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. 2017 Jan 19;15(3):1313–1318. doi: 10.3892/mmr.2017.6131

Figure 1.

Figure 1.

Targeting of MSCs by AAVS-AcGFP1-hFIX. (A) Targeting vector AAVS-AcGFP1-hFIX was composed of the 5′ and 3′ homologous arms, the promoterless GFP expression cassette IRES2-AcGFP1 and the hFIX expression cassette CMV-hFIX-pA under the control of the CMV promoter. The recognition site for ZFN was located at the AAVS1 locus of the first intron of the PPP1R12C gene. ‘primer up’ and ‘primer dn’ were the primers used to validate the site-specific integration clones, which were located at the pA and outside the 3′ end of the 3′ homologous arm, respectively. P1 and P2 were the probes used for the Southern blot to identify the site-specific integration clones. (B) ZFN expression in transfected MSCs. (C and D) Efficiency of GFP expression, as determined using by flow cytometry. (E) Polymerase chain reaction detection for the site-specific integration events. IRES1, internal ribosome entry 2; GFP, green fluorescent protein; CMV, cytomegalovirus; hFIX, human blood coagulation factor IX; AAVS1, adeno-associated virus integration site 1; ZFN, zinc finger nuclease; MSCs, mesenchymal stem cells; D, donor; Z, ZFN; TI, targeted integration.