scholarly journals Characterization of regulatory elements in the 5'-flanking region of the rat GPIIb gene by studies in a primary rat marrow culture system

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3385-3393 ◽  
Author(s):  
KL Block ◽  
K Ravid ◽  
QH Phung ◽  
M Poncz
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Regulatory Element ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Start Site ◽  
Specific Expression ◽  
Flanking Region ◽  
High Level ◽  
Marrow Cells

Abstract Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 52-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 52-flanking region from -39 to -912 bp, localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region -450 to -426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454 site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA454 but had different 50-bp deletions from -450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 52-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rate bone marrow cells. In particular, our study points to the importance of the GATA454 for high levels of GPIIb expression in developing megakaryocytes.

scholarly journals Characterization of regulatory elements in the 5'-flanking region of the rat GPIIb gene by studies in a primary rat marrow culture system

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3385-3393 ◽  
Author(s):  
KL Block ◽  
K Ravid ◽  
QH Phung ◽  
M Poncz
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Regulatory Element ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Start Site ◽  
Specific Expression ◽  
Flanking Region ◽  
High Level ◽  
Marrow Cells

Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 52-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 52-flanking region from -39 to -912 bp, localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region -450 to -426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454 site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA454 but had different 50-bp deletions from -450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 52-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rate bone marrow cells. In particular, our study points to the importance of the GATA454 for high levels of GPIIb expression in developing megakaryocytes.

scholarly journals Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 89-97 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
D Orlic
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Peripheral Blood Cells ◽  
Equivalent Number ◽  
High Level ◽  
Stimulating Factor ◽  
Marrow Cells

Abstract We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G- CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

scholarly journals The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

2007 ◽  
Vol 54 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Wiesława Widłak ◽  
Natalia Vydra ◽  
Volha Dudaladava ◽  
Dorota Scieglińska ◽  
Bolesław Winiarski ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Gc Box ◽  
Specific Expression Pattern ◽  
High Level

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

scholarly journals Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 89-97 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
D Orlic
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Peripheral Blood Cells ◽  
Equivalent Number ◽  
High Level ◽  
Stimulating Factor ◽  
Marrow Cells

We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G- CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

scholarly journals Mice Transgenic for the Human CGM6 Gene Express Its Product, the Granulocyte Marker CD66b, Exclusively in Granulocytes

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 663-672 ◽  
Author(s):  
Anne-Marie Eades-Perner ◽  
John Thompson ◽  
Herman van der Putten ◽  
Wolfgang Zimmermann
Keyword(s):  
Bone Marrow ◽  
Northern Blot Analysis ◽  
Fetal Liver ◽  
Transcriptional Start Site ◽  
Specific Marker ◽  
Start Site ◽  
Specific Expression ◽  
Adult Mice ◽  
Cea Gene ◽  
Translational Start

Abstract The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counterpart is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.

scholarly journals High-level reconstitution of respiratory burst activity in a human X- linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1834-1840 ◽  
Author(s):  
C Ding ◽  
A Kume ◽  
H Bjorgvinsdottir ◽  
RG Hawley ◽  
N Pech ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Respiratory Burst ◽  
Oxidase Activity ◽  
Bone Marrow Cells ◽  
Granulomatous Disease ◽  
Respiratory Burst Activity ◽  
High Level ◽  
Respiratory Burst Oxidase ◽  
Marrow Cells

The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.

scholarly journals A gene transfer strategy for making bone marrow cells resistant to trimetrexate

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2579-2587 ◽  
Author(s):  
HT Spencer ◽  
SE Sleep ◽  
JE Rehg ◽  
RL Blakley ◽  
BP Sorrentino
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Retroviral Vectors ◽  
Hematopoietic Progenitor ◽  
High Level ◽  
Variant Enzyme ◽  
Marrow Cells

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y- containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.

Transgenic targeting with regulatory elements of the humanCD34 gene

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4410-4419 ◽  
Author(s):  
Hanna S. Radomska ◽  
David A. Gonzalez ◽  
Yutaka Okuno ◽  
Hiromi Iwasaki ◽  
Andras Nagy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Bone Marrow Cells ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Human Cd34 ◽  
Cell Assays ◽  
Flanking Region ◽  
Responsive Element

The human CD34 gene is expressed on early progenitor and stem cells in the bone marrow. Here we report the isolation of the human CD34 locus from a human P1 artificial chromosome (PAC) library and the characterization and evaluation of this genomic fragment for expression of reporter genes in stable cell lines and transgenic mice. We show that a 160-kb fragment spanning 110 kb of the 5′ flanking region and 26 kb of the 3′ flanking region of theCD34 gene directs expression of the human CD34gene in the bone marrow of transgenic mice. The expression of human CD34 transgenic RNA in tissues was found to be similar to that of the endogenous murine CD34 gene. Colony-forming cell assays showed that bone marrow cells staining positive for human CD34 consist of early progenitor cells in which expression of CD34 decreased with cell maturation. In order to test the construct for its ability to express heterologous genes in vivo, we used homologous recombination in bacteria to insert the tetracycline-responsive transactivator protein tTA. Analysis of transgenic human CD34-tTA mice by cross breeding with a strain carrying Cre recombinase under control of a tetracycline-responsive element demonstrated induction of Cre expression in mice in a pattern consistent with the expression of the human CD34 transgene.

scholarly journals Mice Transgenic for the Human CGM6 Gene Express Its Product, the Granulocyte Marker CD66b, Exclusively in Granulocytes

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 663-672
Author(s):  
Anne-Marie Eades-Perner ◽  
John Thompson ◽  
Herman van der Putten ◽  
Wolfgang Zimmermann
Keyword(s):  
Bone Marrow ◽  
Northern Blot Analysis ◽  
Fetal Liver ◽  
Transcriptional Start Site ◽  
Specific Marker ◽  
Start Site ◽  
Specific Expression ◽  
Adult Mice ◽  
Cea Gene ◽  
Translational Start

The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counterpart is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.

scholarly journals High-level reconstitution of respiratory burst activity in a human X- linked chronic granulomatous disease (X-CGD) cell line and correction of murine X-CGD bone marrow cells by retroviral-mediated gene transfer of human gp91phox

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1834-1840 ◽  
Author(s):  
C Ding ◽  
A Kume ◽  
H Bjorgvinsdottir ◽  
RG Hawley ◽  
N Pech ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Respiratory Burst ◽  
Oxidase Activity ◽  
Bone Marrow Cells ◽  
Granulomatous Disease ◽  
Respiratory Burst Activity ◽  
High Level ◽  
Respiratory Burst Oxidase ◽  
Marrow Cells

Abstract The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the gene encoding gp91phox, a 91-kD membrane glycoprotein that is the larger subunit of the respiratory burst oxidase cytochrome b. In this study, a new retroviral vector for expression of human gp91phox, MSCV-h91Neo, based on murine stem cell virus vectors, was evaluated using a human X-CGD myeloid cell line (X-CGD PLB-985 cells) and murine bone marrow cells. Expression of recombinant gp91phox in transduced X-CGD PLB-985 cells was substantially improved compared with levels achieved previously using a different retroviral construct, and respiratory burst oxidase activity was fully reconstituted in the majority of clones analyzed. Expression of gp91phox transcripts was also observed in primary and secondary murine colony-forming unit- spleen derived from transduced bone marrow cells. Furthermore, respiratory burst activity was restored to granulocyte-monocyte progeny of transduced X-CGD mice bone marrow cells cultured in vitro. This observation is the first reported use of gene transfer to correct the enzymatic defect in murine CGD phagocytes and is also consistent with the high conservation of the oxidase complex among different species. Taken together, these data suggest that the MSCV-h91Neo vector may be useful for gene replacement therapy in X-linked CGD, in which high- level reconstitution of phagocyte oxidase activity may be important for full correction of phagocyte microbicidal function.

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