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. 2014 Apr;24(4):501-4.
doi: 10.1038/cr.2014.15. Epub 2014 Feb 7.

Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing

Xiaoping Li  1 , Yi Yang  1 , Lei Bu  2 , Xiaogang Guo  3 , Chengcheng Tang  1 , Jun Song  4 , Nana Fan  3 , Bentian Zhao  3 , Zhen Ouyang  3 , Zhaoming Liu  3 , Yu Zhao  3 , Xiaoling Yi  3 , Longquan Quan  1 , Songcai Liu  1 , Zhenguo Yang  1 , Hongsheng Ouyang  1 , Y Eugene Chen  5 , Zhong Wang  5 , Liangxue Lai  6
Affiliations

Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing

Xiaoping Li et al. Cell Res. 2014 Apr.
No abstract available

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Figures

Figure 1
Figure 1
Characterization of pRosa26 and highly efficient gene knock-in and replacement at the pRosa26 locus. (A, B) pRosa26 was expressed in a variety of organ tissues as determined by RT-PCR (A) and quantitative RT-PCR (B). For RT-PCR, the designed primers annealed in exon 1 and exon 2 and amplified a correctly spliced product of 485 bp. Porcine GAPDH was used as a control (234 bp). For qPCR, primers were specific for exon 2. PCR product of the porcine ACTB gene was used as the reference control. Data were presented as the average expression levels from three individual RT/qPCR reactions. (C) pRosa26 promoter-driven tdTomato expression in different cell lines (pRosa26 promoter region was shown in Supplementary information, Figure S1B and S1C). Transient transfection of pRosa26-tdTomato and pCMV-tdTomato vectors was performed in the indicated cell lines. (D) A diagram for TALEN-mediated knock-in of Neo-polyA-iEGFP into the pRosa26 locus. Grey triangles, wild-type (WT) loxP; white triangles, loxP2272 site; SA, splice acceptor. (E) Cre-mediated recombination activates EGFP expression by two mechanisms: Cre induces inversion of the iEGFP flanked by two loxP2272 sites (upper left) followed by excision of Neo flanked by two loxP sites; or Cre induces inversion of both Neo and iEGFP flanked by two loxP sites (upper right) followed by excision of Neo between two loxP2272 sites. (F) RMCE replaces EGFP with tdTomato. (G) Morphologically normal piglets were born from SCNT with the pRosa26-iEGFP PFFs. (H) PCR analysis confirmed the correct homologous recombination at the pRosa26 locus in the 7 piglets generated by SCNT. The pRosa26-iEGFP donor cells were used as positive control (P) and WT pig genomic DNA and water were used as negative controls. Primer pairs were shown in D. (I) EGFP activation by Cre in fibroblasts isolated from the ear tissues of cloned piglets shown in G. Cells were infected with Cre-lentivirus and the EGFP expression was observed after 48 h. (J) Embryos with constitutively activated EGFP expression were generated from SCNT of the pRosa26-EGFP PFFs obtained with a Cre plasmid transient transfection. Shown were an E35 pRosa26-EGFP embryo and its section. WT is an E45 WT embryo. (K) SCNT-generated pRosa26-tdTomato piglets through an RMCE strategy as shown in F.
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