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George W. Smith, W. David Culp, Jr., Grace Byfield, Tal Kafri, John Flannery, Mary Elizabeth Hartnett; Targeting Mueller Cell-derived VEGF With Short Hairpin RNA. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4865.
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© ARVO (1962-2015); The Authors (2016-present)
Mueller cells often overexpress angiogenic factors in pathologic conditions. We designed HEK reporter cell lines that constitutively express either VEGF120 or VEGF164 to test the degree of silencing of VEGF using short hairpin RNAs, fashioned as microRNAs, with the goal of silencing Mueller cell-derived VEGF in vivo.
HEK cells were transduced with lentiviral vectors containing either rat VEGF120 to target VEGFA or rat VEGF164 cDNA and a GFP reporter to assess efficiency of several VEGF shRNA silencing plasmids. The lentiviral transfer vectors of the reporter HEK cells contained two CMV promoters, one driving production of viral elements and the other expression of the desired VEGF splice variant followed by an IRES element and blasticidin/GFP selection/reporter. The GFP in this construct allows for visualization of transfection efficiency while the blasticidin allows for selection of only VEGF expressing cells. The shRNA target sequences (two each for VEGFA and VEGF164) were cloned into expression plasmids containing a red fluorescent reporter gene driven by the CMV promoter. Both HEK VEGF reporter cell lines were transfected with each of the silencing plasmids and flow cytometry was used to determine the percent silencing of VEGF protein expression. Similarly, four commercially available shRNAs each packaged into a lentiviral transfer vector were used to transduce HEK rat VEGF120 and VEGF164 reporter cell lines. VEGFA mRNA silencing was measured 48 hours later by real-time PCR.
Two VEGF164 shRNAs silenced VEGF164 protein expression by 13 and 36 percent and did not silence protein expression in the VEGF120 cell line. Two VEGFA shRNAs silenced VEGF120 protein expression by 13 and 33 percent and VEGF164 by 38 and 47 percent. In HEK rat VEGF164 cells, the four shRNAs silenced VEGFA mRNA by 34, 64, 46, and 30 percent, while in HEK rat VEGF120 cells they silenced VEGFA mRNA by 0, 74, 11, and 50 percent.
Selective silencing of VEGF splice variants or VEGFA is possible using shRNAs on HEK rat VEGF reporter cell lines. Silencing specific VEGF splice variants may prove useful when inhibiting a growth factor like VEGF, which has physiologic and pathologic effects depending on concentration and signaling.
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