Park Ki-Eun, Park Chi-Hun, Powell Anne, Martin Jessica, Donovan David M, Telugu Bhanu P
Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA.
Animal Bioscience and Biotechnology Laboratory, USDA-ARS, Beltsville, MD 20705, USA.
Int J Mol Sci. 2016 May 26;17(6):810. doi: 10.3390/ijms17060810.
The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.
猪是基因工程应用中理想的大型动物模型。相对较短的妊娠期和较大的产仔数使猪成为产生和繁殖基因修饰的有利模型。家猪在解剖学、生理学、体型和预期寿命方面也有密切的相似性，使其成为模拟人类疾病的理想动物。然而，通常在产生所需的基因修饰（如短序列或转基因的靶向敲入）方面的技术困难阻碍了该领域的进展。在本研究中，我们研究并比较了CRISPR/Cas核糖核蛋白在猪体细胞中对普遍表达的COL1A基因下游的假attP位点进行靶向敲入工程中的相对效率，并通过体细胞核移植（SCNT）产生了活胎儿。通过利用这些敲入的假attP位点，我们证明了随后phiC31整合酶介导的绿色荧光蛋白（GFP）转基因整合到该位点。这项工作首次为猪体细胞中CRISPR/Cas介导的敲入创建了优化方案，同时为未来的转基因整合和产生转基因动物创建了一个稳定的平台。
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