Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China.
PLoS One. 2013 May 1;8(5):e62457. doi: 10.1371/journal.pone.0062457. Print 2013.
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.
抗生素选择标记基因已被广泛用于生成转基因动物。一旦获得了转基因动物，选择标记就不再需要，但会引起公众对生物安全的关注。本研究旨在制备用于体细胞核移植（SCNT）的无抗生素选择标记的转基因细胞。PhiC31 整合酶用于将转基因盒插入牛基因组中的“安全港”。然后，在荧光双报告基因的监测下，使用 Cre 重组酶切除选择标记。通过直观地跟踪从红色到绿色荧光的表型转换，很容易通过荧光激活细胞分选检测和分选无抗生素选择标记的细胞。为了安全起见，本研究中使用了 phiC31 mRNA 和细胞通透性 Cre 蛋白。当用作 SCNT 的供核时，这些安全港整合的无标记转基因细胞与非转基因细胞相比，支持 SCNT 胚胎相似的发育能力。胚胎移植后，生成了无抗生素选择标记的转基因牛，并在其哺乳期检测到牛奶中的抗细菌重组人β-防御素-3。因此，这种方法为生产无抗生素选择标记的转基因家畜提供了一种快速且安全的替代方法，从而成为促进转基因家畜健康发展和福利的有价值的工具。
