ABSTRACT
C57BL/6 (B6)-derived embryonic stem (ES) cells are not widely used to generate knockout mice despite the advantage of a well-defined genetic background because of poor developmental potential. We newly established serum- and feeder-free B6 ES cells with full developmental potential by using leukemia inhibitory factor (LIF) and 6-bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase-3 (GSK3) inhibitor. BIO treatment significantly increased the expression levels of 364 genes including pluripotency markers such as Nanog and Klf family. Unexpectedly, by aggregating or microinjecting those ES cells to each eight-cell-stage diploid embryo, we stably generated germline-competent ES-derived mice. Furthermore, founder mice completely derived from female XO, heterozygous, or homozygous mutant B6 ES cells were directly available for intercross breeding and phenotypic analysis. We hereby propose that serum- and feeder-free B6 ES cells stimulated with LIF plus GSK3 inhibitor are valuable for generating mouse models on B6 background.
Subject(s)
Embryonic Stem Cells/drug effects , Indoles/pharmacology , Oximes/pharmacology , Totipotent Stem Cells/drug effects , Animals , Cell Culture Techniques , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Female , Gene Expression Regulation, Developmental/drug effects , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Leukemia Inhibitory Factor/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Inbred Strains , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Receptors, Leptin/genetics , Receptors, Leptin/metabolism , Totipotent Stem Cells/cytology , Totipotent Stem Cells/metabolismABSTRACT
Nectin-1 is a Ca2+-independent Ig-like cell-cell adhesion molecule and an alphaherpesvirus receptor that binds to virion glycoprotein D by the first Ig-like domain. We have investigated the antiviral potentials of soluble forms of porcine nectin-1 to PRV infection by generating transgenic mice expressing different types of fusion protein. Previously, we reported that mice transgenic for a chimera that carried the entire ectodomain of porcine nectin-1 fused to the Fc portion of porcine IgG1 were more resistant than those transgenic for a chimera that carried the first Ig-like domain fused to the Fc portion. Recently, we generated transgenic mice expressing a fusion protein made of the first Ig-like domain fused to the Fc portion of human IgG1, and reported that they showed a microphthalmia. Here, two transgenic mouse lines expressing the fusion protein were challenged with PRV for comparing their resistances with those of transgenic mice expressing different types of fusion protein. Surprisingly, both transgenic mouse lines showed a high resistance to the viral infection, especially via the i.n. route. Significant resistance of the embryonic fibroblasts was also observed. Altogether, these findings indicated that the fusion protein consisting of the first Ig-like domain fused to the human Fc portion provided a marked resistance against PRV infection to the transgenic mice.
Subject(s)
Cell Adhesion Molecules/immunology , Herpesvirus 1, Suid/immunology , Immunity, Innate , Immunoglobulin Fc Fragments/immunology , Pseudorabies/immunology , Animals , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cells, Cultured , Fibroblasts/immunology , Fibroblasts/virology , Herpesvirus 1, Suid/genetics , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Mice , Mice, Inbred C57BL , Nectins , Protein Structure, Tertiary , Pseudorabies/prevention & control , Pseudorabies/virology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Sus scrofaABSTRACT
Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D by the first immunoglobulin (Ig)-like domain. The possibility of making animals resistant to pseudorabies virus (PRV) infection has been investigated by generating transgenic mice expressing soluble forms of porcine nectin-1. Previously, transgenic mice were generated that expressed a fusion protein made of the entire ectodomain of nectin-1 fused to the Fc portion of human IgG, or the first Ig-like domain fused to the Fc portion of porcine IgG. Here, the contribution of the second and third Ig-like domains of nectin-1 was analysed by generating transgenic mice expressing the entire ectodomain of nectin-1 fused to the porcine Fc portion. Transgenic mice expressing each of three different fusion proteins were challenged with PRV for comparison of their resistance. Altogether, mice transgenic for a chimera that carried the entire ectodomain were more resistant than those transgenic for a chimera that carried the first Ig-like domain.
Subject(s)
Antiviral Agents/pharmacology , Cell Adhesion Molecules/genetics , Herpesvirus 1, Suid/drug effects , Herpesvirus 1, Suid/genetics , Animals , Cell Adhesion Molecules/immunology , Immunoglobulin Fc Fragments/immunology , Mice , Mice, Transgenic , Nectins , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , SwineABSTRACT
An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a transgene encoding a soluble form of nectin-1, also known as herpesvirus entry mediator C. Nectin-1 is an alpha-herpesvirus receptor that binds to virion glycoprotein D. Nectin-1 mediates entry of PRV, herpes simplex virus types 1 and 2, and bovine herpesvirus type 1. To assess the antiviral potential of an ectopic expression of the nectin-1 ectodomain in vivo, six transgenic mouse lines expressing a soluble form of nectin-1, consisting of an extracellular domain of porcine nectin-1 and the Fc portion of human IgG1, were generated. All of the transgenic mouse lines showed nearly complete resistance to PRV infection by means of both i.p. and intranasal routes. These results suggest that the introduction into farm animals of a transgene encoding a soluble form of nectin-1 would offer a potent biological approach to generating alpha-herpesvirus-resistant livestock.
Subject(s)
Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/physiology , Pseudorabies/prevention & control , Receptors, Virus/genetics , Receptors, Virus/physiology , Animals , Base Sequence , Cell Adhesion Molecules/chemistry , DNA, Viral/genetics , DNA, Viral/isolation & purification , Herpesviridae/pathogenicity , Herpesvirus 1, Suid/genetics , Herpesvirus 1, Suid/isolation & purification , Herpesvirus 1, Suid/pathogenicity , Humans , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Biological , Nectins , Polymerase Chain Reaction , Pseudorabies/immunology , Pseudorabies/virology , Receptors, Virus/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility , Sus scrofaABSTRACT
Herpesvirus entry mediator (HVEM) is a member of the tumor necrosis factor (TNF) receptor family used as a cellular receptor by virion glycoprotein D (gD) of herpes simplex virus (HSV). Both human and mouse forms of HVEM can mediate entry of HSV-1 but have no entry activity for pseudorabies virus (PRV). To assess the antiviral potential of HVEM in vivo, three transgenic mouse lines expressing a soluble form of HVEM (HVEMIg) consisting of an extracellular domain of murine HVEM and the Fc portion of human IgG1 were generated. All of the transgenic mouse lines showed marked resistance to HSV-1 infection when the mice were challenged intraperitoneally with HSV-1, but not to PRV infection. The present results demonstrate that HVEMIg is able to exert a significant antiviral effect against HSV-1 infection in vivo.
Subject(s)
Herpes Simplex/immunology , Herpesvirus 1, Human/pathogenicity , Mice, Transgenic , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Animals , Cells, Cultured , Chlorocebus aethiops , Fibroblasts , Herpes Simplex/physiopathology , Herpes Simplex/virology , Humans , Mice , Mice, Inbred C57BL/embryology , Receptors, Tumor Necrosis Factor, Member 14 , Vero CellsABSTRACT
Porcine herpesvirus entry mediator C (HveC) is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). Porcine HveC mediates entry of pseudorabies virus (PRV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and bovine herpesvirus type 1 (BHV-1). In order to assess the antiviral potential of a soluble form of porcine HveC, Vero cells were transformed with the chimeric gene expressing a fusion protein (PHveCIg) consisting of an extracellular domain of porcine HveC and the Fc portion of human IgG1. The transformed cell lines expressing PHveCIg showed marked resistance to PRV infection. Resistance to infection by other alphaherpesviruses (HSV-1 and BHV-1) was also observed in the transformed cell line. The present results demonstrate that a soluble form of porcine HveC is able to exert a significant antiviral effect against pseudorabies virus and other alphaherpesvirus infection in vitro.
Subject(s)
Herpesvirus 1, Suid/pathogenicity , Pseudorabies/prevention & control , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Virus/metabolism , Animals , Cattle , Cell Line , Cell Line, Transformed , Chlorocebus aethiops , Herpesvirus 1, Bovine/pathogenicity , Herpesvirus 1, Human/pathogenicity , Herpesvirus 1, Suid/drug effects , History, 20th Century , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/chemistry , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Member 14 , Receptors, Virus/genetics , Solubility , Swine , Vero Cells , Viral Plaque AssayABSTRACT
The latency-associated transcript (LAT) promoter of pseudorabies virus (PRV) is unique among viral promoters in that it remains active in trigeminal ganglia during the latent state. It is not known which the viral or host proteins regulate expression of the PRV LAT gene in latently infected neurons. To determine whether host transcriptional proteins in neurons can regulate the PRV LAT promoter in vivo, three transgenic mouse lines containing the PRV LAT promoter (LAP; LAP1 and LAP2) linked to the chloramphenicol acetyltransferase (CAT) gene were generated. All of the transgenic mouse lines, in the absence of the viral proteins, displayed strong expression of the transgene in trigeminal ganglia in addition to other neuronal tissues such as cerebral cortex, cerebellum, hippocampus and olfactory bulb. Expression of the transgene in neurons of trigeminal ganglia was demonstrated by in situ hybridization. These data provide direct evidence that neuronal transcription factors are sufficient to activate the PRV LAP in vivo and that the promoter is neuron-specific.
