scholarly journals Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 876-881 ◽  
Author(s):  
RA Hock ◽  
AD Miller ◽  
WR Osborne
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Adenosine Deaminase ◽  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
K562 Cells ◽  
Moloney Murine Leukemia Virus ◽  
Hematopoietic Cell Lines ◽  
Murine Leukemia

Abstract Adenosine deaminase (ADA) deficiency is associated with a fatal severe combined immunodeficiency. Because most patients do not have a suitable marrow donor, the introduction of a normal ADA gene into the patient's marrow cells is a potentially useful alternative therapy. To identify vectors that provide optimal gene expression in human hematopoietic cells, we investigated retroviral vectors containing the ADA gene under the transcriptional control of the promoter/enhancers of Moloney murine leukemia virus, the simian virus 40 early region, the cytomegalovirus immediate-early gene, the lymphotropic papovavirus, and the human beta- globin gene. ADA expression from these vectors was monitored in the ADA- human histiocytic lymphoma cell line DHL-9, and in the multipotential chronic myeloid leukemia cell line K562. ADA expression in infected K562 cells was also measured after induction of megakaryoblastic differentiation by phorbol ester, and after induction of erythroid differentiation by sodium n-butyrate or hemin. In these hematopoietic cell lines, the vectors that contained ADA controlled by either the Moloney murine leukemia virus promoter (LASN) or the cytomegalovirus promoter (LNCA) expressed ADA at much higher levels than the other vectors tested. Furthermore, in K562 cells infected with LASN and LNCA vectors, induction of terminal differentiation resulted in the same or higher level expression of ADA. These cell lines have permitted the evaluation of transduced gene expression in proliferating and differentiating hematopoietic cells that provide a model for bone marrow-targeted gene therapy.

scholarly journals Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 876-881 ◽  
Author(s):  
RA Hock ◽  
AD Miller ◽  
WR Osborne
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Adenosine Deaminase ◽  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
K562 Cells ◽  
Moloney Murine Leukemia Virus ◽  
Hematopoietic Cell Lines ◽  
Murine Leukemia

Adenosine deaminase (ADA) deficiency is associated with a fatal severe combined immunodeficiency. Because most patients do not have a suitable marrow donor, the introduction of a normal ADA gene into the patient's marrow cells is a potentially useful alternative therapy. To identify vectors that provide optimal gene expression in human hematopoietic cells, we investigated retroviral vectors containing the ADA gene under the transcriptional control of the promoter/enhancers of Moloney murine leukemia virus, the simian virus 40 early region, the cytomegalovirus immediate-early gene, the lymphotropic papovavirus, and the human beta- globin gene. ADA expression from these vectors was monitored in the ADA- human histiocytic lymphoma cell line DHL-9, and in the multipotential chronic myeloid leukemia cell line K562. ADA expression in infected K562 cells was also measured after induction of megakaryoblastic differentiation by phorbol ester, and after induction of erythroid differentiation by sodium n-butyrate or hemin. In these hematopoietic cell lines, the vectors that contained ADA controlled by either the Moloney murine leukemia virus promoter (LASN) or the cytomegalovirus promoter (LNCA) expressed ADA at much higher levels than the other vectors tested. Furthermore, in K562 cells infected with LASN and LNCA vectors, induction of terminal differentiation resulted in the same or higher level expression of ADA. These cell lines have permitted the evaluation of transduced gene expression in proliferating and differentiating hematopoietic cells that provide a model for bone marrow-targeted gene therapy.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3385-3391 ◽  
Author(s):  
Stephane Barrette ◽  
Janet L. Douglas ◽  
Nancy E. Seidel ◽  
David M. Bodine
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Lentivirus Vector ◽  
Hematopoietic Stem ◽  
Lentivirus Vectors ◽  
Murine Leukemia

Abstract The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3385-3391 ◽  
Author(s):  
Stephane Barrette ◽  
Janet L. Douglas ◽  
Nancy E. Seidel ◽  
David M. Bodine
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Lines ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Lentivirus Vector ◽  
Hematopoietic Stem ◽  
Lentivirus Vectors ◽  
Murine Leukemia

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter

1987 ◽  
Vol 7 (5) ◽  
pp. 1797-1806
Author(s):  
R A Bosselman ◽  
R Y Hsu ◽  
J Bruszewski ◽  
S Hu ◽  
F Martin ◽  
...  
Keyword(s):  
Cell Line ◽  
Sequence Homology ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Helper Cell ◽  
Factors Affecting ◽  
Cis Acting ◽  
Virus Expression ◽  
Murine Leukemia

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

scholarly journals Cellular Transcription Factor ZASC1 Regulates Murine Leukemia Virus Transcription

2010 ◽  
Vol 84 (15) ◽  
pp. 7473-7483 ◽  
Author(s):  
James W. Bruce ◽  
Michael Hierl ◽  
John A. T. Young ◽  
Paul Ahlquist
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Cell Line ◽  
Binding Sites ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cellular Transcription Factor ◽  
Cellular Transcription ◽  
Ltr Promoter ◽  
Murine Leukemia

ABSTRACT To identify cellular processes involved in retroviral infection, we employed a high-volume forward genetic screen of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a clonal cell line that exhibited approximately 10-fold reduced gene expression from MLV vectors following infection despite supporting normal levels of MLV reverse transcription and integration. The defect in this cell line was specific for the MLV long terminal repeat (LTR) promoter, as normal levels of reporter gene expression were obtained from both an internal cytomegalovirus (CMV) promoter contained within an LTR-defective MLV vector and LTR expression from an avian sarcoma and leukosis virus (ASLV) vector. Complementation and shRNA knockdown experiments demonstrated that the defective gene in these cells is ZASC1 (ZNF639), a transcription factor with strong links to cancer and inherited ataxias. We demonstrated that ZASC1 is a sequence-specific DNA binding protein with three closely related binding sites located within the MLV LTR promoter, but it does not bind to the ASLV promoter. Mutating these putative ZASC1 binding sites significantly reduced levels of MLV gene expression. While wild-type ZASC1 activated expression from the MLV promoter, a green fluorescent protein-ZASC1 fusion protein showed dominant-negative inhibition of MLV gene expression. These studies identify the cellular transcription factor ZASC1 as an activator of MLV gene expression and provide tools that should be useful in studying the links between ZASC1 and human diseases.

scholarly journals High-Throughput RNA Sequencing-Based Virome Analysis of 50 Lymphoma Cell Lines from the Cancer Cell Line Encyclopedia Project

2014 ◽  
Vol 89 (1) ◽  
pp. 713-729 ◽  
Author(s):  
Subing Cao ◽  
Michael J. Strong ◽  
Xia Wang ◽  
Walter N. Moss ◽  
Monica Concha ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cancer Cell Line ◽  
Lymphoma Cell ◽  
Viral Gene ◽  
Barr Virus ◽  
Murine Leukemia

ABSTRACTUsing high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.IMPORTANCEViruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

scholarly journals CrFK Feline Kidney Cells Produce an RD114-Like Endogenous Virus That Can Package Murine Leukemia Virus-Based Vectors

1998 ◽  
Vol 72 (9) ◽  
pp. 7685-7687 ◽  
Author(s):  
Jörg G. Baumann ◽  
Walter H. Günzburg ◽  
Brian Salmons
Keyword(s):  
Cell Line ◽  
Kidney Cell ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Infectivity ◽  
Kidney Cells ◽  
Kidney Cell Line ◽  
Endogenous Virus ◽  
Murine Leukemia

ABSTRACT The feline kidney cell line CrFK is used extensively for viral infectivity assays and for study of the biology of various retroviruses and derived vectors. We demonstrate the production of an endogenous, RD114-like, infectious retrovirus from CrFK cells. This virus also is shown to efficiently package Moloney murine leukemia virus vectors.

scholarly journals A Chimeric Ty3/Moloney Murine Leukemia Virus Integrase Protein Is Active In Vivo

1998 ◽  
Vol 72 (5) ◽  
pp. 4297-4307 ◽  
Author(s):  
Sandra L. Dildine ◽  
James Respess ◽  
Doug Jolly ◽  
Suzanne B. Sandmeyer
Keyword(s):  
Cell Line ◽  
Murine Leukemia Virus ◽  
Genomic Dna ◽  
Integration Site ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Packaging Cell Line ◽  
Terminal Domain ◽  
Murine Leukemia

ABSTRACT This report describes the results of experiments to determine whether chimeras between a retrovirus and portions of Ty3 are active in vivo. A chimera between Ty3 and a Neor-marked Moloney murine leukemia virus (M-MuLV) was constructed. The C-terminal domain of M-MuLV integrase (IN) was replaced with the C-terminal domain of Ty3 IN. The chimeric retroviruses were expressed from an amphotrophic envelope packaging cell line. The virus generated was used to infect the human fibrosarcoma cell line HT1080, and cells in which integration had occurred were selected by G418 resistance. Three independently integrated viruses were rescued. In each case, the C-terminal Ty3 IN sequences were maintained and short direct repeats of the genomic DNA flanked the integration site. Sequence analysis of the genomic DNA flanking the insertion did not identify a tRNA gene; therefore, these integration events did not have Ty3 position specificity. This study showed that IN sequences from the yeast retrovirus-like element Ty3 can substitute for M-MuLV IN sequences in the C-terminal domain and contribute to IN function in vivo. It is also one of the first in vivo demonstrations of activity of a retrovirus encoding an integrase chimera. Studies of chimeras between IN species with distinctive integration patterns should complement previous work by expanding our understanding of the roles of nonconserved domains.

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