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. 2014 May 23;4:5043. doi: 10.1038/srep05043

Figure 3. TALEN-mediated genome editing.

Figure 3

(a) Top: Schematic of the endogenous IL2RG locus. HindIII, HindIII restriction sites used for Southern blot analysis; TM, transmembrane domain. Middle: Schematic of the targeting vector. The targeting vector contained Venus cDNA, lox-P-flanked Neomycin resistance cassette, and DT-A negative selectable marker. The 5′ homology arm upstream of the IL2RG start codon was cloned upstream of Venus cDNA, and the 3′ homology arm downstream of the IL2RG transmembrane sequence (exon 6) was cloned downstream of the Neomycin resistance cassette (Neor). 5′ probe, Probe used for Southern blot analysis. Bottom: Schematic of the targeted IL2RG locus. A novel HindIII restriction site would introduced when the targeted knock-in was successful. (b) Flow cytometric analysis of Venus in Jurkat cells with targeted knock-in of IL2RG. Each Jurkat cell clone was analyzed for YFP fluorescence expressed from knocked-in Venus cDNA. WT, wild-type Jurkat cells; KI 1-4, individual clones with targeted knock-in; KI 5, Venushi; MFI, Mean Fluorescence Intensity of Venus. (c) Southern blot analysis of Jurkat cells with targeted knock-in of IL2RG. HindIII digestion resulted in a 3788 bp band from the WT endogenous IL2RG locus and a 3515 bp band (containing Venus cDNA and the Neomycin resistance cassette) from the targeted knock-in. Targeting vector was used as positive control for targeted knock-in, and genomic DNA isolated from WT Jurkat cells was used as negative control.