Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases - PubMed (original) (raw)

. 2011 Jul 19;108(29):12013-7.

doi: 10.1073/pnas.1106422108. Epub 2011 Jul 5.

Bjoern Petersen, Yolanda Santiago, Anna-Lisa Queisser, Joseph W Carnwath, Andrea Lucas-Hahn, Lei Zhang, Xiangdong Meng, Philip D Gregory, Reinhard Schwinzer, Gregory J Cost, Heiner Niemann

Affiliations

Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases

Janet Hauschild et al. Proc Natl Acad Sci U S A. 2011.

Erratum in

Abstract

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Design and characterization of zinc-finger nucleases. (A) Schematic of ZFNs binding to the GGTA1 locus. (B) Surveyor nuclease assay of ZFN cleavage in transformed PK(15) cells at 37 °C and 30 °C. The 553-bp PCR amplicon is cleaved into ∼375- and ∼178-bp fragments. (C) SELEX specificity profile of the SBS 23713 zinc-finger protein. (D) SELEX specificity profile of the SBS 23714 zinc-finger protein. For both SBS 23713 and 23714 the fraction of correctly specified bases for each position is shown above the x axis.

Fig. 2.

Fig. 2.

1.7% of the transfected cells (E38, not used for selection; dark gray) are located in the area where Gal− cells (black line) are located (Gal+ cells, light gray line).

Fig. 3.

Fig. 3.

Live-born piglet C2F1 (Lia), 3 wk old.

Fig. 4.

Fig. 4.

GGTA1 sequencing results of wild type (WT), six cloned fetuses (C1F1–6), and cloned piglets (C2F1 and C2F2). Recloned piglets carry mutations identical to the donor nuclei (C1F5 and -F6). Gray boxes indicate binding sites of ZFNs _GGTA1_-23713 and -23714. -, indicates base deletion. Base insertion is shown with the boxed arrow.

Fig. 5.

Fig. 5.

Protection of ZFN-_GGTA1_-KO fibroblasts from antibody/complement-mediated lysis. 51Cr-labeled fibroblasts from six cloned fetuses (C1F1–F6), from a wild-type control, and Gal-KO fibroblasts generated by conventional knockout (HR-_GGTA1_-KO) were incubated with increasing concentrations of human antibody/complement. The amount of radioactivity released into the supernatant was determined after 4 h by measuring an aliquot of 25 μL. The percentage of specific lysis was calculated as described in Materials and Methods. The data shown were obtained in a single experiment. Similar patterns were observed in a second and third experiment. The HR-_GGTA1_-KO sample is represented by one cell line.

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