scholarly journals Biochemical Correction of X-CGD by a Novel Chimeric Promoter Regulating High Levels of Transgene Expression in Myeloid Cells

2011 ◽  
Vol 19 (1) ◽  
pp. 122-132 ◽  
Author(s):  
Giorgia Santilli ◽  
Elena Almarza ◽  
Christian Brendel ◽  
Uimook Choi ◽  
Chiara Beilin ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Myeloid Cells ◽  
Chimeric Promoter

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

A small chimeric promoter for high prostate-specific transgene expression from adenoviral vectors

The Prostate ◽  
2007 ◽  
Vol 67 (8) ◽  
pp. 829-839 ◽  
Author(s):  
Robert Kraaij ◽  
Laura van der Weel ◽  
Corrina M.A. de Ridder ◽  
Hetty A.G.M. van der Korput ◽  
Joke L.M. Zweistra ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Adenoviral Vectors ◽  
Chimeric Promoter

scholarly journals WHIM syndrome myelokathexis reproduced in the NOD/SCID mouse xenotransplant model engrafted with healthy human stem cells transduced with C-terminus–truncated CXCR4

Blood ◽  
2006 ◽  
Vol 109 (1) ◽  
pp. 78-84 ◽  
Author(s):  
Toshinao Kawai ◽  
Uimook Choi ◽  
Lanise Cardwell ◽  
Suk See DeRavin ◽  
Nora Naumann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Scid Mouse ◽  
Severe Combined Immunodeficiency ◽  
Myeloid Cells ◽  
Xenograft Model ◽  
Healthy Human ◽  
Whim Syndrome ◽  
Mouse Xenograft Model

AbstractWHIM(warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis) syndrome is a rare immunodeficiency caused in many cases by autosomal dominant C-terminal truncation mutations in the chemokine receptor CXCR4. A prominent and unexplained feature of WHIM is myelokathexis (hypercellularity with apoptosis of mature myeloid cells in bone marrow and neutropenia). We transduced healthy human CD34+ peripheral blood–mobilized stem cells (PBSCs) with retrovirus vector encoding wild-type (wt) CXCR4 or WHIM-type mutated CXCR4 and studied these cells ex vivo in culture and after engraftment in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model. Neither wt CXCR4 nor mutated CXCR4 transgene expression itself enhanced apoptosis of neutrophils arising in transduced PBSC cultures even with stimulation by a CXCR4 agonist, stromal cell–derived factor-1 (SDF-1 [CXCL12]). Excess wt CXCR4 expression by transduced human PBSCs enhanced marrow engraftment, but did not affect bone marrow (BM) apoptosis or the release of transduced leukocytes into PB. However, mutated CXCR4 transgene expression further enhanced BM engraftment, but was associated with a significant increase in apoptosis of transduced cells in BM and reduced release of transduced leukocytes into PB. We conclude that increased apoptosis of mature myeloid cells in WHIM is secondary to a failure of marrow release and progression to normal myeloid cell senescence, and not a direct effect of activation of mutated CXCR4.

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3040-3048
Author(s):  
Ahlke Heydemann ◽  
Soren Warming ◽  
Cynthia Clendenin ◽  
Kirsten Sigrist ◽  
J. Peter Hjorth ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Copy Number ◽  
Messenger Rna ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Control Function ◽  
Integration Site ◽  
Myeloid Cells ◽  
Independent Copy

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3040-3048 ◽  
Author(s):  
Ahlke Heydemann ◽  
Soren Warming ◽  
Cynthia Clendenin ◽  
Kirsten Sigrist ◽  
J. Peter Hjorth ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Copy Number ◽  
Messenger Rna ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Control Function ◽  
Integration Site ◽  
Myeloid Cells ◽  
Independent Copy

Abstract The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) humanc-fes genomic fragment was previously shown to containcis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR,c-fes transgenes were analyzed in mice. Myeloid-cell–specific, deoxyribonuclease-I–hypersensitive sites localized to the 3′ boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446–base-pair promoter, integration-site–independent, copy-number–dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

The RUNX1-Regulated Cebpa +37 Kb Enhancer Directs Human CD4 Transgene Expression To Long-Term Hematopoietic Stem Cells and Preferentially To Myeloid Compared With Lymphoid Or Erythroid Progenitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2442-2442
Author(s):  
Alan D. Friedman ◽  
Hong Guo ◽  
Ou Ma ◽  
Leonard I. Zon ◽  
Ilya Shestopalov ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Transgene Expression ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Myeloid Cells ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract We found that adult mice lacking RUNX1 have increased monopoiesis relative to granulopoiesis correlated with reduce Cebpa RNA expression in myeloid progenitors and that RUNX1 binds and activates Cebpa reporters via two sites in the Cebpa promoter and four conserved sites in a 450 bp +37 kb enhancer (Guo et al, Blood, 2012). This enhancer interacts with p300 and contains the H3K4me1 enhancer mark in myeloid cells. Previously reported ChIP-Seq data had demonstrated interaction of RUNX1 with the +37 kb enhancer, along with GATA2, SCL, Gfi-1b, MEIS1, Fli-1, ERG, PU.1, Lyl1, and LMO2. We have now generated transgenic C57BL/6 mice in which the Cebpa +37 kb enhancer positioned upstream of its 845 bp promoter (-720 to +125) directs expression of cytoplasmically truncated human CD4 (hCD4). Two founders with germ line transmission have been identified – both have detectable hCD4 RNA in marrow, at approximately 10-fold increased levels in one line compared to the other, with hCD4 expression detectable by FACS only in the higher expressing line. Analysis of 4-7 mice reveals that hCD4 expression is detected only in a small subset of mature hematopoietic cells, present in 6% of Mac-1+Gr-1+ neutrophils, 31% of Mac-1+Gr-1- monocytes, 4% of CD3+ T cells, 3% of CD19+ or 7% of B220+ B-cells, and 5% of Ter119+ erythroid cells. 6.5+/-1.9% of total marrow mononuclear cells and 13+/-4% of Lin- marrow cells express hCD4. At the progenitor level, hCD4 was evident in 56+/-7% of CMP, 70+/-5% of GMP, 36+/-9% of CLP, and only 1.5+/-1.0% of MEP. There was>100-fold enrichment of myeloid CFU but a 2-fold decrease in B-lymphoid colonies comparing hCD4+ versus hCD4- fractions of total marrow. Staining LSK cells for CD34 and FcγR demonstrated that 91+/-10% of multi-potent progenitors (MPP), 61+/-8% of ST-HSC, and 17+/-8% of LT-HSC express hCD4. Similarly, staining for the SLAM markers demonstrated that 19+/-9% of LSK-CD48-CD150+ LT-HSC express hCD4. Mean fluorescence index of hCD4 staining was about 2-fold higher in LSK, MPP, ST-HSC, CMP, and GMP compared with CLP and LT-HSC and about 3-fold higher than mature blood myeloid cells. 2E6 hCD4+ or hCD4- CD45.2 marrow cells were transplanted into lethally irradiated CD45.1 syngeneic recipients. 5/5 mice receiving hCD4- cells died at 2 weeks, reflecting lack of even progenitor engraftment, whereas 5/5 mice receiving hCD4+ cells were alive at 20 weeks, reflecting expression of the transgene in functional LT-HSC, with secondary transplanted mice viable at 4 week to date. The marrow cells of surviving mice were almost exclusively CD45.2, with hCD4+ expression in Lin- or Lin-c-kit+ marrow cells similar to that of donor mice. Although our second founder does not allow hCD4 detection by FACS, analysis of hCD4 RNA in marrow stem and progenitor subsets for this line is in progress, as is competitive transplantation of hCD4- versus hCD4+ marrow cells from the higher-expressing line. Murine E13.5 fetal liver hematopoietic stem and progenitor cells express hCD4 in a pattern similar to adult marrow. In zebrafish, the murine Cebpa +37 kb enhancer directs ZsGreen expression specifically to the caudal hematopoietic territory (CHT), analogous to the murine fetal liver, at 72 hour post-fertilization. Murine Cebpa +37 kb enhancer:ZsGreen transgene expression, lacking the Cebpa promoter, overlaps in the CHT with that of murine Runx1 +23 kb enhancer:mCherry in compound transgenic fish. The Cebpa and Runx1 enhancers direct similar tdTomato expression from a Sleeping Beauty vector in the K562 cell line, and these vectors are being compared for expression in hESC derived hematopoietic cells. In summary, the Cebpa gene contains an enhancer active in both LT-HSC and myeloid progenitors. Further characterization of its regulation may provide insights into the development of these hematopoietic stem and progenitor subsets and into how Cebpa expression is impaired in myelodysplastic syndromes or in acute myeloid leukemia. In addition, the Cebpa enhancer may prove useful in efforts to optimize LT-HSC formation from hESC. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Doxycycline Regulatable Expression of Cytidine Deaminase Mediates Myeloprotection and Avoids Lymphotoxicity in a Murine Transplant Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2054-2054
Author(s):  
Nico Lachmann ◽  
Sebastian Brennig ◽  
Nils Pfaff ◽  
Heiko Schermeier ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Myeloid Cells ◽  
Hematopoietic Cell ◽  
Significant Protection ◽  
Cell Compartments

Abstract Abstract 2054 Hematopoietic stem cells (HSCs) genetically modified to overexpress drug resistance genes have been advocated to overcome chemotherapy induced myelosuppression. In this context, we demonstrated that overexpression of hCDD from a constitutive spleen focus forming virus (SFFV)-derived promoter protects hematopoietic cells from Ara-C toxicity in vitro and in vivo. However, these studies also indicated substantial lymphotoxicity by high level constitutive CDD expression (Rattmann et al. Blood, 2006). To circumvent this problem, we now have established a Doxycycline (Dox)-inducible (TET-ON) CDD-expression system and have evaluated this system in murine in vitro and in vivo protection assays. In vitro CDD-mediated Ara-C resistance was evaluated in the hematopoietic cell line 32D as well as primary lineage negative (Lin-) clonogenic progenitor cells. In these studies cells were co-transduced with two lentiviral constructs expressing CDD (TET.CDD) or the reverse transactivator protein (rtTA3). In 32D cells, administration of 0.2 to 2.0 μg/ml Dox induced transgene expression to plateau levels within 24 hours. Rapid induction of the transgene also was observed at lower Dox concentrations, although exposure to 0.008 or 0.04 μg/ml led to notably reduced expression levels. Functionality of Dox-inducible hCDD expression was evaluated by exposing TET.CDD transgenic 32D cells to Ara-C. Transduced cells cultured in the presence of 2.0 μg/ml Dox proved completely resistant to Ara-C concentrations of up to 5000 nM, whereas eGFP- or not Dox treated control cells were susceptible to Ara-C exposure from 50 nM onwards. Again, hCDD-mediated drug resistance was dependent on the Dox dosage. When TET.CDD transduced 32D cells were exposed to Dox for 72h, marked protection from Ara-C was shown for Dox concentration of >0.2 μg/ml (LD50 > 2.000 nM Ara-C). After Dox withdrawal transgene expression remained detectable for at least three days. Similar protection was observed in primary hematopoietic cells and progenitor cell derived colonies were protected from Ara-C doses of 300 to 600 nM while untransduced control cells did not yield colony growth at doses of 50 nM Ara-C or higher. In vivo studies were performed by transplanting C57Bl/6 mice with Lin- cells from Rosa26-M2rtTA mice previously transduced with the TET.CDD or a control SIN lentiviral vector. Transgene expression was induced by Dox administration starting four weeks post transplantation. In this model Dox administration induced stable transgene expression in peripheral blood B, T, and myeloid cells peaking 15 days after start of administration and remaining detectable for 21 days after Dox withdrawal. No alterations in peripheral blood cell counts including the lymphocyte count were observed for up to eight weeks of Dox administration and also the relative contribution of gene modified cells to peripheral blood B, CD4+ or CD8+ T, and myeloid cells remained fairly constant during this time period. Even more important, a similar contribution of transduced cells was observed for the myeloid and the lymphoid cell compartment strongly arguing against a major lymphotoxicity. Also, no toxic effects of Dox-regulated hCDD expression was observed in other hematopoietic cell compartments including stem- or progenitor cells in various splenic, thymic and BM-derived hematopoietic cell compartments. Moreover, the TET.CDD vector conveyed significant protection against Ara-C (500 mg/kg, d1-4, i.v.) to the hematopoietic system as measured by granulocyte (0.26 +/−0.25 versus 0.8 +/−0, p=0.02) and platelet counts (584 +/−159 versus 883 +/−194, p=0.02) seven days after treatment. Furthermore, when the long term reconstitution potential of TET.CDD transduced Lin- cells was evaluated by secondary transplantation, robust, Dox-dependent transgenic eGFP expression was observed in peripheral blood B, CD4+ and CD8+ T, as well as myeloid cells of secondary recipients. Taken together, our data demonstrate efficient Dox-inducible hCDD expression in 32D and primary murine bone marrow cells in vitro as well as in a murine in vivo bone marrow transplant gene transfer model. Most importantly, in the latter model Dox-inducible CDD expression not only allowed for significant protection from Ara-C induced myelotoxicity but also abrogated the lymphotoxicity observed previously with high and constitutive hCDD expression. Disclosures: No relevant conflicts of interest to declare.

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