A Site-specific Recombinase-based Method To Produce Antibiotic Selectable Marker Free Transgenic Cattle

Transgenic farm animals are important materials for biomedical and agricultural research.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 butraises public concerns regarding biological safety. The aim of this study was to establish asafe and efficient method to prepare competent antibiotic selectable marker free transgeniccells for somatic cell nuclear transfer (SCNT), which may lay the foundation for breeding ofantibiotic selectable marker free transgenic animals. The main contents of this research are asfollows:1. Construction and function evaluation of general expression carrier based onpseudo attP site integrationFull-length attB and other vector components were generated using splicing by overlapextension (SOE) PCR, and then the general expression carrier pARNG was constructed.Functional evaluation was performed on attB site and fluorescent double reporter of pARNG,and the results showed that the synthetic attBwas competentin site-specific integrationmediated by phiC31integrase, and the fluorescent double reporter could be used to monitortransfected cells and Cre-mediated excision.2. Generation of transgenic bovine fetal fibroblasts using phiC31integrase mRNAPhiC31integrase (Int) mRNA and inactive mutant phiC31integrase (mtInt) mRNA weregenerated by in vitro transcription. In order to optimize phiC31mRNA mediated transfection,bovine fetal fibroblasts were electroporated with the phiC31integrase activity reporter vectorpBP-stop-eGFP in the presence of phiC31mRNA. The optimized dosage was determined as1μg by using flow cytometry analysis (FACS) and immunofluorescence assay against γ-H2AX.An attB-containing human β-defensin-3(HBD3) mammary gland expression vectorpARNG-HBD3was constructed and electroporated into bovine fetal fibroblasts with phiC31mRNA. A total of seven pseudo attP site were detected and analyzed in stably-transfected cellcolonies,28%of which were detected to integrate at BFF2hot spot. In addition, all published pseudo attP sites in bovine genome were re-analyzed according to the criteria of “genomicsafe harbor”. Finally, transgenic cell colonies with single-copy integration ofHBD3gene intoBFF2genomic safe harbor were obtained.3. Excision of the antibiotic selectable marker from transgenic cells usingcell-permeant Cre recombinaseProkaryotic expression and purification of His-NLS-TAT-Cre recombinant protein wasperformed. The Cre recombinase activity reporter fetal fibroblast cell line BFF2-L2GFP wasgenerated to optimize conditions of His-NLS-TAT-Cre mediated protein transduction.Immunofluorescence results showed that His-NLS-TAT-Cre was mainly localized in nucleolus.Excision of antibiotic selectable marker was verified by Southern blot in the EGFP positivecells sorted by FACS after His-NLS-TAT-Cre protein transduction on pARNG-HBD3stably-transfected cell colonies. RT-PCR results showed that phiC31mRNA mediatedinsertion and His-NLS-TAT-Cre protein mediated excision did not alter the expression offlanking genes. In addition, transgenic cells generated by this procedure had the normalchromosome count (2n=58+XX).4. Generation of cloned transgenic cattle that express human β-defensin-3Antibiotic selectable marker free cells from different clones were used as nuclei donorsfor production of transgenic cattle. Finally, twelve transgenic cattle were obtained. Byevaluation of transgenic milk samples from five transgenic cows, which were lactatingnormally in the observation period, the expression of HBD3was detected, ranged from3.9to10.4μg/mL. The transgenic cows’ ability to resist infection by S. aureus and E. coli wasconfirmed by intramammary infusion of viable bacterial cultures. Notably, the higherexpressing transgenic cows, which derived from the evaluated BFF2safe harbor integratedtransgenic cells, never became infected after S. aureus or E. coli infusion.All of above results indicated that combination of phiC31integrase mRNA,cell-permeant Cre protein and fluorescent double reporterprovided a safe and efficientalternative to prepare competent antibiotic selectable marker free transgenic cells for SCNTand sequentially produced antibiotic selectable marker free mastitis-resistant transgenic cattle.