title: A possible strategy to produce pigs resistant to porcine reproductive and respiratory syndrome virus.
abstract: The purpose of this study was to enhance the production of transgenic cloned embryos with porcine reproductive and respiratory syndrome virus (PRRSV) shRNA expression cassettes. To construct transgenic vector with expression targeting against PRRSV, PRRSV shRNA expression cassettes were inserted into pEGFP-N1 and the ability of resulting recombinant plasmid pEGFP-G1 inhibiting virus replication was examined in Marc-145 cells. Results showed that PRRSV replication could be significantly inhibited by pEGFP-G1 in Marc-145 cells compared with the control. The pEGFP-G1 plasmid was used to deliver a transgene expressing EGFP and the PRRSV shRNA into porcine fetal fibroblasts (PFF). Fluorescent-positive cells were used as nuclear donors for somatic cell nuclear transfer (SCNT) to produce shRNA-EGFP transgenic cloned embryos. The effects of trichostatin A (TSA) on production of transgenic cloned embryos were investigated. Reconstructed embryos were designed into 4 groups: Donor cells of Group A were treated with 50nM TSA for 24h before SCNT. Reconstructed embryos of Group B were treated with 50nM TSA for 24h after activation. Both donor cells and reconstructed embryos in Group C were treated with TSA and Group D were the control without TSA treatment. The results showed no difference (p>0.05) in cleavage rates among the 4 groups; however, blastocyst developmental rates of Group B and C (30.9% and 42.0%, respectively) were higher than for Group A and D (21.2% and 22.1%, respectively) with Group C highest among groups (p<0.05). Interestingly, EGFP expression intensity of transgenic cloned blastocysts of Group A was the highest. Our results provide promising evidence toward a new approach for production of transgenic cloned pigs with resistance to PRRSV and possibly a wide variety of other porcine diseases.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu 210095, China.
authors.forename: Biping
authors.initials: B
authors.lastname: Luo
authors.forename: Shiqiang
authors.initials: S
authors.lastname: Ju
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Rong
authors.initials: R
authors.lastname: Rui
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D002522:Chlorocebus aethiops; D019976:Cloning, Organism; D004622:Embryo, Mammalian; D049452:Green Fluorescent Proteins / Q000235:genetics; D006877:Hydroxamic Acids / Q000494:pharmacology; D053652:Nuclear Transfer Techniques; D019318:Porcine Reproductive and Respiratory Syndrome / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D013552:Swine; D014709:Vero Cells; D014779:Virus Replication
chemical_list: D006877:Hydroxamic Acids; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C012589:trichostatin A
keywords: Embryos; PRRSV; Pig; SCNT; TSA; shRNA