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. 2011 Jun;21(6):979-82.
doi: 10.1038/cr.2011.70. Epub 2011 Apr 19.

Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

Dongshan Yang, Huaqiang Yang, Wei Li, Bentian Zhao, Zhen Ouyang, Zhaoming Liu, Yu Zhao, Nana Fan, Jun Song, Jiangtian Tian, Feng Li, Jifeng Zhang, Lin Chang, Duanqing Pei, Y Eugene Chen, Liangxue Lai

Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

Dongshan Yang et al. Cell Res. 2011 Jun.
No abstract available

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Figures

Figure 1
Figure 1
ZFN-mediated Ppar-γ gene disruption in pigs. (A) Binding sites (underlined) of the zinc-finger nucleases produced. (B) A schematic diagram of the Ppar-γ gene structure. (C) Sequencing diagram of a mutant with a mono-allelic insertion, as indicated by an arrow, at the ZFN-targeting site. The upper line shows the wild-type sequence while the lower line shows the mutant sequence. The diagram shows a double curve after insertion. (D) DNA and amino acid sequences of Ppar-γ proximal to the ZFN target site in cell clone #15 (“*” in red indicates stop codon sites after a frame shift resulted from an insertion at the 56 codon of Ppar-γ1). (E) Piglets confirmed to be Ppar-γ heterozygous knockout. (F) PPAR-γ western blotting. In the left panel, piglets #2 and #3 are mutant piglets while piglets #1, #4 and #5 are wild-type controls. Piglets #1 and #2 are littermates while piglets #3, #4, and #5 are littermates. In the right panel, HeLa cells transfected with wild-type Ppar-γ1 cDNA (HeLa+wt) or with mutated Ppar-γ1 cDNA (HeLa+mut) were used for PPAR-γ western blotting. The untransfected cells were used as the control (HeLa). γ 1 and γ 2 represent the two isoforms of PPAR-γ and GAPDH was used as the internal control. (G) The efficiency of ZFN-mediated targeting in pigs. The yeast assay data were provided by the ZFN manufacturer. ND: not determined; NA: not applicable; MC assay: mammalian cultured cell line-based assay; Muts: Mutants.
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