Fig. 2

Characterization of HSP–ZFN in rice. a The CCR5-target construct in pPZP200 binary vector contains GFP, HPT and NPT genes. Each of which is controlled by 35S promoter and nos 3′ terminator. The HPT gene is flanked by 33 bp CCR5 sequences (gray bars). Location of EcoR1 (E) sites and the fragment sizes are shown. b Predicted structure of ZFN-induced precise excision of HPT cassette with indels in between (dotted bar). PCR primer positions and predicted fragment sizes (in kb) are shown below each structure. c Southern blot analysis of rice lines transformed with pBP5. Genomic DNA was cut with EcoRI and hybridized with P³² labeled GFP or NPT probes. Fragment sizes are given in kb. d PCR analysis using primers located in CCR5-target sites (GFP–NPT) or ZFN gene (HSP–ZFN) on genomic DNA isolated from F2 plants derived from crosses between CCR5-target lines and HSP–ZFN lines. F1 parent, and CCR5-target and ZFN lines are also shown. e PCR across CCR5 sites in the retransformed callus clones and the regenerated plants obtained by retransformation of HS–ZFN line #7 (Fig. 1d) with pBP5. The room temperature (RT) or heat-shocked (HS) samples of the selected calli clones (1–4) are shown with the regenerated plants obtained from them. ZFN line #7 serves as the negative control. f PCR across CCR5 sites in the retransformed clones derived from the retransformation of CCR5-target lines with pHSP:ZFN construct. Target line and wild-type (WT) are included as controls. g Depiction of indels created by targeting of the two CCR5 sites in the target site as determined by aligning the DNA sequences of selected ≤ 1.3 kb bands with pBP5 reference. Deletions sizes are given in each diagram