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Results for “transducer selectivity”

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At least 109 records · Page 6Linked to original sources
PubMed1999

Transduction of dendritic cell progenitors with a retroviral vector encoding viral interleukin-10 and enhanced green fluorescent protein allows purification of potentially tolerogenic antigen-presenting cells.

BACKGROUND: Dendritic cells (DC) are important antigen-presenting cells that play critical roles in the initiation and modulation of immune responses. Genetic engineering of DC to express immunosuppressive molecules is a novel approach to the inhibition of allograft rejection. Retroviral delivery of viral interleukin (vIL)-10 to replicating myeloid DC progenitors (DCp) impairs their T-cell stimulatory capacity and promotes the induction of antigen-specific T-cell hyporesponsiveness. However, transduction efficiency with retroviral vectors is comparatively low. Enhanced green fluorescent protein (EGFP) is important both as a marker of gene transduction and for the selection of transduced cells. Our aims were to construct a retroviral vector encoding both vIL-10 and EGFP, to positively select transduced DC, and to assess the impact of these highly purified, vIL-10-secreting antigen-presenting cells on allogeneic T-cell responses. METHODS: DCp propagated from bone marrow of C57BL10 (H2b) mice in granulocyte-macrophage colony-stimulating factor (GM-CSF)+IL-4 were transduced with a retroviral vector encoding both vIL-10 and EGFP by centrifugal enhancement. Gene transfer efficiency was determined by flow cytometry. Transduced cells were flow sorted, and vIL-10 secretion was quantified by ELISA. DC function was assessed by the ability of the cells to induce naive allogeneic (C3H; H2k) T-cell proliferation and cytotoxic T lymphocyte generation. RESULTS: Retrovirally transduced DC expressed both vIL-10 and EGFP gene products. Approximately 20% of unsorted cells expressed EGFP, as determined by flow cytometry. vIL-10 was produced at a mean rate of 31 ng/40 hr/10(6) cells. After sorting, the incidence of EGFP+ DC was increased dramatically to at least 95%, and the production of vIL-10 was increased approximately three- to fourfold, to a mean of 107 ng/40 hr/10(6) cells. These highly purified, vIL-10-secreting DC exhibited markedly diminished capacity to induce allogeneic T-cell proliferative and cytotoxic responses. CONCLUSIONS: DCp retrovirally transduced to express both vIL-10 and EGFP can be rapidly identified and sorted to high levels of purity. The availability of highly enriched preparations of vIL-10-transduced DC facilitates studies of their immunoregulatory function and may enhance their therapeutic potential in transplantation or autoimmune disease.

Animals↗
PubMed1999

Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors.

Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.

3T3 Cells↗
PubMed1998

A simple and efficient purification of transduced cells by using green fluorescent protein gene as a selection marker.

BACKGROUND: Simple and efficient method for the selection of transduced cells would greatly facilitate the clinical utilization of retrovirus vectors. We developed a therapeutic bicistronic retrovirus vector for Gaucher disease, MFG-GC-GFP, which contains the human glucocerebrosidase (GC) gene and the green fluorescent protein (GFP) gene of the jellyfish Aequorea victoria as a vital selection marker, and investigated its applicability as gene therapy for Gaucher disease. METHODS AND RESULTS: A packaging cell line, GP + envAM12, was transfected with MFG-GC-GFP and, thus, produced a high titer recombinant virus (1.0 x 10(6) c.f.u./mL) in the culture supernatant. The expression level of GFP was correlated with the virus production in cells. The recombinant virus infected skin fibroblasts from a Gaucher patient and a sorted fraction of the cells expressing GFP by flow cytometry exhibited almost a six-fold higher activity of GC than normal fibroblasts. CONCLUSIONS: These data indicate that MFG-GC-GFP enables the one-step purification of a transduced fraction of target cells and is, therefore, considered to be a useful therapeutic vector for the experimental gene therapy of Gaucher disease.

Animals↗
PubMed2006

STAT3-dependent pathfinding and control of axonal branching and target selection.

Signal transducers and transcription factors are used in common for developmental cell migration, vasculogenesis, branching morphogenesis, as well as neuronal pathfinding. STAT3, a transcription factor, has been shown to function in all of these processes except neuronal pathfinding. Here, it is shown that STAT3 also facilitates this process. Elimination of STAT3 signaling results in half of zebrafish CaP motoneurons stalling along their ventral pathfinding trajectory. Conversely, constitutive activation leads to precocious branching and redefines CaP axons as a responding population to dorsal guidance cues, resulting in bifurcated axons innervating normal ventral targets as well as additional dorsal muscle groups. These results are consistent with and highlight a fundamental role for STAT3 as a factor promoting cellular responses to guidance cues, not only in nonneural cells but also in pathfinding neurons.

Animals↗
PubMed2000

Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice.

The MDR1 (multidrug resistance) gene, transferred to hematopoietic cells, is expected to protect them from anticancer chemotherapy and may serve as a selectable marker, restoring gene expression in vivo. Appropriate selection strategies, however, need to be established. To investigate whether preselection ex vivo affects chemoresistance, murine bone marrow cells were retrovirally transduced with high-titer or, as a model for suboptimal gene expression, low-titer retroviruses and exposed to daunomycin or colchicine for 48-96 h. Selection significantly increased chemoresistance of clonogenic progenitor cells. In tissue culture, the entire target population was rendered highly drug resistant after MDR1 transfer with high-titer viruses. If transduction was performed under suboptimal conditions, drug selection increased the frequency of chemoresistant colonies up to 40% over the number of unselected cells. Colchicine and daunomycin were equally efficient in increasing drug resistance ex vivo, but colchicine-preselected cells rescued lethally irradiated mice under conditions where daunomycin-selected bone marrow cells failed to do so. Hence, while hematopoietic cells can be protected by MDR1, the selection strategy is critical for repopulation of bone marrow with transduced cells. Preselection in culture before transplantation significantly increased P-gp expression and chemoresistance in vivo in mice reconstituted with transduced bone marrow cells. This study may help to facilitate the use of MDR1 as a selectable marker in gene therapy of the hematopoietic system. Gene Therapy (2000) 7, 348-358.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

L-histidinol provides effective selection of retrovirus-vector-transduced keratinocytes without impairing their proliferative potential.

Retroviral vectors carrying the neomycin phosphotransferase (neo) gene have been shown to confer G418 resistance to canine keratinocytes at relatively high frequency. To investigate the usefulness of keratinocytes as potential target cells for gene therapy, we used a retroviral vector (LASN) that contains both human adenosine deaminase (hADA) and neo genes. We show here that LASN-transduced canine keratinocytes expressed high levels of hADA, a human protein of therapeutic relevance. Selection of LASN-transduced keratinocytes in medium containing G418 resulted in a population of cells that expressed even higher levels of hADA, about 80-fold higher than the endogenous canine ADA level. However, the G418-selected cells had a reduced proliferative potential and altered morphology indicative of terminal differentiation. To test whether L-histidinol is more beneficial for selection of keratinocytes than G418, we constructed two retroviral vectors that contain both the neo and the histidinol dehydrogenase (hisD) genes. Cocultivation of primary keratinocytes with lethally irradiated PA317 retrovirus packaging cells that produce these vectors gave rise to 12-53% drug-resistant colonies in either G418 or L-histidinol. In contrast to G418, selection of transduced keratinocytes in L-histidinol had no apparent effect on the proliferative potential or morphology of drug-resistant cells containing the vectors. Given the utility of this selection system, two hisD-based generic constructs containing cloning sites for cDNA expression from either the retroviral promoter or from an internal human cytomegalovirus immediate early promoter were constructed. Our results suggest that hisD will be a useful selectable marker for use in studies of keratinocyte differentiation and for transfer of genes into keratinocytes for the purposes of gene therapy.

Adenosine Deaminase↗
PubMed2003

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.

Animals↗
PubMed1998

[Construction and application of retroviral vector carrying green fluorescent protein].

OBJECTIVE: To construct retroviral vector carrying rapidly selective marker. METHODS: The recombination retroviral vector GCGFPPXSN was constructed by cloning the green fluorescent protein (GFP) cDNA into the retroviral vector containing putative internal ribosome entry sites GCXPXSN and transferred in ecotropic packaging cell line PE501 by electroporation method. The supernatants of the PE501GCGFPPXSN were used to infect the amphotropic packaging cell line PA317. The G418 resistant clones were selected in 4 weeks and were detectable by fluorescence microscopy or by fluorescence-activated cell sorting(FACS). RESULTS: A recombination retroviral vector GCGFPPXSN carrying rapidly selective marker GFP was constructed. GFP expression in packaging cell line PA317-GCGFPPXSN transferred by GCGFPPXSN was detected by fluorescence microscopy of FACS. PA317-GCGFPPXSN grew to a titer of 1.2 x 10(5) cpu/ml. By cocultivating retroviral vector producing cells and T lymphocytes, expression of GFP was observed in T lymphocytes 2 days after the end of the cocultivation. T lymphocytes expressing GFP were separated. CONCLUSION: The mammalian cell can be efficient gene transfected by retroviral vector carrying GFP. The use of GFP for cell marking represents an important advantage over conventional strategies which typically involve the use of neomycin resistance. GFP, in fact, allows a rapid in vitro selection of transduced cell by FACS. The selection requires only two-day culture with this retroviral vector, compared with 10-14 day culture with a classical retroviral vector.

Cell Line↗
PubMed1998

Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells.

Transfer of drug resistance genes to hematopoietic cells is being studied as a means to protect against the myelosuppression associated with cancer chemotherapy and as a strategy for the in vivo selection and amplification of genetically modified cells. The goal of this study was to test if retroviral-mediated gene transfer of a dihydrofolate reductase (DHFR) variant (L22Y) could be used for in vivo selection of transduced myeloid cells and to determine what proportion of transduced cells was required for protection from myelosuppression. Based on previous work suggesting that selection with antifolates may also require inhibition of nucleoside transport mechanisms, mice transplanted with DHFR-transduced bone marrow cells were treated with trimetrexate and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine riboside phosphate. In vivo selection of transduced myeloid progenitors was seen in the bone marrow and in circulating mature peripheral blood cells following drug treatment. These results show that the novel combination of the L22Y-DHFR cDNA, trimetrexate and nitrobenzylmercaptopurine riboside phosphate can be used to select for transduced myeloid cells, and that this approach warrants further study in large animal models. A bicistronic vector containing a human CD24 reporter gene was used to determine the number of modified cells needed for chemoprotection. Partial protection from neutropenia was seen when greater than 10% of myeloid cells expressed the vector, and high levels of protection were obtained when the proportion exceeded 30%. These results suggest that gene transfer may be useful for myeloprotection in certain pediatric cancers, but that more efficient gene transfer will be required to apply this approach to adult cancer patients.

Adult↗
PubMed1999

In vivo methotrexate selection of murine hemopoietic cells transduced with a retroviral vector for Gaucher disease.

The studies described were performed to investigate whether in vivo selection of retrovirus-transduced hemopoietic cells is feasible starting from a low percentage of transduced hemopoietic stem cells (PHSCs). The vector used is an amphotropic bicistronic retroviral vector carrying a cDNA for human lysosomal glucocerebrosidase (hGC) for treatment of Gaucher disease and a methotrexate (MTX) resistant mutant cDNA encoding human dihydrofolate reductase (DHFR). We tested the effect of MTX selection in mice that were either myeloablated or not before infusion of transduced cells. In addition, we determined whether repeated administration of transduced bone marrow cells has an additional effect on the percentage of hGC expressing cells. The results obtained have shown that, in myeloablated mice transplanted once with transduced bone marrow and treated twice weekly with 10 mg/kg of MTX for a total of 6 months, a two- to three-fold increased numbers of hGC expressing cells could be detected in both peripheral blood and bone marrow as compared with non-MTX treated mice. In mice transplanted with transduced bone marrow once every 2 weeks for a total of four times, percentages of hGC expressing cells were not significantly increased as compared with mice transplanted once. In non-ablated mice neither MTX selection nor multiple infusions of transduced bone marrow resulted in detection of hGC expressing cells 6 months after transplantation, indicating that the success of in vivo selection using MTX is highly dependent on the ratio of transduced hemopoietic stem cells transplanted versus residing and untransduced stem cells.

Animals↗
PubMed1999

In vitro selection of an allosteric ribozyme that transduces analytes to amplicons.

We have selected an allosteric ribozyme ligase from a random sequence population that is activated up to 10,000-fold by oligonucleotide effectors. The ribozyme conforms to a classic two-state model for allostery in which the equilibrium between inactive and active conformers is dramatically altered by the presence of effector ligands. In the presence of the effector the allosteric ribozyme ligase generates templates that can subsequently be amplified using conventional amplification technologies, such as RT-PCR. Thus, the allosteric ribozyme can transduce (or convert) analytes into amplicons. We demonstrate two potential diagnostic applications of the selected allosteric ribozyme ligase: 'counting' short oligonucleotide effectors by RT-PCR, and counting a non-nucleic acid effector, ATP, by ligation.

Allosteric Site↗
PubMed1996

Retroviral-mediated gene transduction of human alkyltransferase complementary DNA confers nitrosourea resistance to human hematopoietic progenitors.

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy due to low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in myeloid precursors. We have shown that high-efficiency myeloproliferative sarcoma virus (vM5MGMT)-mediated transduction of the human MGMT cDNA into murine bone marrow (BM) cells leads to high MGMT expression and increased progenitor resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) in vitro immediately after infection and after BM transplantation. These experiments were designed to increase MGMT expression in human hematopoietic progenitors. CD34(+) BM cells were isolated over an immunoaffinity column (CEPRATE, CellPro, Inc.), resulting in a mean 66-fold enrichment in clonogenic progenitors (colony-forming unit granulocyte-macrophage + burst-forming unit erythroid + colony-forming unit granulocyte erythroid macrophage = megakaryocyte), with an average progenitor yield of 58 +/- 11.5% and a final population that was 54% CD34(+). Seventy % of progenitors derived from CD34(+) cells were transduced after coculture with AM12-vM5MGMT retroviral producers. vM5MGMT-transduced progenitors were over 2-fold more resistant to concentrations of BCNU between 30 and 50 micrometer than were concurrently LacZ-transduced progenitors (P < 0.003). In vitro selection of transduced, cytokine-stimulated CD34(+) cells with 20 micrometer BCNU resulted in survival of 4.7% of MGMT+ clonogenic progenitors compared to 0.05% of LacZ+ progenitors. These studies indicate that MGMT-transduced human hematopoietic progenitors have increased resistance to nitrosoureas, and in a clinical transplant setting, this strategy may reduce alkylating agent myelosuppression.

Antigens, CD34↗
PubMed2004

Cloning and expression of canine O6-methylguanine-DNA methyltransferase in target cells, using gammaretroviral and lentiviral vectors.

The human O(6)-methylguanine-DNA methyltransferase (MGMT) gene and its mutants have been used for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O(6)-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing an immune response, we have cloned the canine MGMT drug resistance gene. Comparison of canine and human MGMT-coding regions indicates that there is about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT is also longer, by nine amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT are highly conserved in the canine sequence. To determine whether mutation of the proline residue at position 144 to lysine in the canine MGMT would provide a similar advantage for selection of transduced cells as the human mutant, Moloney murine leukemia virus and human immunodeficiency type 1 vectors encoding the corresponding mutant MGMT were created and used to express separately canine and human MGMTs in cultured cells. Drug resistance assays using BCNU alone or BCNU with BG demonstrated that the wild-type and mutant canine MGMTs provided resistance to the selection agents that was comparable to the human MGMT counterparts.

Amino Acid Sequence↗
PubMed1999

Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy.

Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.

Gene Transfer Techniques↗
PubMed2005

Tolerance by selective in vivo expansion of foreign major histocompatibility complex-transduced autologous bone marrow.

BACKGROUND: Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector. METHODS: To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice. RESULTS: After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days. CONCLUSION: Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.

Animals↗
PubMed1986

Selection of lambda Spi- transducing phages using the P2 old gene cloned onto a plasmid.

The old gene product of the P2 prophage interferes with plaque formation by lambda wild type phage but allows lambda phages whose red and gam genes have been deleted to form small, visible plaques (the lambda Spi- phenotype). The old gene product also kills Escherichia coli recB or recC mutants. We have cloned the old gene into the high-copy-number plasmid pBR322, where it prevents plaque formation by both lambda Spi+ and lambda Spi- phages. We transferred a DNA fragment that carries the old gene to the low-copy-number plasmid pSC101 and found that lambda Spi- phages can be selected on strains that carry this plasmid. The plasmid-borne old gene kills E. coli recB mutants, providing a selection for old- mutants.

Bacteriophage lambda↗
PubMed2000

In vitro selection of lentivirus vector-transduced human CD34+ cells.

Human CD34(+) cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34(+) hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34(+) cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)(+) cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.

Antigens, CD34↗
PubMed1993

Retrovirus-mediated gene transfer into canine thyroid using an ex vivo strategy.

We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells. Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropin-containing media, and transduced with amphotropic retroviral vectors carrying Escherichia coli beta-galactosidase or Tn7 neomycin-resistance genes. Up to 30% of cells were transduced with retroviral vectors containing the neomycin resistance gene, and transduced cells could be selected with G418. Significantly, transduced and selected cells exhibited the morphology of thyroid follicular cells and continued to express thyroglobulin. To assess the viability of cultivated and transduced cells for transplantation, cells were stained with the vital fluorescent dye DiI, recovered by trypsinization, and transplanted into the contralateral thyroid lobe of autologous animals. Engraftment was demonstrated by fluorescence microscopy and identification of proviral sequences 7-10 days after transplantation. Proviral transcripts were evident using coupled reverse transcription and the polymerase chain reaction using total RNA from transplanted glands. Thyroid follicular cells may represent an attractive target for gene therapy due to their proliferative potential, their large protein synthetic and secretory capacity, and their susceptibility to regulation. The thyroid might be a target for therapy of congenital or acquired thyroid diseases as well as disorders requiring regulated expression of proteins in the circulation. This work demonstrates the feasibility of ex vivo gene delivery to thyroid follicular cells that may be used in future investigations.

Animals↗