CDH5p-GFP-ZEO lentiviral vector was constructed incrementally. First, the CDH5 promoter was cloned into a pENTR5′-TOPO vector as previously demonstrated [24 (link)]. Then, GFP-T2A-Zeocin was amplified from pGreenZEO (System Biosciences) and subcloned into pENTR/D-TOPO. Finally, the pENTR5′CDH5p and pENTR/D-GFP-ZEO were cloned into the pDEST R4R3 Vector II (Thermo Fisher Scientific) using Gateway™ LR Clonase™ II Enzyme mix (Thermo Fisher Scientific). Note that the pDEST R4R3 Vector II includes a blasticidin resistance cassette for selection. The resulting vector was submitted to the Viral Vector Core Facility at the University of Iowa for lentiviral packaging. The CDH5p-GFP-ZEO lentiviral vector is available on Addgene (catalog # 122970). Figure 1 b illustrates a simplified plasmid map.
Pdest r4r3 vector 2
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3 protocols using pdest r4r3 vector 2
1
Construction of CDH5p-GFP-ZEO Lentiviral Vector
2
Lentiviral Packaging Vectors for Choroidal Endothelial Cells
3
Cloning Human CA4 Promoter and Zeocin Resistance Gene
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The human carbonic anhydrase 4 (CA4) promoter, which in the posterior eye is specific to the choriocapillaris, was cloned into the pENTR5'‐TOPO vector using the Gateway Cloning strategy (Thermo Fisher Scientific; Carlsbad, CA; Cat. No. K591‐10) 21 , 22 . The Gateway Cloning strategy was also used to clone a Zeocin resistance gene into the pENTR/D‐TOPO vector. Both entry vectors were subsequently cloned into the pDEST R4R3 Vector II (Thermo Fisher Scientific; Cat. No. 46‐2130, Supporting Information Figure 1 ) with the Gateway LR Clonase II Plus Enzyme Mix (Thermo Fisher Scientific; Cat. No. 12538‐120) and transformed in One Shot TOP10 Chemically Competent Escherichia coli (ThermoFisher Scientific; Cat. No. C4040‐03).
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