Use of retroviral vectors in studying gag-specific CTL in cats infected with feline immunodeficiency virus /
Retroviral vectors were used to transfer the gag gene of feline immunodeficiency virus (FIV) into feline cell lines and feline primary T lymphocytes. The gag gene was first cloned into the retroviral vector pB2NH4 to produce pB2NH4G. Several clones of B2NH4G vector virus-producer cells that produce...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742161921&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Retroviral vectors were used to transfer the gag gene of feline immunodeficiency virus (FIV) into feline cell lines and feline primary T lymphocytes. The gag gene was first cloned into the retroviral vector pB2NH4 to produce pB2NH4G. Several clones of B2NH4G vector virus-producer cells that produced B2NH4G vector virus with a titer of at least I X 105 cfu/nil were generated with the T-CREP amphotropic packaging cell line. Transduction of feline cell lines with the B2NH4G vector virus was verified with PCR using FIV gag-specific primers. Northern blot analysis with FIV gag-specific probe detected full length vector RNA but not the gag niRNA. With NeoR-specific probe, the detection of an RNA species of a smaller size than that of the predicted NeoR RNA indicated that the FIV gag MRNA may be spliced. An alternative retroviral vector, PLGSN, was constructed after deleting the splicing donor site upstream of the FIV gag open reading frame (ORF). Feline cells including the primary feline T lymphocytes were transduced with LGSN vector virus. Western blot indicated that the FIV core polyprotein was expressed in the transduced cells. Using the retroviral vector transduced, autologous feline T cells as antigen presenting stimulator cells (APSC), cytotoxic T lymphocytes (CTL) that specifically lysed autologous FIV infected targets were induced in vitro from PBMC of FIV-infected but not uninfected cats. CTL from at least two of the FIV infected cats also specifically lysed core antigenexpressing target cells. Similar CTL were not induced by cells transduced with Retroviral vectors were used to transfer the gag gene of feline immunodeficiency virus (FIV) into feline cell lines and feline primary T lymphocytes. The gag gene was first cloned into the retroviral vector pB2NH4 to produce pB2NH4G. Several clones of B2NH4G vector virus-producer cells that produced B2NH4G vector virus with a titer of at least I X 105 cfu/@ were generated with the T-CREP amphotropic packaging cell line. Transduction of feline cell lines with the B2NH4G vector virus was verified with PCR using FIV gag-specific primers. Northern blot analysis with FIV gag-specific probe detected full length vector RNA but not the gag niRNA. With NeoR-specific probe, the detection of an RNA species of a smaller size than that of the predicted NeoR RNA indicated that the FIV gag MRNA may be spliced. An alternative retroviral vector, PLGSN, was constructed after deleting the splicing donor site upstream of the FIV gag open reading frame (ORF). Feline cells including the primary feline T lymphocytes were transduced with LGSN vector virus. Western blot indicated that the FIV core polyprotein was expressed in the transduced cells. Using the retroviral vector transduced, autologous feline T cells as antigen presenting stimulator cells (APSC), cytotoxic T lymphocytes (CTL) that specifically lysed autologous FIV infected targets were induced in vitro from PBMC of FIV-infected but not uninfected cats. CTL from at least two of the FIV infected cats also specifically lysed core antigenexpressing target cells. Similar CTL were not induced by cells transduced with a Retroviral vectors were used to transfer the gag gene of feline immunodeficiency virus (FIV) into feline cell lines and feline primary T lymphocytes. The gag gene was first cloned into the retroviral vector pB2NH4 to produce pB2NH4G. Several clones of B2NH4G vector virus-producer cells that produced B2NH4G vector virus with a titer of at least I X 105 cfu/ml were generated with the T-CREP amphotropic packaging cell line. Transduction of feline cell lines with the B2NH4G vector virus was verified with PCR using FIV gag-specific primers. Northern blot analysis with FIV gag-specific probe detected full length vector RNA but not the gag niRNA. With NeoR-specific probe, the detection of an RNA species of a smaller size than that of the predicted NeoR RNA indicated that the FIV gag MRNA may be spliced. An alternative retroviral vector, PLGSN, was constructed after deleting the splicing donor site upstream of the FIV gag open reading frame (ORF). Feline cells including the primary feline T lymphocytes were transduced with LGSN vector virus. Western blot indicated that the FIV core polyprotein was expressed in the transduced cells. Using the retroviral vector transduced, autologous feline T cells as antigen presenting stimulator cells (APSC), cytotoxic T lymphocytes (CTL) that specifically lysed autologous FIV infected targets were induced in vitro from PBMC of FIV-infected but not uninfected cats. CTL from at least two of the FIV infected cats also specifically lysed core antigenexpressing target cells. Similar CTL were not induced by cells transduced with a retroviral vector without the FIV gag sequence. The lysis was NMC-restricted and the in vitro induction resulted in an apparent increase of the percentage of CD8+ cells. 'Me successful expression of FIV core polyprotein in primary feline T lymphocytes and the detection of FIV-specific and FIV gag-specific CTL in infected cats using the transgenic cells may serve as a model for studying CTL against individual FIV proteins and may provide useful information for examining HIV-specific CTL in HIV infected humans. |
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| Item Description: | Vita. "Major Subject: Veterinary Microbiology". |
| Physical Description: | x, 146 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |