Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
J Surg Res. 2013 May 1;181(1):e39-45. doi: 10.1016/j.jss.2012.06.035. Epub 2012 Jul 3.
Genetic modification of the pig has been hampered by inefficiency of homologous recombination and unavailability of pig embryonic stem cells. Engineered zinc finger nuclease (ZFN)-mediated genetic modification in somatic cells combined with somatic cell nuclear transfer (SCNT) technology provides a new approach for targeted modification in pig genome. In this study, we used a ZFN pair to disrupt porcine α-1,3, galactosyltransferase (GGTA1) gene in liver-derived cells (LDC). ZFN-treated LDC were used as nuclear donors to produce fetuses and piglets via SCNT. All cloned fetuses and piglets showed biallelic knockout of GGTA1 gene.
A ZFN pair was designed to target exon 8 of pig GGTA1 gene. LDC were transfected with GGTA1 ZFN plasmids. SURVEYOR assay was used to evaluate the ZFN activity in LDC. GGTA1 gene knockout cells (GTKO) were obtained by counter-selection and used as nuclear donors for SCNT. The cloned fetuses and piglets were characterized by DNA sequencing. Expression of α-Gal epitope was further examined by flow cytometry and confocal microscopy.
SURVEYOR assay revealed 6.48% ZFN activity in LDC. GTKO cells were obtained by counter-selection 10 d after ZFN transfection. A total of six fetuses and 13 piglets were produced by SCNT. All fetuses and piglets had biallelic mutations in the ZFN targeted region and were negative for α-Gal epitope.
Biallelic GGTA1 gene disruption in LDC was generated efficiently by ZFN. GTKO fetuses were produced from ZFN-treated LDC by SCNT. GTKO piglets were obtained by SCNT of ZFN-treated LDC or recloning of fetal fibroblasts from GTKO fetuses. With longer lifespan and robust growth rate, LDC has the potential to endure multiple genetic modifications in vitro without going to SCNT, which could accelerate the production of genetically modified pig organs for xenotransplantation.
猪的基因修饰受到同源重组效率低和猪胚胎干细胞不可用的阻碍。工程化锌指核酸酶 (ZFN) 介导的体细胞基因修饰与体细胞核移植 (SCNT) 技术相结合，为猪基因组的靶向修饰提供了一种新方法。在这项研究中，我们使用一对 ZFN 破坏肝源性细胞 (LDC) 中的猪 α-1,3,半乳糖基转移酶 (GGTA1) 基因。用 ZFN 处理的 LDC 作为核供体通过 SCNT 产生胎儿和仔猪。所有克隆的胎儿和仔猪均显示 GGTA1 基因的双等位基因敲除。
设计了一对 ZFN 靶向猪 GGTA1 基因的外显子 8。用 GGTA1 ZFN 质粒转染 LDC。通过 SURVEYOR 测定评估 LDC 中的 ZFN 活性。通过反选择获得 GGTA1 基因敲除细胞 (GTKO)，并将其用作 SCNT 的核供体。通过 DNA 测序对克隆的胎儿和仔猪进行特征分析。通过流式细胞术和共聚焦显微镜进一步检查 α-Gal 表位的表达。
SURVEYOR 测定显示 LDC 中的 ZFN 活性为 6.48%。ZFN 转染后 10 天通过反选择获得 GTKO 细胞。通过 SCNT 共产生了六头胎儿和 13 头仔猪。所有胎儿和仔猪在 ZFN 靶向区域均具有双等位基因突变，且均不表达 α-Gal 表位。
LDC 中的 GGTA1 基因双等位基因敲除可通过 ZFN 高效产生。通过 SCNT 从 ZFN 处理的 LDC 中产生 GTKO 胎儿。通过 SCNT 处理的 LDC 或 GTKO 胎儿的胎儿成纤维细胞的再克隆获得 GTKO 仔猪。LDC 的寿命更长，生长速度更快，在体外无需进行 SCNT 即可耐受多次基因修饰，这可能会加速用于异种移植的基因修饰猪器官的生产。
