Western blot analysis was performed to investigate the expression of HIV-1 envelope protein in transfected TELCeB6/pTr712 cells and in virus particles isolated from the supernatant of these cells. Selection of hygromycin-resistant colonies after transfection of plasmid pREP4 (Invitrogen) was carried out in medium containing 200 μg/ml of hygromycin B (Sigma), yielding the cell line TELCeB6/pTr712-K14. Online ISSN 1091-6490. It also was shown that heterologous proteins with short intracellular domains can be incorporated by MuLV-derived capsid particles if expressed at high levels (26). CD4-expressing cells can be enriched, cultured, and transduced with a gene transfer vector in vitro and reintroduced into a patient. The most efficient way to stably introduce genes into target cells uses retroviral vectors. To increase the viral yield, stable clones of TELCeB6 packaging cells expressing the truncated envelope gene pTr712 were selected after cotransfection with the selectable marker gene pREP4(hygr). Please note: Your browser does not support the features used on Addgene's website. Cocultivation was continued for 24 hr after which the cells were photographed. Upon staining with 5-bromo-4-chloro-3-indolyl β-d-galactosidase (X-Gal) of the cells exposed to the cell supernatants, no blue colonies (indicative of MFG-nlslacZ gene expression) were detected, indicating the absence of detectable gene transfer (Table 1). Forty-seven of the colonies showed at least 10% HIV-1 env-positive cells (Fig. In a control experiment, the related envelope glycoprotein gene of the human spuma retrovirus clone 2 (HSRV-2), also was tested. More information: pLNCX contains elements derived from Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV), and is designed for retroviral gene delivery and expression. We describe here the generation of a retroviral gene transfer vector particle with specificity for human CD4-expressing cells. The retrovirus MuLV has a positive, single-stranded sense RNA that replicates via reverse transcription. In agreement with this hypothesis, a failure to accommodate a long heterologous C-terminus also has been demonstrated in the case of HIV-1. A study suggests that an asteroid impact, not volcanic activity, was the main cause of nonavian dinosaur extinction. Because the HIV envelope surface (SU) glycoprotein gp120-SU mediates the specific infection of CD4+ lymphocytes, and because capsid particles derived from MuLV can efficiently package and transfer therapeutic genes, it seemed desirable to derive a gene transfer vector combining both properties. and J.S. TELCeB6 cells (5) expressing the vector MFG-nlsLacZ (12) were kindly provided by Y. Takeuchi (Institute of Cancer Research, London) and F. L. Cosset (Centre National de la Recherche Scientifique, Lyon, France), HeLa-CD4+ cells (ADP047; ref. pLNCX contains elements derived from Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV), and is designed for retroviral gene delivery and expression. Plasmid pTr712 encodes a truncated variant of the TM protein due to the insertion of a stop codon at amino acid position 712 by site-directed mutagenesis. Replicating MLVs have a positive sense, single-stranded RNA (ssRNA) genome that replicates through a DNA intermediate via the process of reverse transcription. Many also share a conserved RNA structural element called a core encapsidation signal. Cells were washed again and incubated with a 1:1,000 diluted anti-HIV-1 serum. H902 hybridoma cells were obtained from Dr. B. Chesebro through the AIDS Research and Reference Reagent Programme. Most transfer plasmids available from Addgene were drived from MoMLV (Moloney Murine Leukemia Virus) or MSCV (Murine Stem Cell Virus) sequences. (A) TELCeB6/pTr712 cells + HeLa cells. Why the short form of the envelope TM protein is able to become part of the virus particle and the long form is not is a matter for speculation. 22). The host range of a retrovirus (i.e., its specificity of infection) is determined by the viral surface envelope protein and by the receptors expressed by target cells. TELCeB6 cells expressing the amphotropic MuLV envelope protein or ecotropic envelope proteins did not form syncytia with CD4-expressing cells (data not shown). The transcript of the MFG-nlslacZ gene can be packaged into virus particles. ↵† B.S.S. Retroviral vectors with an expanded host range and increased stability were generated. The specificity of infection of CD4+ cells by the viral pseudotypes also was confirmed in experiments with C8166 T cells (Fig. Lane 1, TELCeB6; lane 2, TELCeB6/pTr712. What strain of bacteria does my stab contain? The virus was considered significant since it was shown to induce leukemia in inoculate mice . Learn more, Download our file to copy and paste plasmid data, Open collection of AAV data generously shared by scientists, Basic analysis for a user-entered sequence; includes restriction sites and map, Digital collection of empty plasmid backbones from publications and commercially available sources. Infections were carried out by 3-hr incubation of the target cells with 1 ml of undiluted or diluted supernatants containing retroviral particles. Cocultivation was continued for 24 hr before they were photographed. What do I need to know about the customs and importation process for my country? Addgene is open for ordering and depositing; find up-to-date details here. Rabies virus was pseudotyped with the HIV-1-derived envelope glycoproteins (27) with titers of up to 4 × 103 cfu/ml. Our attempts to incorporate the full-length HIV-1 envelope protein into the viral particles were not successful. It has been shown that MuLV core particles can be pseudotyped with envelope proteins of simian sarcoma associated virus (3), feline leukemia virus subgroup B (4), and feline endogenous virus RD114 (5), for example. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. 2 B and D). Finally, antigen-containing cells were visualized by addition of substrate buffer (H2O2 plus 3-amino-9-ethylcarbazol; Sigma). Systems, Research In contrast to HIV, which selectively infects CD4-expressing human cells, retroviral particles derived from amphotropic MuLV infect human cells without cell-type specificity. Most ongoing clinical trials are based on procedures using modified versions of the Moloney murine leukemia virus (MuLV) for gene transfer. Most ongoing clinical trials are based on procedures using modified versions of the Moloney murine leukemia virus (MuLV) for gene transfer. Expression of the envelope proteins in the transfected cells was demonstrated by immunostaining of the cells with antisera specific for the viral envelope proteins of HIV-1 (data not shown). The presence of proteins derived from the MuLV core particles was demonstrated by Western blot analysis using an antiserum specific for the MuLV p30-Gag protein (Fig. Detection of HIV-1 Env expression in TELCeB6/pTr712 cells and concentrated supernatants. The cells were stained for β-galactosidase expression 2 days after infection (16). Neutralization of pseudotyped vectors by incubation with anti-HIV-1 serum. Proceedings of the National Academy of Sciences, Earth, Atmospheric, and Planetary Sciences, Cause of end-Cretaceous dinosaur extinction, Opinion: For now, human challenge studies for a SARS-CoV-2 vaccine are unethical, Copyright © 1997, The National Academy of Sciences of the USA. We would like to thank D. Kahlenberg and T. Kearns for excellent technical assistance, M. Selbert for expert automatic DNA sequencing, and S. Norley, M. Baier, S. Ottmann, and M. Grez for constructive discussions. TELCeB6 cells express a MuLV-derived gag-pol gene construct and a MFG-nlslacZ gene containing a MuLV-derived packaging signal. No blue colonies were observed after infection of the parental, CD4−-HeLa cells (Fig. 2 A and C). Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. While i… Retroviridae; subfamily oncovirinae type C, enveloped, icosahedral core, virions 100 nm in diameter, diploid, single stranded, linear RNA genome. Upon transfection into a packaging cell line, pLNCX can transiently express, or integrate and stably express, a transcript containing Y+ (the extended viral packaging signal) a selectable marker, and the … Neural signatures of complex cognitive processing can occur in the absence of visual awareness, according to a case study of a person with distorted visual perception. The expression of the surface HIV-1 envelope glycoprotein was studied by cocultivation of TELCeB6/pTr712 cells with HeLa-CD4+ cells or dexamethasone-induced BJAB-CD4i cells (15) and examination of syncytia formation (Fig. After dexamethasone treatment, a 10-fold increase in infection was observed with virus from TELCeB6/pTr712 supernatants whereas untransfected BJAB cells could not be infected. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. One milliliter of supernatant was used for the infection of the human CD4-expressing cells (HeLa-CD4+) or the CD4− cells (293), which are permissive for infection by HIV-1 and human spuma retrovirus, respectively. Contaminating cells in the viral supernatants had been previously removed by passage through 0.45-μm filters. 3A, lane 1). 9 and 10) encompassing variant HIV-1 env genes and a neomycin resistance gene, the construct pCMV-env/neo encoding the env gene of human spuma retrovirus clone 2 (HSRV-2), and plasmid pCRUCA encoding the env gene of amphotropic MLV were described elsewhere (9, 11). Carp can disperse their eggs via the digestive system of ducks, and dispersal of a single Prussian carp egg could establish a new population, according to a study.

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