Ghahfarokhi Milad Khorramian, Dormiani Kianoush, Mohammadi Ali, Jafarpour Farnoosh, Nasr-Esfahani Mohammad Hossein
Division of Biotechnology, Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.
Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran.
Sci Rep. 2017 Nov 13;7(1):15432. doi: 10.1038/s41598-017-15648-3.
Integration target site is the most important factor in successful production of transgenic animals. However, stable expression of transgene without disturbing the function of the host genome depends on promoter methylation, transgene copy number and transcriptional activity in integration regions. Recently, new genome-editing tools have made much progress, however little attention has been paid to the identification of genomic safe harbors. The aim of the present study was to evaluate the effect of insertion site, promoter and copy number of transgene on the production of embryos from cattle fibroblast cells following somatic cell nuclear transfer (SCNT). So, three donor vectors were constructed with EGFP gene under control of different promoters. Each vector was integrated into safe and non-safe harbors in the genome using phiC31 integrase. Transgenic clones with a single copy of each vector were isolated. Each clone was analyzed to find site and frequency of integration, expression level and promoter methylation before SCNT, as well as transgene expression level and blastocyst formation rate after SCNT. The data obtained demonstrated that BF5, as a safe harbor, not only showed a stable expression, but also the rate of in vitro-produced embryos from BF5-clones are similar to that of non-transfected cells.
整合靶位点是成功生产转基因动物的最重要因素。然而，转基因的稳定表达且不干扰宿主基因组的功能取决于启动子甲基化、转基因拷贝数以及整合区域的转录活性。最近，新的基因组编辑工具取得了很大进展，然而对基因组安全位点的鉴定却很少受到关注。本研究的目的是评估转基因的插入位点、启动子和拷贝数对体细胞核移植（SCNT）后牛成纤维细胞胚胎生产的影响。因此，构建了三种供体载体，其中EGFP基因受不同启动子控制。使用phiC31整合酶将每个载体整合到基因组中的安全和非安全位点。分离出每个载体单拷贝的转基因克隆。在SCNT之前，对每个克隆进行分析以确定整合位点和频率、表达水平和启动子甲基化，以及SCNT之后的转基因表达水平和囊胚形成率。获得的数据表明，BF5作为一个安全位点，不仅表现出稳定的表达，而且BF5克隆的体外生产胚胎率与未转染细胞相似。
