Construction of Ipr1 expression vector and development of cloned embryos in vitro

SummaryThe purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP–EGFP–Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.

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In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

Reproduction Fertility and Development ◽

10.1071/rd00021 ◽

2000 ◽

Vol 12 (2) ◽

pp. 1 ◽

Cited By ~ 49

Author(s):

Sangho Roh ◽

Hosup Shim ◽

Woo-suk Hwang ◽

Jong-taek Yoon

Keyword(s):

Green Fluorescent Protein ◽

Nuclear Transfer ◽

Fluorescent Protein ◽

Blastocyst Formation ◽

In Vitro Development ◽

Donor Cells ◽

Fetal Fibroblasts ◽

Green Fluorescent ◽

Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

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Directed Fiber Outgrowth from Transplanted Embryonic Cortex-Derived Neurospheres in the Adult Mouse Brain

Neural Plasticity ◽

10.1155/2009/852492 ◽

2009 ◽

Vol 2009 ◽

pp. 1-7

Author(s):

Vesna Radojevic ◽

Josef P. Kapfhammer

Keyword(s):

Neural Progenitor Cells ◽

Fluorescent Protein ◽

Neuronal Progenitor ◽

Axonal Projections ◽

Axonal Connections ◽

Donor Cells ◽

The Central Nervous System ◽

Green Fluorescent ◽

Host Brain

Neural transplantation has emerged as an attractive strategy for the replacement of neurons that have been lost in the central nervous system. Multipotent neural progenitor cells are potentially useful as donor cells to repopulate the degenerated regions. One important aspect of a transplantation strategy is whether transplanted cells are capable of fiber outgrowth with the aim of rebuilding axonal connections within the host brain. To address this issue, we expanded neuronal progenitor from the cortex of embryonic day 15 ubiquitously green fluorescent protein-expressing transgenic mice as neurospheres in vitro and grafted them into the entorhinal cortex of 8-week-old mice immediately after a perforant pathway lesion. After transplantation into a host brain with a lesion of the entorhino-hippocampal projection, the neurosphere-derived cells extended long fiber projections directed towards the dentate gyrus. Our results indicate that transplantation of neurosphere-derived cells might be a promising strategy to replace lost or damaged axonal projections.

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185 CELLULAR VIABILITY AND EXPRESSION OF GREEN FLUORESCENT PROTEIN IN BOVINE FETAL FIBROBLASTS FOLLOWING TRANSFECTION OF SYNTHETIC mRNA INCLUDING MODIFIED BASES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab185 ◽

2014 ◽

Vol 26 (1) ◽

pp. 207

Author(s):

T. L. Adams ◽

S. E. Farmer ◽

J. A. Sarmiento-Guzmán ◽

K. R. Bondioli

Keyword(s):

Fluorescent Protein ◽

In Vitro Transcription ◽

Cellular Reprogramming ◽

Cellular Viability ◽

Treatment Groups ◽

Fetal Fibroblasts ◽

Green Fluorescent ◽

Synthetic Mrna ◽

Number Of Cells

Synthetic RNA transfection has been an invaluable tool in understanding the mammalian genome because of its ability to deliver exogenous protein without mutagenic effects caused by double-stranded DNA. A common problem associated with the introduction of exogenous mRNA into mammalian cells is the stimulated interferon response. This innate immune response can be avoided with the addition of modified bases during the in vitro transcription process of synthetically derived mRNA. The bases cytidine triphosphate (CTP) and uridine triphosphate (UTP) are replaced with 5-methylcytidine-5′-triphosphate (5-Methyl-CTP) and pseudouridine-5′-triphosphate (Pseudo-UTP) during in vitro transcription. Cellular reprogramming is achieved by the delivery of this mRNA into the cytoplasm. Previous cellular reprogramming experiments lacking modified bases resulted in increased toxicity and a decrease in cellular viability, which lead to the incorporation of modified bases. In the first experiment, bovine fetal fibroblasts were transfected with modified synthetic mRNA encoding green fluorescent protein (GFP) to evaluate the effects on cellular viability and fluorescence. The cellular viability was measured by counting a final number of cells after seeding a constant number of cells in all treatment groups. The control group consisted of bovine fetal fibroblasts cultured in normal growth medium. A no-RNA (NR) group was held under the same conditions with the addition of the transfection reagent, Lipofectamine (Invitrogen, Carlsbad, CA, USA), to account for toxicity resulting from the transfection reagent alone. The cells were transfected every other day for 12 days and were evaluated on days 3, 6, 9, and 12 for viability and fluorescence by flow cytometry. There was no difference in viability of all cells treated with synthetic mRNA encoding GFP when compared to controls (P = 0.9). There was a significant difference in fluorescence on all time points when compared to controls (Day 3, P = 0.004; Day 6, P = 0.004; Day 9, P = 0.007; Day 12, P = 0.04). The second experiment consisted of bovine fetal fibroblasts transfected with modified synthetic mRNA encoding pluripotency factors. The controls were identical to the previous experiment, but treatment groups were transfected with modified synthetic mRNA encoding either three factors (3F: OCT4, SOX2, KLF4) or four factors (4F: OCT4, SOX2, KLF4, c-MYC). The treated cells were transfected every other day and evaluated on Day 24 for cellular viability. There was no difference in cellular viability in all treatment groups when compared to controls (P = 0.2). The introduction of synthetic mRNA containing modified bases maintains cellular viability when compared to controls. The decreased immune response by the inclusion of modified bases may be advantageous in a variety of applications from the introduction of transcription activator-like effector nuclease (TALEN) or zinc finger nucleases for genomic editing to increased efficiency of the development of induced pluripotent stem cells.

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330 TRICHOSTATIN A IMPROVES THE IN VITRO DEVELOPMENT OF CLONED BOVINE EMBRYOS RECONSTRUCTED WITH TRANSGENIC DONOR CELLS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab330 ◽

2013 ◽

Vol 25 (1) ◽

pp. 313

Author(s):

L. S. A. Camargo ◽

R. J. Otero Arroyo ◽

T. D. Araujo ◽

G. N. Quinelato ◽

C. R. C. Quintao ◽

...

Keyword(s):

Embryo Development ◽

Fluorescent Protein ◽

Trichostatin A ◽

Cell Lineage ◽

Blastocyst Stage ◽

Bovine Embryos ◽

Donor Cells ◽

Transgenic Cells ◽

Cells Cultured

Trichostatin A (TSA), a histone deacetylase inhibitor, has been described as a potential modulator of nuclear reprogramming in bovine zygotes reconstructed by somatic cell nuclear transfer (SCNT), but with controversial results (Lee et al. 2011 J. Reprod. Dev. 57, 34–42; Sangalli et al. 2012 Cell Reprogramming 14, 1–13). The effect of TSA in zygotes reconstructed with transgenic cells cultured for long periods is not known. This study aimed to evaluate the effect of TSA on development of bovine embryos reconstructed with donor cells transfected with a green fluorescent protein (GFP)-reporter transgene. Bovine fibroblasts at second passage were transfected with lentiviral vectors carrying the GFP transgene and cultured at 37.5°C under 5% CO2 in air. Transfected cells were cultured for additional 10 passages to establish a cell lineage expressing the protein. In the 12th passage, the cells were frozen in 10% dimethyl sulfoxide plus FCS (Nutricell, Campinas, Brazil) and frozen–thawed cells expressing GFP were used as nucleus donors. In vitro-matured oocytes were enucleated, fused to GFP positive fibroblasts, and activated with ionomycin. Putative zygotes were randomly distributed into 2 groups: SCNT-CONT (n = 55): zygotes were cultured for 4 h in CR2aa medium plus BSA with 6-DMAP followed by 7 h in CR2aa medium plus 2.5% FCS; SCNT-TSA (n = 49): zygotes were cultured in the same conditions described above, but supplemented with 50 nM TSA (Sigma-Aldrich, St Louis, MO). Then, embryos from all groups were cultured in CR2aa supplemented with 2.5% FCS under 5% CO2, 5% O2, and 90% N2 at 38.5°C. Evaluations of cleavage and blastocyst percentages were performed at 72 and 168 h post-activation, respectively, and 4 replicates were carried out. Expression of GFP in embryos at blastocyst stage was visualised using an epifluorescence microscope. Statistical analysis was performed by ANOVA and data are shown as mean ± SEM. No difference (P > 0.05) on cleavage percentage was found between groups (72.9 ± 11.3% and 66.1 ± 14.4% for SCNT-CONT and SCNT-TSA, respectively). The blastocyst percentage calculated based on putative zygotes tended (P = 0.077) to be higher for SCNT-TSA (16.7 ± 4.0%) than for SCNT-CONT (6.8 ± 2.3%). When the blastocyst percentage was calculated based on cleaved embryos, a higher rate (P < 0.05) was achieved in SCNT-TSA (26.7 ± 3.8%) than in SCNT-CONT (10.3 ± 3.6%) group. Blastocysts of both groups expressed GFP, with no difference among embryos. In a previous study, we reported that TSA had no positive effect on in vitro embryo development or gene expression, despite the reduction on apoptosis index [Camargo et al. 2011 Acta Sci. Vet. 39(Suppl.), S442; Camargo et al. 2012 Reprod. Fert. Dev. 24, 121–122). In the present study, however, the treatment with TSA of zygotes reconstructed with transgenic cells cultured for a long time improved embryo development without impairing GFP expression. This result suggests that TSA may be effective in clones reconstructed with transgenic cells. Supported by Embrapa 01.07.01.002, CBAB/CNPq, CAPES and Fapemig.

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20 GENERATION AND CHARACTERIZATION OF TRANSGENIC-CLONED PIGS EXPRESSING THE FAR-RED FLUORESCENT PROTEIN MONOMERIC PLUM

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab20 ◽

2014 ◽

Vol 26 (1) ◽

pp. 124

Author(s):

M. Kobayashi ◽

M. Watanabe ◽

H. Matsunari ◽

K. Nakano ◽

T. Kanai ◽

...

Keyword(s):

Mixed Culture ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Red Fluorescent Protein ◽

Cell Stage ◽

Fetal Fibroblasts ◽

Morula Stage ◽

Clear Identification ◽

Green Fluorescent

Transgenic (Tg) pigs expressing a fluorescent protein are extremely useful for research into transplantation and regenerative medicine. This study aimed to create Tg pigs expressing monomeric Plum (mPlum), a far-red fluorescent protein with a longer wavelength than enhanced green fluorescent protein (EGFP) and humanized Kusabira Orange (huKO), the two fluorescent proteins that have been used previously for Tg pig production. A linearized CAG-mPlum transgene construct was transferred into porcine fetal fibroblasts (PFF) by electroporation. mPlum fluorescence-positive cells were collected using a cell sorter and used as nuclear donors (mPlum-PFF) for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus–oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. Then, SCNT was performed as reported previously (Matsunari et al., 2008). The reconstructed embryos were cultured for 7 days in porcine zygote medium-5 (PZM-5). mPlum fluorescence expression was screened during the early development of the embryos. After 5 or 6 days of culture, the SCNT embryos were surgically transferred to the uterus of a recipient gilt. We first obtained fetuses on Day 36 or 37 of gestation by Caesarean section and the PFF were retrieved from their skin. Fluorescence expression was analysed using fluorescence microscope, and the number of transgene copies in each fetus was determined by Southern blot analysis. We also analysed whether unique spectral properties of mPlum are suitable for multicolor imaging using confocal microscope and flow cytometer. The identification of mPlum-expressing PFF under the mixed culture of PFF expressing EGFP and huKO was examined. The 2 cell lines of PFF expressing EGFP and huKO were previously generated in our laboratory. Rates of normal cleavage and blastocyst formation occurred in the SCNT embryos generated with mPlum-PFF (mPlum embryos) were equivalent to those of SCNT embryos derived from nontransgenic PFF (34/42, 81.0%; 33/42, 78.6% v. 37/40, 92.5%; 30/40, 75.0%). Total cell numbers in mPlum and control blastocysts did not differ significantly (88.3 ± 6.0 v. 99.9 ± 8.8). Fluorescence expression in the mPlum embryos began at the 8-cell stage and became brighter from the morula stage. The gilt into which 103 mPlum embryos were transferred produced 3 fetuses. These fetuses expressed mPlum fluorescence systemically and had 1 to 5 copies of the transgene. Multicolor fluorescence imaging and flow cytometric analyses of a mixed culture of mPlum PFF and PFF expressing EGFP and huKO showed that clear identification and isolation of cells displaying each of the 3 fluorescence signals was possible. These observations demonstrate that the transfer of CAG-mPlum did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of Tg cloned pigs that systemically expressed mPlum. This work was supported by JSPS KAKENHI Grant Number 25293279.

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Production of transgenic canine embryos using interspecies somatic cell nuclear transfer

Zygote ◽

10.1017/s0967199410000651 ◽

2011 ◽

Vol 20 (1) ◽

pp. 67-72 ◽

Cited By ~ 9

Author(s):

So Gun Hong ◽

Hyun Ju Oh ◽

Jung Eun Park ◽

Min Jung Kim ◽

Geon A. Kim ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Fluorescent Protein ◽

Somatic Cells ◽

Embryonic Stem ◽

Transfected Cells ◽

Fetal Fibroblasts ◽

Transgenic Cells

SummarySomatic cell nuclear transfer (SCNT) has emerged as an important tool for producing transgenic animals and deriving transgenic embryonic stem cells. The process of SCNT involves fusion of in vitro matured oocytes with somatic cells to make embryos that are transgenic when the nuclear donor somatic cells carry ‘foreign’ DNA and are clones when all the donor cells are genetically identical. However, in canines, it is difficult to obtain enough mature oocytes for successful SCNT due to the very low efficiency of in vitro oocyte maturation in this species that hinders canine transgenic cloning. One solution is to use oocytes from a different species or even a different genus, such as bovine oocytes, that can be matured easily in vitro. Accordingly, the aim of this study was: (1) to establish a canine fetal fibroblast line transfected with the green fluorescent protein (GFP) gene; and (2) to investigate in vitro embryonic development of canine cloned embryos derived from transgenic and non-transgenic cell lines using bovine in vitro matured oocytes. Canine fetal fibroblasts were transfected with constructs containing the GFP and puromycin resistance genes using FuGENE 6®. Viability levels of these cells were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Interspecies SCNT (iSCNT) embryos from normal or transfected cells were produced and cultured in vitro. The MTT measurement of GFP-transfected fetal fibroblasts (mean OD = 0.25) was not significantly different from non-transfected fetal fibroblasts (mean OD = 0.35). There was no difference between transgenic iSCNT versus non-transgenic iSCNT embryos in terms of fusion rates (73.1% and 75.7%, respectively), cleavage rates (69.7% vs. 73.8%) and development to the 8–16-cell stage (40.1% vs. 42.7%). Embryos derived from the transfected cells completely expressed GFP at the 2-cell, 4-cell, and 8–16-cell stages without mosaicism. In summary, our results demonstrated that, following successful isolation of canine transgenic cells, iSCNT embryos developed to early pre-implantation stages in vitro, showing stable GFP expression. These canine–bovine iSCNT embryos can be used for further in vitro analysis of canine transgenic cells and will contribute to the production of various transgenic dogs for use as specific human disease models.

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Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

Biochemical Journal ◽

10.1042/bj20070770 ◽

2008 ◽

Vol 410 (2) ◽

pp. 291-299 ◽

Cited By ~ 11

Author(s):

Fui-Ching Tan ◽

Qi Cheng ◽

Kaushik Saha ◽

Ilka U. Heinemann ◽

Martina Jahn ◽

...

Keyword(s):

Expression Vector ◽

Fluorescent Protein ◽

Sequence Similarity ◽

Homology Search ◽

Protein Database ◽

Gene Encoding ◽

Reading Frame ◽

Green Fluorescent

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

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Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

Microorganisms ◽

10.3390/microorganisms9051005 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1005

Author(s):

Olga Chervyakova ◽

Elmira Tailakova ◽

Nurlan Kozhabergenov ◽

Sandugash Sadikaliyeva ◽

Kulyaisan Sultankulova ◽

...

Keyword(s):

Fluorescent Protein ◽

Viral Vector ◽

Foot And Mouth Disease ◽

Open Reading Frames ◽

Foreign Gene ◽

Mouth Disease Virus ◽

Foreign Genes ◽

Green Fluorescent ◽

Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

Microorganisms ◽

10.3390/microorganisms9020379 ◽

2021 ◽

Vol 9 (2) ◽

pp. 379

Author(s):

Breanne M. Head ◽

Christopher I. Graham ◽

Teassa MacMartin ◽

Yoav Keynan ◽

Ann Karen C. Brassinga

Keyword(s):

Growth Kinetics ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Disease Incidence ◽

Acanthamoeba Castellanii ◽

Infection Model ◽

Intracellular Infection ◽

Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

Veterinary Research ◽

10.1186/s13567-021-00964-4 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Peng-Fei Fu ◽

Xuan Cheng ◽

Bing-Qian Su ◽

Li-Fang Duan ◽

Cong-Rong Wang ◽

...

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Antiviral Agents ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Economic Losses ◽

Green Fluorescent ◽

Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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