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1.
J Mol Neurosci ; 65(3): 351-358, 2018 Jul.
Article in English | MEDLINE | ID: mdl-30003430

ABSTRACT

Induced pluripotent stem cells (iPS cells) generated from somatic cells through reprogramming hold great promises for regenerative medicine. However, how reprogrammed cells survive, behave in vivo, and interact with host cells after transplantation still remains to be addressed. There is a significant need for animal models that allow in vivo tracking of transplanted cells in real time. In this regard, the zebrafish, a tropical freshwater fish, provides significant advantage as it is optically transparent and can be imaged in high resolution using confocal microscopy. The principal goal of this study was to optimize the protocol for successful short-term and immunosuppression-free transplantation of human iPS cell-derived neural progenitor cells into zebrafish and to test their ability to differentiate in this animal model. To address this aim, we isolated human iPS cell-derived neural progenitor cells from human fibroblasts and grafted them into (a) early (blastocyst)-stage wild-type AB zebrafish embryos or (b) 3-day-old Tg(gfap:GFP) zebrafish embryos (intracranial injection). We found that transplanted human neuronal progenitor cells can be effectively grafted and that they differentiate and survive in zebrafish for more than 2 weeks, validating the model as an ideal platform for in vivo screening experiments. We conclude that zebrafish provides an excellent model for studying iPS cell-derived cells in vivo.


Subject(s)
Blastocyst/cytology , Induced Pluripotent Stem Cells/transplantation , Neural Stem Cells/transplantation , Stem Cell Transplantation/methods , Animals , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/cytology , Neural Stem Cells/cytology , Zebrafish
2.
Cell Transplant ; 21(12): 2603-19, 2012.
Article in English | MEDLINE | ID: mdl-22889456

ABSTRACT

In previous studies, we have demonstrated that spinal grafting of human or rat fetal spinal neural precursors leads to amelioration of spasticity and improvement in ambulatory function in rats with spinal ischemic injury. In the current study, we characterize the survival and maturation of three different human embryonic stem (ES) cell line-derived neural precursors (hNPCs) once grafted into ischemia-injured lumbar spinal cord in rats or in naive immunosuppressed minipigs. Proliferating HUES-2, HUES-7, or HUES-9 colonies were induced to form embryoid bodies. During the nestin-positive stage, the rosettes were removed and CD184(+)/CD271(-)/CD44(-)/CD24(+) population of ES-hNPCs FAC-sorted and expanded. Male Sprague-Dawley rats with spinal ischemic injury or naive immunosuppressed Gottingen-Minnesota minipigs received 10 bilateral injections of ES-NPCs into the L2-L5 gray matter. After cell grafting, animals survived for 2 weeks to 4.5 months, and the presence of grafted cells was confirmed after staining spinal cord sections with a combination of human-specific (hNUMA, HO14, hNSE, hSYN) or nonspecific (DCX, MAP2, CHAT, GFAP, APC) antibodies. In the majority of grafted animals, hNUMA-positive grafted cells were identified. At 2-4 weeks after grafting, double-labeled hNUMA/DCX-immunoreactive neurons were seen with extensive DCX(+) processes. At survival intervals of 4-8 weeks, hNSE(+) neurons and expression of hSYN was identified. Some hSYN-positive terminals formed putative synapses with the host neurons. Quantitative analysis of hNUMA(+) cells at 2 months after grafting showed comparable cell survival for all three cell lines. In the presence of low-level immunosuppression, no grafted cell survival was seen at 4.5 months after grafting. Spinal grafting of proliferating pluripotent HUES-7 cells led to consistent teratoma formation at 2-6 weeks after cell transplantation. These data show that ES-derived, FAC-sorted NPCs can represent an effective source of human NPCs to be used in CNS cell replacement therapies.


Subject(s)
Embryonic Stem Cells/cytology , Neural Stem Cells/transplantation , Spinal Cord Ischemia/therapy , Animals , Antigens, Nuclear/metabolism , Cell Cycle Proteins , Cell Differentiation , Cell Line , Cell Survival , Doublecortin Protein , Embryoid Bodies/physiology , Embryonic Stem Cells/metabolism , Humans , Immunocompromised Host , Ki-67 Antigen/metabolism , Male , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurons/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Rats , Rats, Sprague-Dawley , Spinal Cord Ischemia/metabolism , Spinal Cord Ischemia/pathology , Swine , Swine, Miniature , Transcription Factors/metabolism
3.
Cell Transplant ; 20(8): 1163-78, 2011.
Article in English | MEDLINE | ID: mdl-21669049

ABSTRACT

A major limitation of neural transplantation studies is assessing the degree of host-graft interaction. In the present study, rat hippocampal/cortical embryonic neurons (E18) were infected with a lentivirus encoding enhanced green fluorescent protein (GFP) under control of the neuron-specific synapsin promoter, thus permitting robust identification of labeled neurons after in vivo grafting. Two weeks after transient forebrain ischemia or sham-surgery, GFP-expressing neurons were transplanted into CA1 hippocampal regions in immunosuppressed adult Wistar rats. The survival, distribution, phenotype, and axonal projections of GFP-immunoreactive (IR) positive transplanted neurons were evaluated in the sham-operated and ischemia- damaged CA1 hippocampal regions 4, 8, and 12 weeks after transplantation. In both experimental groups, we observed that the main phenotype of host-derived afferents projecting towards grafted GFP-IR neurons as well as transplant-derived GFP-IR efferents were glutamatergic in both animal groups. GFP axonal projections were seen in the nucleus accumbens, septal nuclei, and subiculum-known target areas of CA1 pyramidal neurons. Compared to sham-operated animals, GFP-IR neurons grafted into the ischemia-damaged CA1 migrated more extensively throughout a larger volume of host tissue, particularly in the stratum radiatum. Moreover, enhanced axonal sprouting and neuronal plasticity of grafted cells were evident in the hippocampus, subiculum, septal nuclei, and nucleus accumbens of the ischemia-damaged rats. Our study suggests that the adult rat brain retains some capacity to direct newly sprouting axons of transplanted embryonic neurons to the correct targets and that this capacity is enhanced in previously ischemia-injured forebrain.


Subject(s)
Axons/metabolism , CA1 Region, Hippocampal/pathology , Dendrites/metabolism , Green Fluorescent Proteins/metabolism , Ischemic Attack, Transient/therapy , Neurons/transplantation , Synapsins/metabolism , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Embryo, Mammalian/cytology , Ischemic Attack, Transient/pathology , Lentivirus/genetics , Male , Neurons, Efferent/cytology , Neurons, Efferent/metabolism , Phenotype , Rats , Rats, Wistar , Staining and Labeling
4.
Mol Ther ; 4(3): 232-8, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11545614

ABSTRACT

We have previously shown that vesicles containing the spike glycoprotein of the vesicular stomatitis virus (VSV-G) can associate efficiently with immature, non-infectious, envelope-deficient retrovirus-like particles assembled by packaging cells to produce infectious, pseudotyped viruses in cell-free conditions in vitro. We have also previously reported that VSV-G can enhance DNA lipofection efficiency by interacting with liposomes to form fusogenic, serum-stable liposomes with enhanced transfection properties. Here, we report that VSV-G can form a complex directly with naked plasmid DNA in the absence of a lipofection reagent and can thereby enhance the transfection efficiency of the naked plasmid vector. Sucrose gradient sedimentation analysis demonstrated that VSV-G can also associate with plasmid DNA and murine leukemia virus (MLV) gag-pol particles to form ternary complexes that co-sediment with high DNA transfecting activity. The increased transfection efficiency with VSV-G was dependent on the presence of the polycation (Polybrene) in the culture medium during transfection. Enhanced transfection was abolished by a neutralizing antibody to VSV-G. These results may be useful in the study of retrovirus assembly, in the further design of hybrid DNA-based retrovirus-like vectors, and in the full in vitro, cell-free assembly of infectious virus-like particles from component parts.


Subject(s)
Leukemia Virus, Murine/metabolism , Membrane Glycoproteins , Plasmids/metabolism , Transfection/methods , Viral Envelope Proteins/metabolism , Virion/metabolism , Animals , Cell Line , Cell-Free System , Genes, Reporter/genetics , Leukemia Virus, Murine/chemistry , Liposomes/metabolism , Luciferases/genetics , Luciferases/metabolism , Plasmids/chemistry , Plasmids/genetics , Viral Envelope Proteins/genetics , Virion/chemistry
6.
J Virol ; 74(22): 10790-5, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11044124

ABSTRACT

In the absence of viral envelope gene expression, cells expressing human immunodeficiency virus type 1 (HIV-1) gag and pol, accessory HIV functions, and a vector genome RNA produce and secrete large amount of noninfectious virus-like particles (VLPs) into the conditioned medium. After partial purification, such HIV-1 VLPs can be made infectious in cell-free conditions in vitro by complex formation with lipofection reagents or with the G protein of vesicular stomatitis virus (VSV-G). The resulting in vitro-modified HIV-1 particles are able to infect nondividing cells. Infectivity of envelope-free HIV VLPs can also be induced by prior modification of target cells through exposure to partially purified VSV-G vesicles. Similarly, infection can be carried out by attachment of envelope-free noninfectious VLPs to unmodified cells followed by subsequent treatment of cells with VSV-G. We interpret these findings to indicate that interaction between a viral envelope and a cell surface receptor is not necessary for the initial virus binding to the cells but is required for subsequent cell entry and infection.


Subject(s)
HIV-1/physiology , Membrane Glycoproteins , Cell Line , Fusion Proteins, gag-pol/genetics , Fusion Proteins, gag-pol/metabolism , Humans , Phosphatidylethanolamines/genetics , Phosphatidylethanolamines/metabolism , RNA, Viral/metabolism , Transfection , Viral Envelope Proteins/genetics , Viral Envelope Proteins/metabolism , Virion/physiology
7.
In Vitro Cell Dev Biol Anim ; 36(6): 395-9, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10949999

ABSTRACT

The inability to stably introduce and express foreign genes has hampered basic research in molluscan species. We cultured cells from dissociated embryos of the Pacific oyster, Crassostrea gigas, and infected these primary cultures with pantropic retroviral vectors containing the envelope glycoprotein of vesicular stomatitis virus. Luciferase transgene expression mediated by different heterologous promoters was demonstrated for at least 9 d after infection of the cells. Surprisingly, the promoter reproducibly mediating the highest level of luciferase expression was the retroviral promoter (U3 region of long terminal repeat) from the Moloney murine leukemia virus. The infection efficiency using a low multiplicity of infection (0.05) was estimated by quantitative polymerase chain reaction to be between 0.1-0.5%. This system will facilitate studies of gene expression and regulation and should be widely applicable to other molluscan species.


Subject(s)
Cells, Cultured , Embryo, Nonmammalian/cytology , Genetic Vectors , Ostreidae/embryology , Retroviridae/genetics , Animals , Cell Adhesion , Cell Culture Techniques , Cell Survival , Embryo, Nonmammalian/virology , Genes, Reporter , Heart/embryology , Luciferases/biosynthesis , Luciferases/genetics , Myocardium/cytology , Osmotic Pressure , Ostreidae/cytology , Ostreidae/virology , Promoter Regions, Genetic , Recombinant Proteins/biosynthesis , Retroviridae/physiology
8.
J Virol ; 73(11): 9632-7, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10516075

ABSTRACT

During the replication cycle of murine leukemia virus (MLV), Pol is normally synthesized as part of a Gag-Pol fusion protein. In this study, the ability of free MLV Pol to be incorporated into virions was examined. When MLV Gag and MLV Pol were coexpressed from separate plasmids in cells, reverse transcriptase (RT) activity associated with Gag core particles at a slightly lower level than did RT activity generated from wild-type Gag-Pol expression. Particles produced in this manner were somewhat less infectious than those produced with wild-type Gag-Pol. A smaller amount of MLV Pol also associated with heterologous human immunodeficiency virus type 1 Gag cores.


Subject(s)
Fusion Proteins, gag-pol/metabolism , Gene Products, pol/metabolism , HIV/physiology , Leukemia Virus, Murine/physiology , Animals , Blotting, Western , Gene Products, gag/metabolism , Humans , Leukemia Virus, Murine/metabolism , Mice , RNA-Directed DNA Polymerase/metabolism , Virion/metabolism , Virus Replication
9.
Gene Ther ; 5(5): 708-11, 1998 May.
Article in English | MEDLINE | ID: mdl-9797877

ABSTRACT

Lipofection involves the introduction of foreign genetic information into mammalian cells through the use of lipophilic reagents that enhance cellular uptake of polynucleotides. Despite the use of currently optimized lipofection conditions, including the use of serum-depleted media, the efficiency of gene transfer is often low. We show here that, in a variety of cell lines, polybrene markedly enhances the efficiency of lipofection under standardized conditions and also compensates the serum-mediated inhibition of lipofection. Although the degree of the polybrene effect depends on the nature of the cell line, these results indicate that individually optimized concentrations of polybrene can be useful for increasing the efficiency of lipofectin-mediated gene transfer in vitro.


Subject(s)
Gene Transfer Techniques , Heparin Antagonists/pharmacology , Hexadimethrine Bromide/pharmacology , Transfection/methods , Animals , Cell Line , Cells, Cultured , Cricetinae , Culture Media, Serum-Free , Dose-Response Relationship, Drug , Mammals , Transfection/drug effects
10.
J Virol ; 72(8): 6356-61, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9658075

ABSTRACT

In the absence of envelope gene expression, retrovirus packaging cell lines expressing Moloney murine leukemia virus (MLV) gag and pol genes produce large amounts of noninfectious virus-like particles that contain reverse transcriptase, processed Gag protein, and viral RNA (gag-pol RNA particles). We demonstrate that these particles can be made infectious in an in vitro, cell-free system by the addition of a surrogate envelope protein, the G spike glycoprotein of vesicular stomatitis virus (VSV-G). The appearance of infectivity is accompanied by physical association of the G protein with the immature, noninfectious virus particles. Similarly, exposure in vitro of wild-type VSV-G to a fusion-defective pseudotyped virus containing a mutant VSV-G markedly increases the infectivity of the virus to titers similar to those of conventional VSV-G pseudotyped viruses. Furthermore, similar treatment of an amphotropic murine leukemia virus significantly allows infection of BHK cells not otherwise susceptible to infection with native amphotropic virus. The partially cell-free virus maturation system reported here should be useful for studies aimed at the preparation of tissue-targeted retrovirus vectors and will also aid in studies of nucleocapsid-envelope interactions during budding and of virus assembly and virus-receptor interactions during virus uptake into infected cells. It may also represent a potentially useful step toward the eventual development of a completely cell-free retrovirus assembly system.


Subject(s)
Membrane Glycoproteins , Moloney murine leukemia virus/pathogenicity , Vesicular stomatitis Indiana virus/metabolism , Viral Envelope Proteins/metabolism , Animals , Cell Line , Cell Line, Transformed , Cell-Free System , Cricetinae , Culture Media , Fusion Proteins, gag-pol/genetics , Fusion Proteins, gag-pol/physiology , HeLa Cells , Humans , Moloney murine leukemia virus/classification , Moloney murine leukemia virus/genetics , RNA, Viral/physiology , Virion
11.
J Virol ; 72(7): 6159-63, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9621082

ABSTRACT

Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.


Subject(s)
Gene Transfer Techniques , Membrane Glycoproteins , Phosphatidylethanolamines/administration & dosage , Viral Envelope Proteins/administration & dosage , Culture Media , DNA, Viral/administration & dosage , Liposomes , Transfection , Viral Envelope Proteins/isolation & purification
12.
Hum Gene Ther ; 8(14): 1645-50, 1997 Sep 20.
Article in English | MEDLINE | ID: mdl-9322866

ABSTRACT

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.


Subject(s)
Adenoviridae/genetics , Carotid Arteries , Connective Tissue , Gene Transfer Techniques , Liposomes/administration & dosage , Membrane Glycoproteins , Retroviridae/genetics , Animals , Cell Division , Endothelium, Vascular , Genetic Vectors/genetics , Lac Operon/genetics , Moloney murine leukemia virus/genetics , Muscle, Smooth/cytology , Plasmids , Rabbits , Vesicular stomatitis Indiana virus/genetics , Viral Envelope Proteins/genetics
13.
Proc Natl Acad Sci U S A ; 94(20): 10803-8, 1997 Sep 30.
Article in English | MEDLINE | ID: mdl-9380714

ABSTRACT

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 10(5) colony-forming units per ml.


Subject(s)
Moloney murine leukemia virus/physiology , Transfection/methods , Virion/physiology , Virus Assembly , Cell Line , Culture Media, Conditioned , Genes, gag , Genes, pol , Indicators and Reagents , Microscopy, Electron , Moloney murine leukemia virus/pathogenicity , Moloney murine leukemia virus/ultrastructure , RNA, Viral , Virion/pathogenicity , Virion/ultrastructure
14.
J Biol Chem ; 270(10): 5191-7, 1995 Mar 10.
Article in English | MEDLINE | ID: mdl-7890629

ABSTRACT

There is strong experimental evidence that oxidized low density lipoprotein (Ox-LDL) plays an important role in atherosclerosis. However, the mechanisms by which Ox-LDL is formed in vivo are unknown. To test whether 15-lipoxygenase (15-LO) could play a role in oxidation of LDL by cells, we expressed 15-LO activity in murine fibroblasts, which do not normally have 15-LO activity, and tested their ability to modify LDL. Using a retroviral vector, we prepared fibroblasts that expressed 2- to 20-fold more 15-LO activity than control fibroblasts infected with a vector containing beta-galactosidase (lacZ). Compared with LDL incubated with lacZ cells, LDL incubated with 15-LO-containing cells were enriched with lipid hydroperoxides. When these LDL samples were subsequently subjected to oxidative stress, they were more susceptible to further oxidative modification, as judged by increased conjugated diene formation and by increased ability to compete with 125I-Ox-LDL for uptake by macrophages. These findings establish that cellular 15-LO can contribute to oxidative modification of LDL, but the quantitative significance of these findings to the in vivo oxidation of LDL remains to be established.


Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Lipid Peroxides/metabolism , Lipoproteins, LDL/metabolism , Animals , Arachidonate 15-Lipoxygenase/biosynthesis , Blotting, Northern , Cell Line , Cells, Cultured , DNA, Complementary , Fibroblasts/metabolism , Gene Expression , Genetic Vectors , Humans , Kinetics , Macrophages, Peritoneal/metabolism , Mice , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Retroviridae , Transfection , beta-Galactosidase/biosynthesis , beta-Galactosidase/metabolism
15.
Gene Ther ; 2(2): 138-42, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7719930

ABSTRACT

Ideal methods for human gene therapy will eventually include direct gene transfer to defective tissues in a patient in vivo. Toward that goal, we have used high titer, pseudotyped retroviral vectors expressing genes for the Escherichia coli beta-galactosidase (lacZ) or hepatitis B virus surface antigen (HBsAg) to infect mouse liver by in vivo direct injection into the liver parenchyma. We have found that a single percutaneous injection of small volumes of vectors into the newborn mouse liver leads to transduction of at least 25-30% of the hepatocytes throughout the liver, as judged by in situ staining of liver sections for beta-gal activity at 4 weeks after injection. We have demonstrated that stable levels of HBsAg were also detected in the circulation of injected mice up to 4 months after HBsAg-vector injection. We suggest that the high efficiency of in vivo transduction in the neonatal liver and subsequent stable transgene expression by high-titer pseudotyped retroviral vectors in the absence of an invasive partial hepatectomy may effectively be applied to gene therapy studies in a number of human liver disease [corrected].


Subject(s)
Genetic Vectors , Hepatitis B Surface Antigens/biosynthesis , Liver , Membrane Glycoproteins , Moloney murine leukemia virus , Recombinant Fusion Proteins/biosynthesis , Transfection/methods , Viral Envelope Proteins , beta-Galactosidase/biosynthesis , Animals , Animals, Newborn , Evaluation Studies as Topic , Gene Expression Regulation, Viral , Genetic Vectors/administration & dosage , Genetic Vectors/chemistry , Genetic Vectors/genetics , Genetic Vectors/pharmacokinetics , Hepatitis B Surface Antigens/blood , Hepatitis B Surface Antigens/genetics , Injections , Liver/metabolism , Liver/virology , Mice , Moloney murine leukemia virus/genetics , Recombinant Fusion Proteins/genetics , Vesicular stomatitis Indiana virus/chemistry , beta-Galactosidase/genetics
16.
Proc Natl Acad Sci U S A ; 91(20): 9564-8, 1994 Sep 27.
Article in English | MEDLINE | ID: mdl-7937806

ABSTRACT

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.


Subject(s)
Genetic Vectors , Liver/metabolism , Retroviridae , Transfection/methods , Animals , Cell Line , Cells, Cultured , Fibrosarcoma , Genetic Therapy/methods , HeLa Cells , Helper Viruses , Humans , Liver/microbiology , Mice , Moloney murine leukemia virus , Rats , Repetitive Sequences, Nucleic Acid , Tumor Cells, Cultured , Vesicular stomatitis Indiana virus , beta-Galactosidase/analysis , beta-Galactosidase/biosynthesis
17.
J Virol ; 66(5): 2952-65, 1992 May.
Article in English | MEDLINE | ID: mdl-1373198

ABSTRACT

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) may prove useful as vectors for gene transfer, particularly to nondividing cells. Cgal delta 3 is an immediate-early gene 3 (IE 3) deletion mutant of HSV-1 that expresses the lacZ gene of Escherichia coli from the human cytomegalovirus immediate-early control region but does not express viral early or late genes. This vector was able to efficiently infect and express lacZ in cells refractory to traditional methods of gene transfer. However, 1 to 3 days postinfection, Cgal delta 3 induced cytopathic effects (CPE) in many cell types, including neurons. In human primary fibroblasts Cgal delta 3 induced chromosomal aberrations and host cell DNA fragmentation. Other HSV-1 strains that caused CPE, tested under conditions of viral replication-inhibition, included mutants of the early gene UL42, the virion host shutoff function, single mutants of IE 1, IE 2, and IE 3, and double mutants of IE 3 and 4 and IE 3 and 5. Inhibition of viral gene expression by UV irradiation of virus stocks or by preexposure of cells to interferon markedly reduced the CPE. We conclude from these studies that HSV-1 IE gene expression is sufficient for the induction of CPE, although none of the five IE gene products appear to be solely responsible. After infection of human fibroblasts with Cgal delta 3 at a low multiplicity of infection, we were able to recover up to 6% of the input virus 2 weeks later by a superinfection-rescue procedure, even though the virally transduced human cytomegalovirus-lacZ transgene was not expressed at this time. It is therefore likely that inhibition or inactivation of viral IE gene expression, either for establishing latency or for the long-term transduction of foreign genes by HSV-1 vectors, is essential to avoid the death of infected cells.


Subject(s)
Genetic Vectors , Herpes Simplex/pathology , Host-Parasite Interactions , Simplexvirus/pathogenicity , Virus Replication , Animals , Cell Death , Chromosome Aberrations , Cytomegalovirus/genetics , DNA Damage , DNA, Recombinant , Escherichia coli/genetics , Evaluation Studies as Topic , Fibroblasts/pathology , Gene Expression Regulation, Viral , Herpes Simplex/genetics , Humans , Interferons/pharmacology , Lac Operon/genetics , Mutation , Simplexvirus/genetics , Simplexvirus/growth & development , Ultraviolet Rays
18.
New Biol ; 4(3): 238-46, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1316145

ABSTRACT

We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein. By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ. The results were striking in that the serum level of cFIX reached 10% of the normal murine levels. Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation. Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX. The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.


Subject(s)
Factor IX/genetics , Simplexvirus/genetics , Transfection , Animals , Factor IX/biosynthesis , Female , Genetic Vectors , Hepatitis B Surface Antigens/genetics , Herpes Simplex/microbiology , Liver/enzymology , Liver/immunology , Liver/microbiology , Mice , Organ Specificity , beta-Galactosidase/genetics
19.
New Biol ; 4(3): 261-7, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1581292

ABSTRACT

Primary cultures of rat hepatocytes embedded and grown to high density in a wafer of reticulated polyurethane have been infected in vitro with a retroviral vector expressing the hepatitis B virus surface antigen (HBsAg). Wafers containing infected cells and implanted directly between the liver lobes of recipient rats became vascularized and firmly attached to the liver parenchyma. Stable levels of HBsAg were detected in the blood circulation for at least 4 months after grafting. Hepatocytes recovered from the recipient rats up to 4 months after implantation were recultured under selective conditions and found to produce high levels of HBsAg.


Subject(s)
Hepatitis B Surface Antigens/genetics , Liver/microbiology , Transfection , Animals , Female , Gene Expression , Genes, Viral , Genetic Vectors , Hepatitis B Surface Antigens/biosynthesis , Liver/immunology , Rats , Rats, Inbred F344 , Retroviridae/genetics
20.
J Lipid Res ; 31(12): 2167-78, 1990 Dec.
Article in English | MEDLINE | ID: mdl-2090710

ABSTRACT

We have reported the use of a retroviral vector to introduce the low density lipoprotein (LDL) receptor gene into receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbit fibroblasts (Miyanohara, A., M. F. Sharkey, D. Steinberg, J. L. Witztum, and T. Friedmann. 1988. Proc. Natl. Acad. Sci. USA. 85: 6538-6542). Because the cDNA for the LDL receptor did not contain the 5' sterol regulatory element that confers sterol-mediated inhibition of LDL receptor transcription, we did not anticipate that LDL receptor activity transduced by this vector would be sterol-responsive. However, we now demonstrate sterol-mediated down-regulation of receptor protein in the infected cells by a mechanism that appears to be mediated at a post-transcriptional level. Down-regulation of LDL receptor activity occurred when infected WHHL cells were preincubated with either LDL or cholesterol plus 25-hydroxycholesterol. Identically organized vectors bearing cDNAs encoding irrelevant genes such as firefly luciferase or bacterial beta-galactosidase exhibited no sterol regulation of reporter gene activity. Insulin receptor activity in WHHL fibroblasts and in WHHL fibroblasts infected with the LDL receptor retroviral vector was also unchanged by sterol. Transfection of LDL receptor-deficient Chinese hamster ovary (CHO) cells with a nonretroviral vector containing the same LDL receptor cDNA also resulted in sterol responsiveness of the transduced LDL receptor. These experiments suggest that the effect of sterol was specific for the LDL receptor transcript. Transgene LDL receptor mRNA levels from the infected cells were unaffected by sterol, indicating that the sterol-mediated reduction in LDL receptor activity did not result from alterations in steady state mRNA levels. These data suggest the existence of post-transcriptional level mechanisms that are responsible for sterol regulation of expression of the transduced LDL receptor gene.


Subject(s)
Receptors, LDL/genetics , Retroviridae/genetics , Transcription, Genetic , Transduction, Genetic , Animals , Blotting, Northern , Cell Line , Cricetinae , Down-Regulation , Genetic Vectors , Humans , Mice , Rabbits , Receptors, LDL/metabolism , Transfection
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