RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells AbstractFoot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, P鈥?lt;鈥?.05). The ear fibroblasts of the transgenic lambs transfected with the PsiCheck2-VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the 鈥?i>Sleeping Beauty鈥?transposon system is an efficient method to produce transgenic animals. IntroductionFoot and mouth disease (FMD) is an acute, febrifacient and highly contagious disease to the mammals which are susceptible for them. It easily and rapidly spreads through the air and causes enormous economic losses1. The culprit responsible for this disease is the foot and mouth disease virus (FMDV), which is a picornavirus and belongs to a prototypical member of the Aphthovirus genus. The virus particle (25鈥?0鈥塶m), which has no envelope, has an icosahedral capsid, a protein containing a single strand of ribonucleic acid (RNA), which positively encodes its genome. When the virus encounters the membrane of a host cell, it binds to a receptor site and triggers an infolding of the membrane. Once the virus enters the host cell, the capsid dissolves, and the RNA is replicated and translated into viral proteins by the cell ribosomes using a cap-independent mechanism driven by the internal ribosome entry site element. The FMDV genome consists of a 5鈥?untranslated region (5鈥?UTR), an open reading frame (ORF), the 3鈥?UTR and poly-A tails2. The P1 region in the ORF encodes four structural proteins called VP1, VP2, VP3 and VP4, respectively, which form the viral capsid3. In particular, VP1 is the pivotal antigenic variation site, most of which is exposed to the virus protein surface that mediates viral infection of cells. In FMD molecular epidemiology research, the VP1 gene nucleotide sequence is often determined and analyzed4.RNA interference (RNAi) is an RNA-dependent gene-silencing process controlled by the RNA-induced silencing complex (RISC), and it is initiated by short, double-stranded RNA molecules in the cytoplasm of a cell that interact with the catalytic RISC component argonaute5. RNAi is highly effective to inhibit VP1 replication in transient assays with small interfering RNA (siRNA)6, and more durable inhibition can be achieved when an antiviral short hairpin RNA (shRNA) is expressed in stably transfected or transduced cell lines7, 8. shRNAs, different from siRNAs, are synthesized in the cell nucleus, further processed and transported to the cytoplasm, and then incorporated into the RISC, where they become active9. They can be transcribed by either RNA polymerase II or III through RNA polymerase II or III promoters on the expression cassette10. Owing to the stability of shRNA, it is increasingly being used to develop antisense therapeutics, that is post-transcriptionally knockdown gene expression11. Compared to traditional vaccines, the RNAi approach is preferred for the inhibition of FMDV infection and replication due to its direct effect on the FMDV genome. By targeting VP1 gene-conserved sequences, previous studies have designed one shRNA that can effectively control FMDV infection by inhibiting VP1 gene duplication and further silencing the expression of this protein6.The SB transposon system is a synthetic DNA transposon designed to introduce precisely defined DNA sequences into the chromosomes of vertebrates to introduce new traits as well as to discover new genes and their functions. It consists of two components: the transposon and the transposase. The transposon is the DNA surrounded by the two-terminal inverted repeat (IR)/direct repeat (DR) elements, and the transposase is the protein that facilitates transposition by binding to the DR regions within the IR/DR elements. Together, these two components act in a cut-and-paste manner to move the entire transposon from the donor plasmid or location to a thymine-adenine (TA) dinucleotide within the recipient DNA fragment12. Previous reports have described an integrated transposon system in which the transposon and transposase are built in separate expression vectors so that other DNA fragments can translocate to the primary transposase sites13. With this system, SB transposons can carry any foreign DNA fragments and integrate them into animal genomes to achieve stable transposon-mediated insertional mutagenesis.In 2015, we successfully bred sixty-one goats, of which seven individuals positively integrated 3D-7414shRNA targeting the 3Dpol gene (which encodes the viral RNA polymerase, a vital element for FMDV replication) in the FMDV genome14. However, few studies have integrated the SB transposon system with RNAi technology in farm animals, so this study investigated the prospect of producing anti-FMD sheep by utilizing VP1-shRNA coupled with the SB transposon system.ResultsVP1-shRNA inhibits the expression of FMDV-VP1The FMDV-VP1 sequences were analyzed, and the shRNA sequences were screened and synthesized. After annealing, the sense and antisense strands were cloned into the pLL3.7 vector (Fig.聽1A), The VP1 gene was obtained by overlapping PCR (Fig.聽1B) and cloned into vector psiCheck2, resulting in a new vector psiCheck2-VP1, which was co-transfected with pLL3.7-NonshRNA or pLL3.7-VP1-shRNA, respectively (4:1), into 293FT. The Dual-Glo Luciferase Assay System was used to explore the activities of firefly luciferase and renilla luciferase. The formula [(renilla/firefly luciferase activity)empty group 鈭?(renilla/firefly luciferase activity)shRNA group]/(renilla/firefly luciferase activity)empty group accounts for the inhibition rate. The results indicated that, the inhibition efficiency of VP1-shRNA was 75.22% higher than that non-shRNA (P鈥?lt;鈥?.05) (Fig.聽1C).Figure 1Inhibitory effects of VP1-shRNA on FMDV-VP1. (A) Schematic map of the pLL3.7-VP1-shRNA expression vector. (B) The VP1 gene amplification using overlapping PCR; 1: a-h, 2: a-g, 3: a-f, 4: a-e, 5: a-d, 6: a-c, 7: a-b, and M: marker. (C) Use of Dual-Glo luciferase to detect the inhibitory effect of targeted genes. 239FT cells were co-transfected with pll3.7-shRNA and psiCheck2 genes with different ratios, and the expression of Dual-Glo luciferase reporter genes was measured after 48鈥塰. Data were expressed as the means鈥壜扁€塖.E.M. (n鈥?鈥?). Columns with different superscripts differ significantly, P鈥?lt;鈥?.05.Full size image Production of transgenic sheep through the SB transposon systemThe SB transposon expression vector pUC-VP1-shRNA was constructed (Fig.聽2A) and was successfully integrated into eight transgenic sheep (Fig.聽2F) by pronuclear microinjection (Fig.聽2B). Southern blot analysis was used to detect the positive transgenic lambs. By analyzing the developed blots to calculate the transgene copy numbers, in which the individuals were identified to carry various copy numbers of the exogenous gene. In the U6-VP1-shRNA group, 160 fertilized eggs were transplanted into 43 recipients and 28 lambs were obtained. However, PCR and Southern blot analysis showed that only one of them was transgenic positive (Figs.聽2C-a-1,D-a-1). In the linearized pLL3.7-VP1-shRNA group, 63 fertilized eggs were transplanted into 14 recipients and 14 lambs were obtained and only one of them was transgenic positive (Fig.聽2C-a-,D-a-2). In the linearized IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roup, 153 fertilized eggs were transplanted into 40 recipients and 23 lambs were obtained and 3 of them were transgenic positive (Figs聽2C-b-3,4,5;D-b-3,4,5). The numbers of exogenous gene copies were calculated, which were 4.38, 2.13, 1.21, 1.19 and 1.82, respectively (Fig.聽2D:1鈥?). The efficiency of producing transgenic sheep with the SB transposon system was 13.04%, which is higher than that of the U6-VP1-shRNA group and the linearized pLL3.7-VP1-shRNA group (P鈥?lt;鈥?.05) (Table聽1).Figure 2Identification of SB transposon-mediated transgenic sheep. (A) Schematic diagram of the inserted part of the pUC-VP1-shRNA expression vector, which was cut from pLL3.7 and ligated into the PUC19-IR/DR vector. (B) Pronuclear microinjection. (a): non-centrifuged fertilized ovine egg and (b): centrifuged fertilized egg (12000鈥壝椻€塯 for 5鈥塵in). (C) Electrophoresis of PCR products and (D) Southern blot analysis to identify the transgenic sheep, respectively. a: U6-VP1-shRNA group and linearized pLL3.7-VP1-shRNA plasmid group. b: IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roup. WT鈥?鈥墂ild type as a negative control; 鈥減鈥? positive control; 鈥?鈥?鈥? transgenic lambs. (E) Inhibitory effects of shRNA on VP1 gene expression in ear fibroblasts of transgenic versus wild type sheep as determined by luciferase reporter assay. Each test was repeated three times for each individual. Tg (鈥?鈥塼ransgenic sheep), N鈥?鈥?; WT (wild type), N鈥?鈥?. (F) A photo of the transgenic lamb. Data were expressed as the means鈥壜扁€塖.E.M. Columns with different superscripts differed significantly, P鈥?lt;鈥?.05.Full size image Figure 3Identification of the SB transposase gene. (A) A diagraph of construction of the SB transposase gene (B) The PCR result of SB transposase gene SB100鈥壝椻€塨ased on the template pCMVSB100 vector. 1: Marker, 2: SB100鈥壝椻€塮ragment.Full size image Table 1 Efficiency of transgenic sheep production by pronuclear microinjection.Full size table In addition, the pronuclei, in IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roup, were better visualized than that in other groups.,In the current study, some fertilized eggs of this group were centrifuged. It was found that after centrifugation, the lipid droplets in the cytoplasm were gathered to one side (Fig.聽2B-b). In the centrifuged group, 132 fertilized eggs were transplanted into 33 recipients and 14 lambs were obtained. PCR and Southern blot analysis showed that one of them was transgenic positive (Fig.聽2C-b-6,D-b-6); the positive rate of the centrifuged group was 7.14%. However, in the un-centrifuged group, 63 fertilized eggs were transplanted into 21 recipient and 13 lambs were obtained, two of them were transgenic positive (Fig.聽2C-b-7,8, D-b-7,8), the positive rate of the un-centrifuged group was 15.38%, higher than that in centrifuged group (P鈥?lt;鈥?.05) (Table聽2). The numbers of exogenous gene copies were calculated, which were 1.79, 1.12 and 1.92, respectively (Fig.聽2D:6鈥?).Table 2 Efficiency of transgenic sheep production by centrifugation relative to the control.Full size table In the IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roup, to study the effect of different ratio of transposon and transposase on the transgenic efficiency, variable concentrations of transposon and transposase were microinjected into ovine zygotes. It was found when the ratio of transposon to transposase was 1:5, the pregnancy rate was 42.42%, and the PCR positive rate of the progeny was 19.05%. However, when this ratio was 5:1, the pregnancy rate was 26.14%, and the PCR positive rate of the progeny was 35.48%. When the ratio was 10:1, the pregnancy rate was 30.33%, and the PCR positive rate of the progeny was 33.33% (Table聽3).Table 3 Effects of different transposon and transposase ratios on the transgenic efficiency of ovine pronuclear microinjection.Full size table Fibroblasts of transgenic sheep ear tissue inhibit VP1 gene expressionTo compare the inhibitory effects of transgenic animals on VIP gene expression with non-transgenic ones (each group has 8 lambs), we transfected the psiCheck2-VP1 into the ear fibroblast cells derived from both group. The inhibition rate was determined by the activities of firefly luciferase and renilla luciferase, which were measured through the Dual-Glo Luciferase Assay System. The results showed that the cells from transgenic animals significantly inhibited the expression of VP1 gene compared with the wild type (P鈥?lt;鈥?.05) (Fig.聽2E).DiscussionFMDV causes an acute vesicular disease in livestock, and vaccination is currently the main preventive method15, 16. To a certain extent, vaccinated animals are resistant to FMDV, but there are many drawbacks, such as the short-lived effectiveness of the resulting antibodies17. In contrast, RNAi is an ideal method for inhibiting FMDV infection and replication because it can directly affect the FMDV genome18, 19. The FMDV genome encodes many genes, including 2B, 3Dpol, VP1, VP2, VP3, VP4, VPg, etc. These genes have been selected as RNAi targets20,21,22,23. VP1 is essential during the life cycle of the virus and plays a key role in its attachment to susceptible cells24, 25. In this study, we screened and constructed the valid shRNA recombinant vectors (Fig.聽1A) targeting the conserved region of VP1 to inhibit its replication and further silence the expression of FMDV. The anti-FMDV shRNA proved to be effective with an inhibition efficiency of 75.22% in 293FT cells compared with Non-shRNA cells (P鈥?lt;鈥?.05) (Fig.聽1C). Furthermore, ear fibroblast cells from the positively integrated VP1-shRNA sheep also significantly inhibited the expression of VP1 gene compared with wild type animals (P鈥?lt;鈥?.05) (Fig.聽2E). In transgenic bovines, it has been reported that the primary epithelium cells with shRNA expressing RNAi-LT4 (VP4) or RNAi-LT6 (VP1) showed greater resistance to FMDV than those in non-transgenic cells20, 26, which is similar to our findings.There are currently many methods for producing transgenic animals, such as pronuclear microinjection, retrovirus infection, somatic cell cloning, etc.; however, these transgenic technologies are still to be improved to increase their efficiency. SB was the first transposon shown to be capable of efficient transposition in vertebrate cells, thus enabling new avenues for genetic engineering in animal models27 that can mediate the stable integration and long-term expression of foreign genes28. SB transposon vectors have been shown to efficiently deliver a wide variety of transgene cassettes29, including shRNA expression cassettes to obtain stable RNAi knockdown cell lines as well as cassettes inducing gain-of-function and loss-of-function gene mutations30. The integrated SB transposon has been shown to be stably transmitted to offspring through germ cells and maintained for several generations in all species tested31. Compared with the viral vector, the SB transposon system has many advantages, such as a simple structure, a less-restricted inserted fragment length, controllable exogenous gene expression and higher transgenic efficiency. Combining RNAi-mediated gene silencing and SB transposition has been used to down-regulate gene expression, and examples include SB-mediated RNAi targeting the lamin A gene32, the multidrug-resistance gene MDR-1 33, the HIV-1 co-receptor genes CCR5 and CXCR4 34, and the huntingtin gene Htt 35, etc. However, previous studies have mainly focused on the cellular level rather than the individual level. Here, we developed a transposon-based vector system that allows the stable expression of shRNA through the microinjection of pronuclear embryos to produce genetically modified ovine species as well as to achieve disease resistance through animal breeding. Thus, a stable knockdown expression system, such as that described here, can overcome the obstacle of transient knockdown of durable proteins by enabling long-lasting production of siRNA oligonucleotides. Our results indicate that the non-viral plasmid-based approach to VP1-shRNA cassette delivery is more efficient with the SB transposon system than without it as to generate transgenic animals, The transgenic positive offspring with SB system was 13.04% (3/23) compared to the others without SB system 3.57% (1/28) and 7.14% (1/14), respectively, P鈥?lt;鈥?.05 (Table聽1). The transgenic positive rate was also higher than that previously reported36.The efficiency of the SB transposon system is affected by many factors including species, cell lines, transposon length, copy number, lateral sequences, transposase, transposon to transposase ratio, etc.37. Zsuzsanna Izsv谩k et al. transfected the SB transposon system into the A6 cell line of Xenopus followed by screening with neomycin and found the efficiency to be only 0.03%, but an 8% transposition rate was reported in the hamster K1 cell38. The authors also showed that transposition efficiency would be reduced by 30% per additional 1 Kb SB transposons in Hela cells38. Yant et al. reported when the ratio of the transposon plasmid to the transposase was 25:1 the highest transposition efficiency was achieved during the transfection of mouse liver cells39, but Geurts et al. found that the highest transposon efficiency was when the transposon to transposase ratio was 5:1 in Hela cell transfection40. In the current study, 35.48% transposon efficiency was achieved when the transposon to transposase ratio was 5:1, which was higher than other ratios of 1:5 or 10:1 (19.05% and 33.33%, respectively, P鈥?lt;鈥?.05) (Table聽3); these results were consistent with those of Geurts et al. but differed from Yant et al. These discrepancies may be explained by species-specific differences in the integration and viability of embryos, gene constructs and microinjection facilities.Pronuclear embryo microinjection technology involves the injection of exogenous genes into the pronuclei of fertilized eggs using a micromanipulator. The pronuclei of late zygotes are located close to one another, usually in the center of the cytoplasm although they may be closer to the pole with dark granules; one of the pronuclei (male) is somewhat larger than the other (female). Pronuclei in sheep, unlike those in mouse, rabbit, or swine, are not visualized, and they morphologically resemble goat and cow pronuclei41. The two pronuclei may not always be simultaneously visible, and one can be located in the lipid granules. Nontransparent cytoplasm is due to the presence of a large amount of lipid granules, which hinders pronuclei visualization and affects microinjection42. However, previous reports have mainly focused on the pronuclei of goats rather than sheep; for example, Freitas et al.42 centrifuged fertilized, one-cell goat embryos at 13400鈥塺pm for 4鈥?鈥塵in to visualize the pronuclei rather than 15600鈥壝椻€塯 for 3鈥?鈥塵in as described by Baldassarre et al.41. In this study, we conducted pronuclear microinjection by centrifuging some fertilized eggs at 12000鈥壝椻€塯 for 5鈥塵in (Fig.聽2B-b) to try to visualize the pronuclei better. In the rest of the fertilized eggs, we were unable to observe the morphology of the pronuclei in detail, thus, the microinjections were performed at the most likely location of the pronuclei. In these cases, it was difficult to control the quality of the injected eggs. For example, by visualizing whether there were significant change in pronucleus morphology due to swelling or other reasons. Even through, the proportion of clearly visible pronuclei increased with centrifugation, the efficiency of transgenic sheep production is reduced (Table聽2), presumably due to skeletal damage to the fertilized egg after centrifugation.ConclusionsIn summary, the combination of Sleeping Beauty transposon expression vector with RNAi and pronuclear microinjection technology significantly increased the genetic positive offspring in sheep. By use of this combination, 8 transgenic sheep expressing VP1-shRNA targeting the VP1 gene in the FMDV genome were generated out of 92 individuals. In addition, we proved that the ear fibroblasts from transgenic lambs could significantly inhibit VP1 replication compared to the cells from the wild type. Moreover, it was found that SB transposon-mediated shRNA had an increased transgenic integration rate over random integration of anti-VP1 shRNA. The DNA-mediated transposition may suggest new ways to further improve this novel integration system.Materials and MethodsChemicalsUnless specified, reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).Animal studies and ethics statementAll transgenic sheep production and sample collection procedures were strictly followed the protocols approved by the Animal Welfare Committee of China Agricultural University (Permit Number: XK662), and this study was carried out in strict accordance with the guidelines and regulations established by this committee. The experimental ewes were aged 1 to 4 years old and were free of reproductive diseases. They were sufficiently fed to achieve good condition at least one month prior to the experiment. The experimental rams weighed 75~85鈥塳g and had a good sex drive and high-quality semen.Vector constructionTarget screening and construction of shRNA recombinant vectors were performed as follows. One candidate shRNA, VP1-shRNA, targeting FMDV (GenBank accession: AY686687.1) was designed with BLOCK-iTRNAi Designer (http://rnaidesigner.lifetechnologies.com/rnaiexpress), and the sequences were as follows: VP1-shRNAsen (5鈥?TGGAGTCTGCGGACCCCGTGACT- TTCAAGAGAAGTCACGGGGTCCGCAGACTCCTTTTTTC-3鈥?, VP1-shRNAanti (5鈥?TCGAG- AAAAAAGGAGTCTGCGGACCCCGTGACTTCTCTTGAAAGTCACGGGGTCCGCAGACT-CCA-3鈥?, Non-shRNA-sense (5鈥?TGGCAGTACGGAACTCCGCTAGGTTCAAGAGACCTAG -CGGAGTTCCGTACTGCCTTTTTTC-3鈥?, and Non-shRNA-anti (5鈥?TCGAGAAAAAAGGCA- GTACGGAACTCCGCTAGGTCTCTTGAACCTAGCGGAGTTCCGTACTGCCA-3鈥?. The sequences were synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (Sangon, China). The two shRNAs were cloned into the pLL3.7 expression vector (Addgene, USA) to obtain pLL3.7-VP1-shRNA and pLL3.7-NonshRNA, which were located after promoter U6, and HpaI and XbaI were the restriction enzyme cutting sites. The construction of the shRNA vector in the SB transposon system was as follows. The IR/DR sequences of the pT2 vector (Sino-US Ltd.) were inserted into the Sma I sites of the pUC19 vector (Addgene, USA), namely, PUC19-IR/DR. The U6-VP1-shRNA, which was cut by Not I and XbaI in pLL3.7, was ligated into the IR/DR of the PUC19-IR/DR vector, resulting in a new vector named pUC-VP1-shRNA. The SB transposase gene SB100鈥壝椻€?Forward primer 5鈥?ATGGGAAAA- TCAAAAGAAATCAGC-3鈥?and Reverse primer 5鈥?CTAGTATTTGGTAGCATTGCC-3鈥?with a product size of 1249鈥塨p) was amplified by PCR based on the pCMVSB100 vector template (Fig.聽3). The SB100鈥壝椻€塵RNA was obtained by in vitro transcription using mMessage mMACHINE T7 Ultra Kit (Ambion, AM1345) according to the manufacturer鈥檚 protocol.Analysis of shRNA inhibition efficiencyThe VP1 gene was chemically synthesized by designing 8 primers (Table聽4) based on the FMDV-conserved sequence and cloned into the psiCheck2 plasmid (Promega, USA) between Xho I and Not I to obtain psiCheck2-VP1. Then, psiCheck2-VP1 was co-transfected with pLL3.7-NonshRNA or pLL3.7-VP1-shRNA, respectively (4:1), into 293FT by Lipofectamine 2000 (Invitrogen, USA); the cells were transfected with pLL3.7 plasmid as an empty control. The 293FT cells were grown in Dulbecco鈥檚 modified Eagle鈥檚 medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA). All transfection experiments were performed in triplicate. The activities of firefly luciferase and renilla luciferase were detected by the Dual-Glo Luciferase Assay System (Promega, USA) at 48鈥塰 post transfection, and the inhibition rate was calculated by the formula [(renilla/firefly luciferase activity)empty group 鈭?(renilla/firefly luciferase activity)shRNA group]/(renilla/firefly luciferase activity)empty group.Table 4 The VP1 gene primers.Full size table Superovulation and artificial insemination of donor sheepThe estrous cycles of the ewes were synchronized by a 14鈥塪ay treatment using a controlled internal drug release device (CIDR; EAZI-BREED庐 CIDR庐 Sheep and Goat Device, Pfizer Animal Health, New Zealand) containing 300鈥塵g of progesterone. The CIDR was placed into the vagina of the donor sheep during their estrous cycles, and after 14 days, follicle stimulating hormone (FSH) (OVAGENTM, ICP, Auckland, New Zealand) was injected for superovulation. The donor sheep were in their estrus 24~48鈥塰 following the terminal FSH injection. Outstanding rams were selected as sperm donors. Their semen was diluted by diluent and examined for motility; sperm was only used for artificial insemination if the motility reached 0.6. Insemination was timed based on follicle development, which was determined by abdominal endoscopy (Karl Storz Endoskope GmbH, Tuttlingen, Germany); the infusion dose was 0.3鈥塵L. The CIDR鈥?鈥塅SH protocol followed that of previous studies with modifications43.Acquisition of pronuclei and gene microinjectionAll sheep were deprived of food for 12鈥塰 before embryo recovery to facilitate surgery and to reduce post-operation intestinal adhesions. The pronuclei, which were flushed from the oviduct with a flushing medium (PBS鈥?鈥?.3% BSA) (Fraktion, 735078; Roche Diagnostics, GmbH, Mannheim, Germany) by peritoneal operation following 18鈥塰 of insemination, were aspirated with a micropipette and placed in a small petri dish containing a holding medium (Immuno-Chemical Products Ltd., Auckland, New Zealand) enriched with 20% serum for use after being examined for quality on a thermal plate at 38鈥壜癈 under a stereomicroscope (SZ61; Olympus, Kawasaki, Japan). The acquired pronuclei were immediately microinjected and then cultured in vitro at 38.5鈥壜癈 in a chamber with 5% CO2 and a humidified atmosphere for 30鈥塵in. Microinjection was performed with an inverted microscope equipped with DIC and a pair of micromanipulators (Narishige, Tokyo, Japan). The DNA to be injected was divided into 3 groups: the U6-VP1-shRNA group (cut from the pLL3.7-VP1-shRNA plasmid), the linearized pLL3.7-VP1-shRNA plasmid group (linearization by AgeI digestion), and the IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roup (IR/DR-U6-VP1-shRNA cut from pUC19-VP1-shRNA and mixed with SB100鈥壝椻€塵RNA at a 5:1 ratio). To facilitate clearness of the pronuclei and promote precipitation of the lipid granules, the centrifuged group (12000鈥壝椻€塯 for 5鈥塵in)44 and un-centrifuged control groups were subset among the IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roups. The final concentration and dose of the DNA or mRNA were 5鈥塶g/渭L and 5 pL, respectively. The gene was injected into each embryo as described previously45.Recipient preparation and embryo transferThe FSH鈥?鈥塁IDR donor synchronization method was used; while injecting prostaglandin (PG) (Sansheng Pharmaceuticals, Ningbo, China) 280 IU, the CIDR was removed half a day before the donor. Twelve hours after injection, the ram test was conducted 3 times a day to confirm that the recipient ewe was in estrus; the surgical site and preparation methods for the recipient were consistent with those of the donors. Whether the corpus luteum of the recipient could be transplanted was determined by peritoneal operation through a 2~3鈥塩m cavity in the abdomen. Approximately 2 to 6 embryos were transplanted into the uterine horn per recipient as previously described46, and early pregnancy was diagnosed by an ultrasonic instrument (Vetko Plus, Noveko, Quebec, Canada) 50 days later.Identification of the DNA level in transgenic sheepThe genomic DNA of the lambs was extracted and analyzed by PCR and Southern blotting as described by Annemarie H眉bers et al.47. The presence of the VP1-shRNA gene was confirmed by PCR amplification with forward primer 5鈥?ATTACCGGTAGATCCAGTTTGGTTAGTA -CCGGG-3鈥?and reverse primer 5鈥?GCTTGGAACCCTTAATATAACTTC-3鈥? and PCR amplification was used to generate a specific digoxigenin-labeled probe (Roche Diagnostics, Mannheim, Germany). The PCR cycles were as follows: 94鈥壜癈 for 5鈥塵in; 32 cycles at 94鈥壜癈 for 30鈥塻, 52鈥壜癈 for 30鈥塻, and 72鈥壜癈 for 30s; and 72鈥壜癈 for 10鈥塵in. The PCR system (10鈥壩糒) contained 5鈥壩糒 of 2鈥壝椻€塃S Taq MasterMix, 1鈥壩糒 of DNA, 3鈥壩糒 of ddH2O, and 0.25鈥壩糓 forward primer and reverse primer, respectively. The PCR products (5鈥壩糒) were electrophoresed in a 1% agarose gel. In the IR/DR-U6-VP1-shRNA鈥?鈥?i>SB100鈥壝椻€塯roup, the probes were prepared by PCR amplification using the primers (Forward primer 5鈥?ATCCGACGCCGCCATCTC-3鈥? Reverse primer 5鈥?ACAATAGTTTTGGCAAGTCAGTT-3鈥? with the DIG-High-labeled substrate and cut by XbaI and HpaI. In the U6-VP1-shRNA and the linearized pLL3.7-VP1-shRNA plasmid groups, the primers and cutting sites for the labeled probe were Forward primer 5鈥?AAACAGCACAA -AAGGAAACTCACCC-3鈥? Reverse primer 5鈥?GATACCGTCGACCTCGAGAAAG-3 鈥? HpaI and XhoI. The sheep genome DNA and the plasmid were digested with the restriction enzyme followed by electrophoresis, transfer blotting, pre-hybridization and hybridization with the labeled probe, and phosphorimager exposure. By analyzing the developed blots of Southern blotting to calculate the transgene copy numbers.Detection of inhibition efficiency in ear fibroblast cells from transgenic sheep against the VP1 geneFibroblast cells of eight positively VP1-shRNA integrated sheep and eight wild type sheep were harvested at the logarithmic growth phase and cultured in Dulbecco鈥檚 modified Eagle鈥檚 medium-F12 (DMEM-F12; Invitrogen, USA) supplemented with 10% fetal bovine serum. 2鈥壝椻€?05 cells per well were seeded in a 12-well plate and cultured in fresh medium without antibiotics to achieve 80鈥?0% confluency on the day of transfection, and psiCheck2-VP1 was then transfected into the fibroblast cells by Lipofectamine 2000. All transfection experiments were performed in triplicate. 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Neurobiology of Aging 35, 1214.e1鈥?214.e6 (2014).Article聽Google Scholar聽 Download referencesAcknowledgementsThis study was supported by the National Transgenic Creature Breeding Grand Project (2010ZX08008-002B; 2014ZX08008-002B; 2016ZX08008003).Author informationAuthor notesShoulong Deng and Guangdong Li contributed equally to this work.AffiliationsBeijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, ChinaShoulong Deng,聽Guangdong Li,聽Kun Yu,聽Xiuzhi Tian,聽Feng Wang,聽Wenting Li,聽Wuqi Jiang,聽Pengyun Ji,聽Hongbing Han,聽Juncai Fu,聽Zhengxing Lian聽 聽Guoshi LiuState Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, ChinaShoulong Deng聽 聽Yixun LiuTianjin Institute of Animal Sciences, Tianjin, ChinaXiaosheng Zhang聽 聽Jinlong ZhangAuthorsShoulong DengView author publicationsYou can also search for this author in PubMed聽Google ScholarGuangdong LiView author publicationsYou can also search for this author in PubMed聽Google ScholarKun YuView author publicationsYou can also search for this author in PubMed聽Google ScholarXiuzhi TianView author publicationsYou can also search for this author in PubMed聽Google ScholarFeng WangView author publicationsYou can also search for this author in PubMed聽Google ScholarWenting LiView author publicationsYou can also search for this author in PubMed聽Google ScholarWuqi JiangView author publicationsYou can also search for this author in PubMed聽Google ScholarPengyun JiView author publicationsYou can also search for this author in PubMed聽Google ScholarHongbing HanView author publicationsYou can also search for this author in PubMed聽Google ScholarJuncai FuView author publicationsYou can also search for this author in PubMed聽Google ScholarXiaosheng ZhangView author publicationsYou can also search for this author in PubMed聽Google ScholarJinlong ZhangView author publicationsYou can also search for this author in PubMed聽Google ScholarYixun LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarZhengxing LianView author publicationsYou can also search for this author in PubMed聽Google ScholarGuoshi LiuView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsShoulong Deng conceived and designed the experiments. Kun Yu, Xiuzhi Tian, Feng Wang, Wuqi Jiang, Pengyun Ji, Wenting Li, Hongbing Han, Juncai Fu, Xiaosheng Zhang, Jinlong Zhang and Yixun Liu performed the experiments, and Guangdong Li analyzed the data, wrote the main text of the manuscript and prepared the figures. All experiments were directed by Guoshi Liu and Zhengxing Lian. All authors reviewed the manuscript.Corresponding authorsCorrespondence to Zhengxing Lian or Guoshi Liu.Ethics declarations Competing Interests The authors declare that they have no competing interests. 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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and PermissionsAbout this articleCite this articleDeng, S., Li, G., Yu, K. et al. RNAi combining Sleeping Beauty transposon system inhibits ex vivo expression of foot-and-mouth disease virus VP1 in transgenic sheep cells. Sci Rep 7, 10065 (2017). https://doi.org/10.1038/s41598-017-09302-1Download citationReceived: 10 April 2017Accepted: 25 July 2017Published: 30 August 2017DOI: https://doi.org/10.1038/s41598-017-09302-1 Soo-Young Yum, Song-Jeon Lee, Sin-Gi Park, In-Gang Shin, Sang-Eun Hahn, Woo-Jae Choi, Hee-Soo Kim, Hyeong-Jong Kim, Seong-Hun Bae, Je-Hyeong Lee, Joo-Yeong Moon, Woo-Sung Lee, Ji-Hyun Lee, Choong-Il Lee, Seong-Jin Kim Goo Jang BMC Genomics (2018) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. 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