Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-11-03
2004-05-25
Lankford, L Blaine (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S235100, C435S352000, C435S455000
Reexamination Certificate
active
06740519
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to immortalized STAT1-deficient mammalian cell lines. STAT1 is a signal transducer and activator of transcription that becomes phosphorylated when cells are treated with type I or type II interferons and leads to induction of specific gene expression, resulting in establishment of the antiviral state and the other known biological responses to interferons, including the inhibition of cell proliferation. Cells which lack this gene product are useful for producing high titers of viral stocks, for producing recombinant viral vectors, for testing samples, especially clinical samples for the presence of virus and for screening candidate compounds or drugs for anti-viral activity.
BACKGROUND OF THE INVENTION
Interferon (IFN) treatment of cells leads to activation of a family of proteins termed signal transducers and activators of transcription, or STATs. The STAT proteins play a role in a cascade of events that leads to transcription of IFN stimulated genes (ISGs). The ISGs then mediate a multitude of well-known cellular responses to IFN such as induction of an antiviral state, inhibition of cellular proliferation, immune modulation, differentiation and resistance to bacterial and parasitic infections.
IFN induction of gene expression occurs through the Jak-STAT pathway. The molecular basis of this signal transduction and transcriptional activation pathway has been extensively studied and is reviewed by Levy (1995) Semin. Virol. 6:181-189 and Bluyssen et al. (1996) Cytokine Growth Factor Rev. 7:11-17.
STATs are constitutively-produced cytoplasmic proteins which are activated by tyrosine phosphorylation upon binding of IFN to its receptor. Cells treated with type I IFN(the family of proteins known as IFN &agr; and IFN &bgr;) phosphorylate STAT1 and STAT2, whereas cells treated with type II IFN (or IFN &ggr;) only phosphorylate STAT1. Evidence strongly suggests that phosphorylation is mediated by the Jak family of protein kinases which are associated with the IFN receptors and autophosphorylate when cells are treated with IFN.
Once activated, the STATs multimerize, translocate to the cell nucleus and form transcription factor complexes which bind to specific sequences of DNA in a manner dependent upon the type of IFN that stimulated the cells. In the absence of IFN stimulation, the STAT proteins do not exhibit complex formation, nuclear localization or DNA binding.
In addition to activation by the IFN system, STAT1 is also activated by a variety of cytokines and growth factors, including IL-6, leukemia inhibitory factor (LIF), oncostatin M, growth hormone, IL-10, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1) and angiotensin II [Durbin et al. (1996) Cell 84: 443-450].
Recently, STAT1 knockout mice were prepared (Durbin, 1996). STAT1 knock out mice are homozygous for a null allele of the murine Stat1 gene, i.e., Stat1
−/−
, and produced by targeted disruption of the Stat1 gene. The cDNA sequence of the murine Stat1 gene is available as GenBank accession number U06924. The disrupted Stat1 gene was cloned into tranfection vector pPNT. That linearized construct was transfected into murine embryonic stem cells (ES cells) and cultured in the presence of G148 and gancyclovir. Homozygous ES cell lines were isolated by culturing with high concentrations of G148.
The Stat1
−/−
animals were obtained by injection of heterozygous Stat1
+/−
ES cells into normal mouse blastocysts and interbreeding to produce the homozygous progeny. The STAT1 knockout mice were born at normal frequency, had no gross developmental defects (as might be expected if the cytokine or growth factor signaling pathways sensitive to STAT1 had been disrupted) but were highly susceptible to viral diseases, including mouse hepatitis virus (MHV), vesicular stomatitis virus (VSV) and influenza virus. There was no transcriptional response to IFN in isolated tissues (splenocytes and macrophages) of STAT1 knockout mice. However, when the macrophages were treated with IL-6, a cytokine, the transcriptional response was normal.
SUMMARY OF THE INVENTION
This invention is directed to immortalized Stat1
+/+
mammalian cell lines preferably of murine or human origin. Such cell lines can be obtained from STAT1 knockout animals or can be prepared by converting cultured cells to homozygosity for a Stat1 null allele, followed by immortalization if necessary. Immortalized cell lines can be obtained spontaneously or by transformation with a transforming agent such as SV40 T antigen or other oncogene.
The cell lines of the invention are preferably endothelial cells, epithelial cells, hematopoetic cells, bone marrow cells, kidney cells or liver cells. Most preferably the cell lines are murine or human fibroblasts and bone marrow cells.
Another aspect of the invention relates to a method of producing a viral stock by (a) infecting immortalized Stat1
−/−
mammalian cells of the invention with a virus, (b) culturing the infected cells under conditions and for a time sufficient to allow replication of that virus and (c) recovering the so-produced virus to obtain the viral stock. The cells can be either adherent cells or non-adherent cells. Infections are typically done at a multiplicity of infection (MOI) of about one or less and can result in viral titers ranging from about 10
2
plaque forming units per milliliter (PFU/mL) to more than 10
6
(PFU/mL) depending on the virus, the MOI and growth conditions.
The cell lines of the invention are particularly useful for producing viral stocks from a wide variety of viruses, including viruses not typically grown in that cell type since the STAT1-deficient cell lines show altered viral tropism. Hence, viral stocks can be prepared, for example, for influenza virus, parainfluenza virus, measles virus, respiratory syncytial virus (RSV), hepatitis viruses, adenovirus, herpes viruses or vesicular stomatitis virus.
Yet another aspect of this invention is directed to a method of producing a recombinant viral vector by (a) infecting or transfecting immortalized Stat1
−/−
mammalian cells with the recombinant viral vector, (b) culturing those cells under conditions and for a time suffcient to allow replication of that vector, and (c) recovering the vector. The method is applicable to recombinant viral DNA and RNA vectors, and is particularly useful for vectors such as adenovirus vectors, retrovirus vectors or sindbis virus vectors. Vectors which can be used in gene therapy can also be prepared by this method.
Still further the present invention provides a sensitive method for detecting the presence, absence or quantity of a virus in a sample by (a) contacting immortalized Stat1
−/−
mammalian cells with a test sample, (b) culturing those cells under conditions and for a time to allow replication of any virus that may be present in the test sample, and (c) recovering, if necessary, and identifying and/or quantitating the virus. The test sample is typically a clinical sample and can be treated to remove particulates or a viral extract can be prepared therefrom and used as the testing sample. Clinical samples include but are not limited to body fluids, body tissues or other bodily materials. The identity of the virus can be determined by immunoassay, polymerase chain reaction or nucleic acid hybridization using a viral-specific reagent. When desired, quantitation of the virus can be accomplished by serial dilution of the test sample and culturing as above to determine the end point of viral infection.
Yet still another aspect of the invention is directed to providing a method for screening compounds for antiviral activity. Immortalized Stat1
−/−
mammalian cells are treated with a candidate compound and infected with a virus against which antiviral activity is sought. The cells can be exposed to the compound for various periods of time prior to, concurrently, or after viral infection. The cells are cultu
Durbin Joan Elizabeth
Garcia-Sastre Adolfo
Levy David
Palese Peter
Kenyon & Kenyon
Lankford L Blaine
Mount Sinai School of Medicine
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