The murine viruses are able to infect only murine cells ecotropic, whereas derivatives of mmlv or human retroviruses like vesicular stomatiitis virus vsv can infect both human and murine cells amphotropic. Amphotropic or gibbon ape leukemia virus retrovirus. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. To transduce primate cells, amphotropic retroviruses are instead used. Amphotropic and ecotropic retroviral vector viruses. Hosts for some groups of retroviruses particularly the avian ctype and mammalian ctype virusesare characterized by a high degree of genetic polymorphism in the availability of receptors for the viruses.
Ecotropic virus definition of ecotropic virus by medical. Pdf cloning of the cellular receptor for amphotropic. Emergent diseases such as aids and adult tcell lymphoma have widened even further the community of investigators directly concerned with retroviruses, a development that has highlighted. Packaging your retrovirus is only the first step to ensuring successful expression of your gene. In the present study, we have examined amphotropic retrovirus receptor amphor and ecotropic retrovirus receptor mrna expression in highly purified populations of mouse and human hsc. Guidelines for working with retroviruses biomed hms. Xenotropic murine leukemia virusrelated virus wikipedia. Most retrovirus vectors are based on mumolv, an ecotropic virus, which infects only murine cells. The ecotropic receptor booster, for example, allows you to efficiently transduce human cells with ecotropic retrovirus or lentivirus that otherwise can only infect rodent cells. A recombinant adenoviral vector that expresses the receptor for amphotropic retrovirus ram was constructed and used to express the receptor cdna in. Xenotropic murine leukemia virusrelated virus xmrv is a retrovirus which was first described in 2006 as an apparently novel human pathogen found in tissue samples from men with prostate cancer. Thus, efficient transduction by an amphotropic retrovirus vector requires highlevel expression of the retrovirus receptor pit2. Select chapter 1 genetic manipulation of the nervous system.
Amphotropic and ecotropic retroviral vector viruses transduce midgestational murine fetal liver cells in a dualchambered cocultivation system skip to main content thank you for visiting. Packaging cell lines useful for the generation of helperfree recombinant retroviruses with amphotropic or ecotropic host ranges, methods of constructing such packaging cell lines and methods of using the recombinant retroviruses to introduce dna of interest into eukaryotic cells, both in vitro and in vivo. Plate and plata cells stably express the gag, pol, and env genes, allowing retroviral packaging with a single plasmid transfection to produce recombinant ecotropic and amphotropic virus, respectively. To achieve infection of human cells, the vectors are propagated in packaging cells that express the envelope of the amphotropic or nonmurine viruses such as 4070a murine leukemia virus, gibbon ape leukemia virus, vsv or the feline endogenous virus. Wo1990002806a1 recombinant retroviruses with amphotropic. Construction of a safe and efficient retrovirus packaging. Phoenixtm retroviral packaging cell lines ampho and eco. The friend virus fv is a strain of murine leukemia virus. Retrovirus entry by endocytosis and cathepsin proteases. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. The level of mrna encoding the amphotropic retrovirus. Lentiviral vectors are based on the fact that hiv virus is an. However, all work involving ecotropic viruses, hazardous genes or unknown viruses must be handled as amphotropic retroviruses.
We have identified the cellular receptor for amphotropic murine. Growth factors increase amphotropic retrovirus binding to. Retropack pt67 is the recommended packaging cell line for creating a stable producer cell line for continuous production of. This specificity is determined by envelope glycoproteins that require specific receptors in the host. Replicationcompetent retrovirus produced by a split. Amphotropic and mink cell focusforming mcf retroviruses.
Retroviruses are classified as either amphotropic or ecotropic on the basis of their species specificity. Introduction of a vector genome into this cell line can be performed simply and rapidly by crossinfection. The ecotropic viruses were the first class of mlvs to be identified. Phoenixtm retroviral packaging cell lines ampho and eco phoenix ampho and eco packaging cell lines are secondgeneration retrovirus producer lines for the generation of helper free amphotropic and ecotropic retroviruses.
Mlv reverse transcriptases are thought to have a slightly higher fidelity than the hiv1 rt. Characterization of recombination events leading to the. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. The ecotropic receptor booster is used to temporarily elevate the density of the ecotropic receptor protein mcat1 on the surface of any target cell that you wish to infect. Transduction of mouse hematopoietic stem cells is more. In addition to producing helperfree ecotropic and amphotropic retroviruses, methods are presented for rapidly pseudotyping retroviral virions with. A method for producing hightiter, helperfree infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line bosc 23 and amphotropic line cak 8, both of which cell lines and their precursor cell lines are disclosed. Because of the advantages over stable packaging cell lines, the bosc 23 and cak 8. Detection of ecotropic replicationcompetent retroviruses.
In all cases, susceptibility to infection is dominant over resistance, implying that the loci directly encode the receptors themselves. The ecotropic retroviruses have a gp70 envelope protein which binds to a specific murine cell surface receptor an amino acid transport protein. Stable expression of the ecotropic retrovirus receptor in. Pmc free article finer mh, dull tj, qin l, farson d, roberts mr. The lines are based on the 293t cell line a human embryonic kidney line transformed with adenovirus e1a and carrying a temperature sensitive t antigen coselected with neomycin. Efficient transduction by an amphotropic retrovirus vector. Retrovirus vector an overview sciencedirect topics.
Amphotropic virus definition of amphotropic virus by. Receptor interference assays demonstrated that the virus was pseudotyped with an ecotropic envelope. Ecotropic and amphotropic retrovirus packaging cell lines have been constructed in which the helper virus genome have been separated onto two plasmids. Retrovirus detection molecular diagnostic services, inc. Studies were undertaken to determine the origin and structure of this rcr.
Amphotropic retroviruses infect most mammalian cells, whereas ecotropic viruses are restricted to murine cells because of specific interaction of a viral glycoprotein and a murine cell surface cationic amino acid transporter. Amphotropic retroviruses have a different envelope. The selected cell line will detect the specific type of retrovirus. Fred hutchinson cancer research center, 1100 fairview avenue north, room c2023, seattle, wa 98109. Adenovirusmediated transfer of the amphotropic retrovirus. Transfect a platinum retrovirus packaging cell line, such as plata amphotropic or plate ecotropic, which stably expresses both mmlv gagpol and an amphotropic or ecotropic envelope protein, with a retroviral expression vector. Pantropic retrovirus may also be produced by transfecting platgp cells with the expression vector and a second vector containing vsvg. Amphotropic retrovirus transduction is inhibited by high. Crude and concentrated viral stocks were then titrated on ht1080 or nih 3t3 cells and analyzed via flow cytometry at 72 hr postinfection to. Direct focus assays using one detector cell line enable direct detection of retrovirus particles.
Murine hsc with low to undetectable levels of amphor mrna and relatively high levels of ecotropic retrovirus receptor mrna were studied. Other institutions, including yale university, allow ecotropic murine virus systems using oncogenic. When these hsc were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus. Us5955331a recombinant retroviruses with amphotropic and. Phoenix is a secondgeneration retrovirus producer lines for the generation of helper free ecotropic and amphotropic retroviruses.
Amphotropic and ecotropic retroviral vector viruses transduce midgestational murine fetal liver cells in a dualchambered cocultivation system skip to. Amphotropic or replicationcompetent murine retroviruses will require bsl2absl2 containment and handling practices. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus induced diseases and efficient exploitation of retroviral vectors. Ecotropism or ecotropic from eco hearth and tropic to turn towards refers to the philosophy that for human culture to be healthy, it must exist as in an ecological niche and thereby relate appropriately with all the fields of forces of nature, organic and inorganic. They are currently used in over 2500 laboratories worldwide for delivery of. Thus we have the means to deliver large libraries of retroviruses into nearly any mammalian cell type, mouse or human. Wo1994019478a1 production of high titer helperfree. Finally, we have tested the titres produced by these lines. The presence of functional amphotropic receptors on the cell surface is necessary for amphotropic retrovirus mediated gene transfer. The lines are based on the 293t cell line a human embryonic kidney line transformed with adenovirus e1a and carrying a temperature sensitive t. Unlike vsvgpseudotyped retrovirus, ecotropic and amphotropic retrovirus is not stable even when stored at 80. Infects mammalian cell types, including human cells and requires that the retrovirale virus contains amphotropic envelope proteins.
University of pittsburgh institutional biosafety committee. Receptor booster technologies are used to temporarily elevate the density of viral receptor proteins on the surface of any target cell that you wish to infect. For over 25 years the study of retroviruses has underpinned much of what is known about information transfer in cells and the genetic and biochemical mechanisms that underlie cell growth and cancer induction. Cloning of the cellular receptor for amphotropic murine retrovirus reveals homology to that for gibbon ape leukemia virus. They comprise some of the most intensely studied retroviruses, including exogenous viruses such as moloney mlv momlv and friend mlv frmlv, as well as the endogenous virus of the akr mouse strain, akr mlv akv. Cellsurface receptors for retroviruses and implications.
Initial reports erroneously linked the virus to prostate cancer and later to chronic fatigue syndrome cfs, leading to considerable interest in the scientific and patient communities, investigation. Selective gene expression hollandfrei cancer medicine. Ecotropic retrovirus is infectious only to rodent cells, while amphotropic retrovirus has a very broad host range including humans. In summary, a new amphotropic packaging cell line was established, which can be infected with ecotropic retroviral vectors due to its expression of the ecotropic retrovirus receptor. As with other retroviruses, the mlvs replicate their genomes with relatively low fidelity. The level of mrna encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction donald orlict, laurie j.
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