title: A site-specific recombinase-based method to produce antibiotic selectable marker free transgenic cattle.
abstract: 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.
authors: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, PR China.
authors: Yuan
authors: Y
authors: Yu
authors: Yongsheng
authors: Y
authors: Wang
authors: Qi
authors: Q
authors: Tong
authors: Xu
authors: X
authors: Liu
authors: Feng
authors: F
authors: Su
authors: Fusheng
authors: F
authors: Quan
authors: Zekun
authors: Z
authors: Guo
authors: Yong
authors: Y
authors: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000900:Anti-Bacterial Agents / Q000378:metabolism; D002417:Cattle / Q000235:genetics*; D060467:Disease Resistance / Q000235:genetics; D005260:Female; D005818:Genetic Engineering; D005819:Genetic Markers; D006801:Humans; D019426:Integrases / Q000235:genetics; D008892:Milk / Q000378:metabolism; D008969:Molecular Sequence Data; D053652:Nuclear Transfer Techniques; D023083:beta-Defensins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; C422950:DEFB103A protein, human; D005819:Genetic Markers; D023083:beta-Defensins; C045073:Cre recombinase; D019426:Integrases
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