Title: Neuropathologies in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 Tat Protein under the Regulation of the Astrocyte-Specific Glial Fibrillary Acidic Protein Promoter and Doxycycline
Abstract: The human immunodeficiency virus type 1 (HIV-1) Tat protein is a key pathogenic factor in a variety of acquired immune deficiency syndrome (AIDS)-associated disorders. A number of studies have documented the neurotoxic property of Tat protein, and Tat has therefore been proposed to contribute to AIDS-associated neurological diseases. Nevertheless, the bulk of these studies are performed in in vitro neuronal cultures without taking into account the intricate cell-cell interaction in the brain, or by injection of recombinant Tat protein into the brain, which may cause secondary stress or damage to the brain. To gain a better understanding of the roles of Tat protein in HIV-1 neuropathogenesis, we attempted to establish a transgenic mouse model in which Tat expression was regulated by both the astrocyte-specific glial fibrillary acidic protein promoter and a doxycycline (Dox)-inducible promoter. In the present study, we characterized the phenotypic and neuropathogenic features of these mice. Both in vitro and in vivo assays confirmed that Tat expression occurred exclusively in astrocytes and was Dox-dependent. Tat expression in the brain caused failure to thrive, hunched gesture, tremor, ataxia, and slow cognitive and motor movement, seizures, and premature death. Neuropathologies of these mice were characterized by breakdown of cerebellum and cortex, brain edema, astrocytosis, degeneration of neuronal dendrites, neuronal apoptosis, and increased infiltration of activated monocytes and T lymphocytes. These results together demonstrate that Tat expression in the absence of HIV-1 infection is sufficient to cause neuropathologies similar to most of those noted in the brain of AIDS patients, and provide the first evidence in the context of a whole organism to support a critical role of Tat protein in HIV-1 neuropathogenesis. More importantly, our data suggest that the Dox inducible, brain-targeted Tat transgenic mice offer an in vivo model for delineating the molecular mechanisms of Tat neurotoxicity and for developing therapeutic strategies for treating HIV-associated neurological disorders. The human immunodeficiency virus type 1 (HIV-1) Tat protein is a key pathogenic factor in a variety of acquired immune deficiency syndrome (AIDS)-associated disorders. A number of studies have documented the neurotoxic property of Tat protein, and Tat has therefore been proposed to contribute to AIDS-associated neurological diseases. Nevertheless, the bulk of these studies are performed in in vitro neuronal cultures without taking into account the intricate cell-cell interaction in the brain, or by injection of recombinant Tat protein into the brain, which may cause secondary stress or damage to the brain. To gain a better understanding of the roles of Tat protein in HIV-1 neuropathogenesis, we attempted to establish a transgenic mouse model in which Tat expression was regulated by both the astrocyte-specific glial fibrillary acidic protein promoter and a doxycycline (Dox)-inducible promoter. In the present study, we characterized the phenotypic and neuropathogenic features of these mice. Both in vitro and in vivo assays confirmed that Tat expression occurred exclusively in astrocytes and was Dox-dependent. Tat expression in the brain caused failure to thrive, hunched gesture, tremor, ataxia, and slow cognitive and motor movement, seizures, and premature death. Neuropathologies of these mice were characterized by breakdown of cerebellum and cortex, brain edema, astrocytosis, degeneration of neuronal dendrites, neuronal apoptosis, and increased infiltration of activated monocytes and T lymphocytes. These results together demonstrate that Tat expression in the absence of HIV-1 infection is sufficient to cause neuropathologies similar to most of those noted in the brain of AIDS patients, and provide the first evidence in the context of a whole organism to support a critical role of Tat protein in HIV-1 neuropathogenesis. More importantly, our data suggest that the Dox inducible, brain-targeted Tat transgenic mice offer an in vivo model for delineating the molecular mechanisms of Tat neurotoxicity and for developing therapeutic strategies for treating HIV-associated neurological disorders. Human immunodeficiency virus type 1 (HIV-1) Tat protein plays an important role in the pathogenesis of a number of acquired immune deficiency syndrome (AIDS)-related disorders.1Rappaport J Joseph J Croul S Alexander G Del Valle L Amini S Khalili K Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat.J Leukoc Biol. 1999; 65: 458-465PubMed Google Scholar, 2Nath A Mattson MP Magnuson DSK Jones M Berger JR Role of viral proteins in HIV-1 neuropathogenesis with emphasis on Tat.NeuroAIDS. 1998; : 1Google Scholar As one of the early HIV-1 proteins translated from the multiply spliced viral RNA transcripts,3Sodroski J Patarca R Rosen C Wong-Staal F Haseltine W Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III.Science. 1985; 229: 74-77Crossref PubMed Scopus (354) Google Scholar Tat transactivates HIV-1 gene expression through interactions with the transactivation responsive element TAR within the HIV-1 long terminal repeat promoter, human cyclin T1, and CDK9.4Wei P Garber ME Fang SM Fischer WH Jones KA A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1041) Google Scholar This involves recruitment of an essential multicomponent factor, termed positive transcription elongation factor b (P-TEFb) to the HIV-1 long terminal repeat promoter, and phosphorylation of the C-terminal domain of RNA polymerase II (Pol II). In addition to being a transactivator of HIV-1 gene expression, Tat has also been documented to exert pleiotropic effects on host cells, through direct modulation of gene expression by Tat uptake from extracellular microenvironment, and/or intracellular signaling elicited by interaction of extracellular Tat protein with cell surface receptors.5Cupp C Taylor JP Khalili K Amini S Evidence for stimulation of the transforming growth factor beta 1 promoter by HIV-1 Tat in cells derived from CNS.Oncogene. 1993; 8: 2231PubMed Google Scholar, 6Ganju RK Munshi N Nair BC Liu ZY Gill P Groopman JE Human immunodeficiency virus Tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells.J Virol. 1998; 72: 6131-6137Crossref PubMed Google Scholar, 7Milani D Zauli G Neri LM Marchisio M Previati M Capitani S Influence of the human immunodeficiency virus type 1 Tat protein on the proliferation and differentiation of PC12 rat pheochromocytoma cells.J Gen Virol. 1993; 74: 2587-2594Crossref PubMed Scopus (40) Google Scholar, 8Zauli G Gibellini D Milani D Mazzoni M Borgatti P La Placa M Capitani S Human immunodeficiency virus type 1 Tat protein protects lymphoid, epithelial, and neuronal cell lines from death by apoptosis.Cancer Res. 1993; 53: 4481-4485PubMed Google Scholar Corroborated with these functions, HIV-1 Tat has been demonstrated as secreted from Tat-expressing cells9Ensoli B Barillari G Salahuddin SZ Gallo RC Wong-Staal F Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients.Nature. 1990; 345: 84-86Crossref PubMed Scopus (793) Google Scholar, 10Chang HC Samaniego F Nair BC Buonaguro L Ensoli B HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region.AIDS. 1997; 11: 1421-1431Crossref PubMed Scopus (391) Google Scholar, 11Zauli G La Placa M Vignoli M Re MC Gibellini D Furlini G Milani D Marchisio M Mazzoni M Capitani S An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity.J Acquir Immune Defic Syndr Hum Retrovirol. 1995; 10: 306-316Crossref PubMed Scopus (68) Google Scholar and HIV-infected cells,12Westendorp MO Frank R Ochsenbauer C Stricker K Dhein J Walczak H Debatin KM Krammer PH Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.Nature. 1995; 375: 497-500Crossref PubMed Scopus (910) Google Scholar, 13Xiao H Neuveut C Tiffany HL Benkirane M Rich EA Murphy PM Jeang KT Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1.Proc Natl Acad Sci USA. 2000; 97: 11466-11471Crossref PubMed Scopus (316) Google Scholar and as being capable of entering cells in a biologically active form.14Frankel AD Pabo CO Cellular uptake of the Tat protein from human immunodeficiency virus.Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2285) Google Scholar, 15Liu Y Jones M Hingtgen CM Bu G Laribee N Tanzi RE Moir RD Nath A He JJ Uptake of HIV-1 Tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.Nat Med. 2000; 6: 1380-1387Crossref PubMed Scopus (329) Google ScholarHIV-1 infects the central nervous system (CNS) of a majority of AIDS patients,16Gabuzda DH Hirsch MS Neurologic manifestations of infection with human immunodeficiency virus. 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Thus, many indirect mechanisms have been explored and/or proposed for HIV-1 infection-induced neuropathogenesis. Those include HIV-1 viral proteins gp120 and Tat, and cellular factors secreted from HIV-infected macrophages/microglia and astrocytes, such as tumor necrosis factor-α, platelet-activating factor, arachidonic acid metabolites, oxygen-free radicals, nitric oxide, excitatory amino acids, and chemokines.22Epstein LG Gendelman HE Human immunodeficiency virus type 1 infection of the nervous system: pathogenetic mechanisms [see comments].Ann Neurol. 1993; 33: 429-436Crossref PubMed Scopus (347) Google Scholar, 23Genis P Jett M Bernton EW Boyle T Gelbard HA Dzenko K Keane RW Resnick L Mizrachi Y Volsky DJ Cytokines and arachidonic metabolites produced during human immunodeficiency virus (HIV)-infected macrophage-astroglia interactions: implications for the neuropathogenesis of HIV disease.J Exp Med. 1992; 176: 1703-1718Crossref PubMed Scopus (494) Google Scholar, 28Bukrinsky MI Nottet HS Schmidtmayerova H Dubrovsky L Flanagan CR Mullins ME Lipton SA Gendelman HE Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease.J Exp Med. 1995; 181: 735-745Crossref PubMed Scopus (313) Google Scholar, 29Gelbard HA Nottet HS Swindells S Jett M Dzenko KA Genis P White R Wang L Choi YB Zhang D Platelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin.J Virol. 1994; 68: 4628-4635Crossref PubMed Google Scholar, 30Giulian D Lachman LB Interleukin-1 stimulation of astroglial proliferation after brain injury.Science. 1985; 228: 497-499Crossref PubMed Scopus (761) Google Scholar, 31Lipton SA Sucher NJ Kaiser PK Dreyer EB Synergistic effects of HIV coat protein and NMDA receptor-mediated neurotoxicity.Neuron. 1991; 7: 111-118Abstract Full Text PDF PubMed Scopus (361) Google Scholar, 32Pulliam L Herndier BG Tang NM McGrath MS Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical alterations in cultured human brains.J Clin Invest. 1991; 87: 503-512Crossref PubMed Scopus (249) Google Scholar, 33Toggas SM Masliah E Rockenstein EM Rall GF Abraham CR Mucke L Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice [see comments].Nature. 1994; 367: 188-193Crossref PubMed Scopus (601) Google Scholar, 34Conant K Garzino-Demo A Nath A McArthur JC Halliday W Power C Gallo RC Major EO Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia.Proc Natl Acad Sci USA. 1998; 95: 3117-3121Crossref PubMed Scopus (501) Google Scholar However, the precise in vivo role of these factors in contributing to HIV-associated CNS injury remains to be defined.A number of studies have implicated Tat protein in HIV-induced neuropathogenesis. Tat is neurotoxic in vitro.1Rappaport J Joseph J Croul S Alexander G Del Valle L Amini S Khalili K Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat.J Leukoc Biol. 1999; 65: 458-465PubMed Google Scholar, 2Nath A Mattson MP Magnuson DSK Jones M Berger JR Role of viral proteins in HIV-1 neuropathogenesis with emphasis on Tat.NeuroAIDS. 1998; : 1Google Scholar When injected into the brain, Tat protein causes histological changes similar to those seen in HIV-demented patients.1Rappaport J Joseph J Croul S Alexander G Del Valle L Amini S Khalili K Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat.J Leukoc Biol. 1999; 65: 458-465PubMed Google Scholar, 35Jones M Olafson K Del Bigio MR Peeling J Nath A Intraventricular injection of human immunodeficiency virus type 1 (HIV-1) Tat protein causes inflammation, gliosis, apoptosis, and ventricular enlargement.J Neuropathol Exp Neurol. 1998; 57: 563-570Crossref PubMed Scopus (143) Google Scholar A recent study has suggested a positive correlation between the levels of Tat mRNA transcript and HIV- and simian-human immunodeficiency virus-induced encephalitis.36Hudson L Liu J Nath A Jones M Raghavan R Narayan O Male D Everall I Detection of the human immunodeficiency virus regulatory protein Tat in CNS tissues.J Neurovirol. 2000; 6: 145-155Crossref PubMed Scopus (197) Google Scholar The mechanisms proposed for Tat neurotoxicity include direct depolarization of neurons, increased levels of intracellular calcium, increased production/release of proinflammatory cytokines, increased infiltration of macrophages/monocytes, activation of excitatory amino acid receptors, and increased apoptosis.2Nath A Mattson MP Magnuson DSK Jones M Berger JR Role of viral proteins in HIV-1 neuropathogenesis with emphasis on Tat.NeuroAIDS. 1998; : 1Google Scholar However, the argument has always been whether Tat is present within the CNS or cerebrospinal fluid in HIV-infected individuals at sufficient concentrations, to directly exhibit the acute neurotoxicity observed in most of those studies. Meanwhile, independent studies have shown that at lower concentrations Tat exhibits no acute neurotoxic activity, and instead transactivates gene expression and induces cell proliferation, differentiation, adhesion, and morphological changes.7Milani D Zauli G Neri LM Marchisio M Previati M Capitani S Influence of the human immunodeficiency virus type 1 Tat protein on the proliferation and differentiation of PC12 rat pheochromocytoma cells.J Gen Virol. 1993; 74: 2587-2594Crossref PubMed Scopus (40) Google Scholar, 37Helland DE Welles JL Caputo A Haseltine WA Transcellular transactivation by the human immunodeficiency virus type 1 Tat protein.J Virol. 1991; 65: 4547-4549Crossref PubMed Google Scholar, 38Ensoli B Buonaguro L Barillari G Fiorelli V Gendelman R Morgan RA Wingfield P Gallo RC Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.J Virol. 1993; 67: 277-287Crossref PubMed Google Scholar, 39Kolson DL Buchhalter J Collman R Hellmig B Farrell CF Debouck C Gonzalez-Scarano F HIV-1 Tat alters normal organization of neurons and astrocytes in primary rodent brain cell cultures: rGD sequence dependence.AIDS Res Hum Retroviruses. 1993; 9: 677-685Crossref PubMed Scopus (62) Google Scholar In agreement with these observations, our recent studies suggest that Tat may affect neuronal function through direct uptake of extracellular Tat protein and subsequent disruption of neuronal metabolic balance of low-density lipoprotein receptor-related protein physiological ligands.15Liu Y Jones M Hingtgen CM Bu G Laribee N Tanzi RE Moir RD Nath A He JJ Uptake of HIV-1 Tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.Nat Med. 2000; 6: 1380-1387Crossref PubMed Scopus (329) Google Scholar In addition, an overwhelming majority of the studies on Tat neurotoxicity has only focused on Tat interaction with neurons, and has not taken into account the interaction among neurons, astrocytes, and other brain cells. Nevertheless, it is certain that Tat interaction with astrocytes and other brain cells including endothelial cells also has adverse effects on neuronal survival and function.34Conant K Garzino-Demo A Nath A McArthur JC Halliday W Power C Gallo RC Major EO Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia.Proc Natl Acad Sci USA. 1998; 95: 3117-3121Crossref PubMed Scopus (501) Google Scholar, 40Taylor JP Cupp C Diaz A Taylor JP Cupp C Diaz A Chowdhury M Khalili K Jimenez SA Amini S Activation of expression of genes coding for extracellular matrix proteins in Tat-producing glioblastoma cells.Proc Natl Acad Sci USA. 1992; 89: 9617-9621Crossref PubMed Scopus (89) Google Scholar, 41Hofman FM Chen P Incardona F Zidovetzki R Hinton DR HIV-1 Tat protein induces the production of interleukin-8 by human brain-derived endothelial cells.J Neuroimmunol. 1999; 94: 28-39Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 42Zidovetzki R Wang JL Chen P Jeyaseelan R Hofman F Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways.AIDS Res Hum Retroviruses. 1998; 14: 825-833Crossref PubMed Scopus (76) Google Scholar, 43Kutsch O Oh J Nath A Benveniste EN Induction of the chemokines interleukin-8 and IP-10 by human immunodeficiency virus type 1 Tat in astrocytes.J Virol. 2000; 74: 9214-9221Crossref PubMed Scopus (154) Google ScholarThus, it has become increasingly evident that the bona fide mechanisms of Tat neurotoxicity should be addressed in the context of a whole organism. There are several Tat transgenic mouse models available,44Choi J Liu RM Kundu RK Sangiorgi F Wu W Maxson R Forman HJ Molecular mechanism of decreased glutathione content in human immunodeficiency virus type 1 Tat-transgenic mice.J Biol Chem. 2000; 275: 3693-3698Crossref PubMed Scopus (145) Google Scholar, 45Garza Jr, HH Prakash O Carr DJ Aberrant regulation of cytokines in HIV-1 TAT72-transgenic mice.J Immunol. 1996; 156: 3631-3637PubMed Google Scholar, 46Vellutini C Horschowski N Philippon V Gambarelli D Nave KA Filippi P Development of lymphoid hyperplasia in transgenic mice expressing the HIV Tat gene.AIDS Res Hum Retroviruses. 1995; 11: 21-29Crossref PubMed Scopus (31) Google Scholar, 47Vogel J Hinrichs SH Reynolds RK Luciw PA Jay G The HIV Tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice.Nature. 1988; 335: 606-611Crossref PubMed Scopus (458) Google Scholar but none of them is suitable for studying Tat neurotoxicity. In those models Tat expression occurs constitutively throughout development and in all or most of tissues. As a result, any phenotypic abnormalities observed in the brain or a particular brain region could be caused by, or complicated by, abnormalities that occur in the brain any time during development or that exist in any other tissues or organs of the animal. In the present study we modified the doxycycline (Dox)-regulated gene expression strategy48Gossen M Bujard H Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.Proc Natl Acad Sci USA. 1992; 89: 5547-5551Crossref PubMed Scopus (4227) Google Scholar with a brain-specific promoter, ie, the astrocyte-specific glial fibrillary acid protein (GFAP) promoter,33Toggas SM Masliah E Rockenstein EM Rall GF Abraham CR Mucke L Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice [see comments].Nature. 1994; 367: 188-193Crossref PubMed Scopus (601) Google Scholar, 49Johnson WB Ruppe MD Rockenstein EM Price J Sarthy VP Verderber LC Mucke L Indicator expression directed by regulatory sequences of the glial fibrillary acidic protein (GFAP) gene: in vivo comparison of distinct GFAP-lacZ transgenes.Glia. 1995; 13: 174-184Crossref PubMed Scopus (74) Google Scholar, 50Mucke L Abraham CR Ruppe MD Rockenstein EM Toggas SM Mallory M Alford M Masliah E Protection against HIV-1 gp120-induced brain damage by neuronal expression of human amyloid precursor protein.J Exp Med. 1995; 181: 1551-1556Crossref PubMed Scopus (88) Google Scholar and generated the Dox-inducible and brain-targeted Tat transgenic mouse model. Characterization of phenotypes and neuropathologies of the transgenic mice concludes that Tat protein is a critical pathogenic factor in HIV-associated neuropathogenesis. More importantly, our data suggest that the unique Tat transgenic mouse model is very valuable for understanding the molecular mechanisms of Tat neurotoxicity, and for developing therapeutics for treating HIV-associated neurological diseases.Materials and MethodsPlasmid ConstructionThe murine GFAP promoter in C-3123 plasmid (a gift from Dr. Lennart Mucke, University of California, San Francisco, CA49Johnson WB Ruppe MD Rockenstein EM Price J Sarthy VP Verderber LC Mucke L Indicator expression directed by regulatory sequences of the glial fibrillary acidic protein (GFAP) gene: in vivo comparison of distinct GFAP-lacZ transgenes.Glia. 1995; 13: 174-184Crossref PubMed Scopus (74) Google Scholar) was released by restriction digestion with EcoRI and BamHI, and cloned into the pTeton (Clontech, Palo Alto, CA) in place of the cytomegalovirus promoter. The recombinant plasmid was designed as pTeton-GFAP. The standard polymerase chain reaction (PCR) cloning technique was used to construct pTRE-Tat86 plasmid. Briefly, HIV-1 Tat gene encoding 86 amino acids from both the first and second exons of HIV-1 isolate HXB2 was obtained by PCR using pBD-Tat8615Liu Y Jones M Hingtgen CM Bu G Laribee N Tanzi RE Moir RD Nath A He JJ Uptake of HIV-1 Tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.Nat Med. 2000; 6: 1380-1387Crossref PubMed Scopus (329) Google Scholar as the template and primers 5′-GGA ATT CAC CAT GGA GCC AGT AGA TCC T-3′ (EcoRI site underlined) and 5′-CGG GAT CCC TAT TCC TTC GGG CCT GT-3′ (BamHI site underlined), and then cloned into EcoRI- and BamHI-digested pTRE vector backbone (Clontech). Similarly, we also constructed a control plasmid pTRE-CAT expressing the chloramphenicol acetyltransferase (CAT) reporter gene. All recombinant plasmids were verified by sequencing.Cell Cultures, Transfection, and the CAT AssayHeLa and U87.MG cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37°C with 5% CO2. HeLa (1 × 106 cells/100 mm plate) and U87.MG (0.5 × 106 cells/100 mm plate) cells were transfected with plasmid DNAs as indicated using the standard calcium phosphate method. Transfected cells were cultured in the presence of Dox (1 μg/ml) for 48 hours, and harvested for the CAT reporter gene assay using the phase extraction and direct scintillation counting method as previously described.51He J Furmanski P Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA.Nature. 1995; 373: 721-724Crossref PubMed Scopus (321) Google Scholar For U87.MG/iTat stable cells line, we first transfected U87.MG cells with the regulator plasmid pTeton-GFAP, and selected in the presence of 200 μg/ml of G418 for the stable transfectants, called U87.MG/GFAP cells. The stable transfectants were then functionally characterized by transient transfection of the expressor plasmid pTRE-CAT. The cell line with the highest transactivation of the CAT expression in the presence of Dox was selected and transfected with the expressor plasmid pTRE-Tat86. The selection was performed in the presence of 50 μg/ml of hygromycin (Life Technologies, Inc., Gaithersburg, MD) because a plasmid expressing the hygromycin-resistant gene (Clontech) was included in the transfection. The resulting stable cell line was designated U87.MG/iTat cells.Creation of Inducible Tat Transgenic MiceInducible Tat transgenic mouse colonies (GT-tg) were obtained by generation of two separate transgenic lines Teton-GFAP mice (G-tg) and TRE-Tat86 mice (T-tg), and then cross-breeding of these two lines of transgenic mice. Briefly, a DNA fragment (2238 bp) containing the Teton-GFAP gene, along with downstream simian virus 40 splicing and polyadenylation sequences, was released by XhoI and PvuII digestion of the pTeton-GFAP plasmid and purified by agarose gel electrophoresis, and microinjected into fertilized eggs of F1 females obtained from mating between C3HeB and FeJ mice (Jackson Laboratories, Bar Harbor, ME). Founder transgenic animals were crossed with C57BL/6 mice to generate stable G-tg transgenic lines. Similarly, T-tg transgenic lines were obtained using a DNA fragment (1189 bp) released by XhoI and PvuII digestion of the pTRE-Tat86 plasmid. Found