVp-MCS Expression Vector (Cell Biolabs) using the Gibson assembly system (New England Biolabs). For pAAV.CMV.EGFP using pAAV-GFP vector (Cell Biolabs), viral vectors AAV2/2 were generated and purified following a method described previously.48 (link) Each vector (1.0 × 1012 genome copy [gc]/mL) was injected at 2.0 μL per injection into the vitreous of an anaesthetized mouse. The dose was the same in all experiments involving intravitreal injection of AAV in this study.
Egfp cdna
Manufactured by Takara Bio
EGFP cDNA is a complementary DNA (cDNA) sequence encoding the enhanced green fluorescent protein (EGFP) from the jellyfish Aequorea victoria. EGFP is a widely used reporter protein that emits green fluorescence when exposed to blue or ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Paav mcs promoterless expression vector, by Cell Biolabs (2 mentions)
Paav gfp vector, by Cell Biolabs (1 mentions)
Gibson assembly system, by New England Biolabs (1 mentions)
Nf κb response element, by Promega (1 mentions)
Pcdh cmv mcs ef2 puro, by System Biosciences (1 mentions)
X tremegene hp dna transfection reagent, by Merck Group (1 mentions)
3 protocols using egfp cdna
1
Constructing Targeted Gene Expression Vectors
2
AAV-Mediated Reporter Gene Transduction
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For the reporter experiments, a 720-bp fragment of EGFP cDNA (Clontech, Mountain View, CA) was sub-cloned into a pAAV-MCS Promoterless Expression Vector (Cell Biolabs Inc., San Diego, CA). Promoter fragments of the antioxidant response element (Promega Corp., Madison, WI), ATF6 response element (Promega Corp., Madison, WI), hypoxia response element (Promega Corp., Madison, WI), Mcp-1 promoter (560 bp immediately upstream of the initiation codon ATG; Accession number U12470)48 (link), NF-κB response element (Promega Corp., Madison, WI), p53 response element (p53 RE; Promega Corp., Madison, WI), and SMAD-binding element (Promega Corp., Madison, WI) were each sub-cloned into AAV reporter vectors without a promoter. AAV2/2 containing the reporter constructs were generated and purified following the method described previously49 (link). Each virus (1 × 1012 gc/mL) was injected (2 μL/injection) into the vitreous of a mouse.
3
Lentiviral-mediated EGFP expression
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