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

et al. (2013) A Site-Specific Recombinase-Based Method to Produce Antibiotic Selectable Marker Free Transgenic Cattle. PLoS ONE 8(5): e62457. doi:10.1371/journal.pone.0062457 A Site-Specific Recombinase-Based Method to Produce Antibiotic Selectable Marker Free Transgenic Cattle Yuan Yu. 0 Yongsheng Wang. 0 Qi Tong 0 Xu Liu 0 Feng Su 0 Fusheng Quan 0 Zekun Guo 0 Yong Zhang 0 Xiuhcun (Cindy) Tian, University of Connecticut, United States of America 0 Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University , Yangling, Shaanxi , People's Republic of China 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 b-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. - Funding: This work was supported by the National Major Project for Production of Transgenic Breeding (No. 2011ZX08007-004) and the National High Technology Research and Development Program of China (863 Program) (No. 2011AA100303). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Transgenic farm animals are important materials for biomedical and agricultural research [1,2]. However, the present approaches to generate transgenic animals are still hampered by low efficiency [3], variable expression levels of the transgene [4], and the residual antibiotic-resistance gene that is required to select transgenic cells but provokes public concerns regarding biological safety. Thus, efficient and safe methods are urgently needed to improve the current situation. Currently, somatic cell nuclear transfer (SCNT) has been proven to be the most effective protocol for the production of transgenic animals [5,6]. Therefore, preparation of competent transgenic donor cells is a key step for successful SCNT. Many methods are available to produce transgenic donor cells, and the traditional method relies on random integration of the transgene of interest. However, random integration into chromosomes suffers from low stable integration [7], and variable expression levels of the genes due to positional effects and the number of inserted copies [811]. Homologous recombination provides site specificity, but at a very low efficiency [12]. Furthermore, many virus-based gene transfer approaches are limited by their preference for integration into the gene-coding region [13,14], which is a safety risk of transgenic animal production. Thus, an efficient and safe gene delivery approach is important for transgenic cell preparation. PhiC31 integrase, the Streptomyces phage-derived recombinase, has been developed as a non-viral gene therapy tool, because it has the ability to integrate a transgene-containing plasmid carrying an attB site into pseudo attP sites in mammalian genomes [15,16]. This enzyme has been previously shown to integrate genes effectively and prolong transgene expression in several mammalian cell culture systems including those for human keratinocytes [17], muscle-derived stem cells and myoblasts [18], and a human T cell line [19]. In addition, it has been recently reported that phiC31mediated integration events usually occur in genomic safe harbors in mammalian cells [20], the regions of the genome where the integrated material is adequately expressed without perturbing endogenous gene structure or function, following a process that is amenable to precise mapping and minimizing occult genotoxicity [21], which makes phiC31 integrase an ideal tool for gene delivery and transgenic animal production. Furthermore, the integration of antibiotic-resistance genes into transgenic animals may cause many problems such as disturbing the expression of neighboring genes [22], confounding the evaluation of food safety of these transgenic animals, and increasing worldwide public concern regarding the release of such antibiotics resistance genes into the environment. Recent studies have shown that selective marker genes can be successfully knocked out from transgenic cells using the Cre/loxP system [23 26]. However, because of the reversibility of Cre recombinase, labor-intensive procedures must be performed to identify complete excision events among randomly picked colonies. Although a lot of effort is being invested into solving this issue, an efficient and reliable method has yet to be developed. Here, we demonstrate an efficient and safe approach to produce transgenic cattle, which consists of single-copy integration of human b-defensin-3 (HBD3) gene into a genomic safe harbor and visual removal of the antibiotic-resistance marker. In addition, this procedure can be prospectively applied for breeding other antibiotic marker-free disease-resistant transgenic animals as well as production of human recombinant pharmaceuticals in transgenic cattle. Generation of Transgenic Cells using phiC31 Integrase mRNA Delivery of the human b-defensin-3 transgene into bovine fetal fibroblasts was performed by co-electroporation of phiC31 integrase mRNA produced by in vitro transcription, and a transgenic plasmid, pARNG-HBD3. The pARNG-HBD3 plasmid was an attB-containing human b-defensin-3 mammary gland expression vector (Fig. 1). A fluorescence double reporter was constructed to monitor Cre-mediated recombination in living cells before and after Cre recombination by expression of two different fluorescent proteins. The fluorescence double reporter construct contained the ubiquitous active CMV IE promoter that drove (...truncated)