PLoS ONE · 2013 · 85 citations · 35 references
Life SciencesBiotechnologyGeneticsGenetic EngineeringGenome EditingTransgenic Animal ModelsEmbryo ManipulationGenetic Improvement Of LivestockSomatic Cell Nuclear TransferTransgene DesignTransgenesis StrategiesZinc Finger NucleasesCloning By Somatic Cell Nuclear TransferGene Editing In PigsGene KnockoutInterleukin-2 Receptor Gamma Chain DeficiencyTransgenic PigsZinc Finger Nuclease EditingZinc Finger Nuclease Knock-inZinc Finger Nuclease Knockout
Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration.
35
Production of α-1,3-Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning
Liangxue Lai, Donna Kolber‐Simonds, Kwang‐Wook Park et al. · Science · 2002 · 1.3K citations