A Site-Specific Recombinase-Based Method to Produce Antibiotic Selectable Marker Free Transgenic Cattle

Antibiotic selectable marker genes have been widely used to generate transgenic animals. Once transgenic animals have been obtained, the selectable marker is no longer necessary but raises public concerns regarding biological safety. The aim of this study was to prepare competent antibiotic selectable marker free transgenic cells for somatic cell nuclear transfer (SCNT). PhiC31 intergrase was used to insert a transgene cassette into a “safe harbor” in the bovine genome. Then, Cre recombinase was employed to excise the selectable marker under the monitoring of a fluorescent double reporter. By visually tracking the phenotypic switch from red to green fluorescence, antibiotic selectable marker free cells were easily detected and sorted by fluorescence-activated cell sorting. For safety, we used phiC31 mRNA and cell-permeant Cre protein in this study. When used as donor nuclei for SCNT, these safe harbor integrated marker-free transgenic cells supported a similar developmental competence of SCNT embryos compared with that of non-transgenic cells. After embryo transfer, antibiotic selectable marker free transgenic cattle were generated and anti-bacterial recombinant human β-defensin-3 in milk was detected during their lactation period. Thus, this approach offers a rapid and safe alternative to produce antibiotic selectable marker free transgenic farm animals, thereby making it a valuable tool to promote the healthy development and welfare of transgenic farm animals.

抗生素筛选标记基因已被广泛应用于转基因动物的制备。一旦获得转基因动物，筛选标记基因便不再必需，却会引发公众对生物安全性的广泛担忧。本研究旨在制备适用于体细胞核移植（somatic cell nuclear transfer, SCNT）的无抗生素筛选标记转基因细胞。研究人员利用PhiC31整合酶将转基因表达盒插入牛基因组的“安全港”位点；随后在荧光双报告系统的监测下，借助细胞渗透性Cre蛋白切除筛选标记基因。通过可视化追踪红色至绿色荧光的表型转换，研究人员可通过荧光激活细胞分选轻松检测并分选出无抗生素筛选标记的转基因细胞。为保障生物安全性，本研究使用了PhiC31信使核糖核酸（messenger RNA, mRNA）与细胞渗透性Cre蛋白。当将这些整合于安全港位点的无标记转基因细胞用作体细胞核移植的供核细胞时，其支持克隆胚胎发育的能力与非转基因细胞相当。胚胎移植后，成功获得无抗生素筛选标记的转基因肉牛，并在其泌乳期的乳汁中检测到了抗菌重组人β防御素-3。综上，该方法为制备无抗生素筛选标记的转基因畜禽提供了一种快速且安全的替代方案，因而可作为推动转基因畜禽健康发展与福利提升的实用工具。