scholarly journals Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3304-3315 ◽  
Author(s):  
Marti F.A. Bierhuizen ◽  
Yvonne Westerman ◽  
Trudi P. Visser ◽  
Wati Dimjati ◽  
Albertus W. Wognum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Identification of Leukemia Stem Cells Associated with AML1-ETO through Characterization of Oncogene-Induced Changes in Cell Surface Antigen Profiles on Hematopoietic Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1357-1357
Author(s):  
Carmen S Ballestas ◽  
Hyung-Gyoon Kim ◽  
Claude Scott Swindle ◽  
Christopher Klug
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Cell Surface ◽  
Progenitor Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Cell Phenotype ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract Acute myelogenous leukemia (AML) is a heterogenous group of myeloid malignancies that are characterized by the clonal outgrowth of immature myeloid progenitor cells. For most subtypes of AML, mutations that give rise to the leukemic phenotype occur in the hematopoietic stem/progenitor cell (HSC) subset as demonstrated by studies showing that only primitive CD34+CD38− bone marrow cells could function as leukemia-initiating cells (LSC) when transferred into immunodeficient NOD-SCID mice. One rather significant challenge has been that LSC share many of the same cell-surface markers as their normal counterparts in bone marrow, thus making it difficult to functionally characterize and purify this important subset of leukemic cells from bulk bone marrow samples. To rapidly identify novel antigens that are mutation-specific and induced specifically on LSC and not on normal HSC, we have transduced highly purified HSC isolated from mouse bone marrow with retroviral vectors that co-expressed AML1-ETO along with a green fluorescent protein (GFP) reporter gene. HSC of the cell-surface phenotype c-Kit+Lin−Sca-1+Flt3− (KLSF) were transduced for 18 hours in vitro and then were resorted for GFP+ cells by flow cytometry (FACS). Purified cells were then lysed for mRNA isolation and cDNA synthesis for the generation of a probe that was then hybridized to Affymetrix oligonucleotide arrays (430 2.0 GeneChip arrays). HSC transduced with a retroviral vector that only expressed GFP were used as controls to identify genes that would normally be expressed in the HSC subset. Importantly, since retroviral vectors only integrate into cycling cells, all sorted GFP+ cells from the independent transductions would represent cycling cells, which minimizes any gene expression differences due to differential frequencies of resting versus actively cycling HSC. Changes in expression of cell-surface proteins observed at the mRNA level were then validated at the protein level using FACS. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and GFP from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for the cell-surface marker of interest. For one marker, CD55, we noted a 100-fold increase in cell-surface expression specifically on HSC that express AML1-ETO and not on normal HSC. These results indicate that short-term retroviral expression of specific AML-associated mutations in HSC followed by microarray analysis of transduced cells may provide a rapid means of prospectively identifying leukemia-initiating cells in bulk patient bone marrow samples and that CD55 may be a useful therapeutic and diagnostic marker for patient samples that express the AML1-ETO chromosomal translocation. Figure 1. CD55 expression distinguishes normal HSC from HSC with the AML1-ETO translocation. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and the green fluorescent protein (GFP) from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for CD55. Cells that expressed AML1-ETO are shown below as GFP+ gated cells. Note that cells that express AML1-ETO express CD55 at approximately 100-fold greater levels on the cell-surface than GFP-negative (AML1-ETO-negative) bone marrow cells. MPC=myeloid progenitor cells of the phenotype c-Kit+ Lin− Sea-1− cells. HSC are defined c-Kit+ Lin− Sca-1+ cells. Figure 1. CD55 expression distinguishes normal HSC from HSC with the AML1-ETO translocation. Bone marrow cells were isolated from an animal that was transplanted with cells expressing AML1-ETO and the green fluorescent protein (GFP) from a retroviral vector. Cells were stained for the HSC/progenitor cell phenotype (KLS) as well as for CD55. Cells that expressed AML1-ETO are shown below as GFP+ gated cells. Note that cells that express AML1-ETO express CD55 at approximately 100-fold greater levels on the cell-surface than GFP-negative (AML1-ETO-negative) bone marrow cells. MPC=myeloid progenitor cells of the phenotype c-Kit+ Lin− Sea-1− cells. HSC are defined c-Kit+ Lin− Sca-1+ cells.

scholarly journals Characterization of Transplanted Green Fluorescent Protein+Bone Marrow Cells into Adipose Tissue

Stem Cells ◽  
2008 ◽  
Vol 26 (2) ◽  
pp. 330-338 ◽  
Author(s):  
Koji Tomiyama ◽  
Noriko Murase ◽  
Donna Beer Stolz ◽  
Hideyoshi Toyokawa ◽  
Daniel R. O'Donnell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adipose Tissue ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Green Fluorescent ◽  
Marrow Cells

scholarly journals Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1777-1786 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard J. Smeyne ◽  
Richard A. Ashmun ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
High Titer ◽  
Hematopoietic Cells ◽  
Green Fluorescent Protein Gene ◽  
Retroviral Gene Transfer ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

Persistence of eGFP Marked Bone Marrow Cells in Long-Term Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2111-2111
Author(s):  
Ingo H. Pilz ◽  
Manfred Schmidt ◽  
Claudia Ball ◽  
Hanno Glimm ◽  
Fritz von Weizsäcker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkylating Agent ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Egfp Expression ◽  
Hind Limbs ◽  
Long Term Follow Up ◽  
Green Fluorescent

Abstract To study transplanted unperturbed and mobilized long-term hematopoiesis after selection with an alkylating agent, bone marrow (BM) from 5 C57BL/6J mice was pooled, repeatedly transduced with retroviruses encoding the alkylating agent resistance protein O6-Methylguanine-DNA and enhanced green fluorescent protein (eGFP) as an easily traceable marker. Between 1 to 9x105 transfected BM cells were transplanted into 15 myeloablatively irradiated sex-mismatched C57BL/6J mice. Subsequently, 3 to 4 selection rounds with BCNU/O6-BG were carried out, enriching eGFP marked hematopoiesis in these mice up to 70–90%. Between 1 and 7x107BM cells of different mice were transplanted according to marrow location into groups of 5 sex-matched Bri44[1] mice. Two mice each received BM from the hind limbs, two from the pelvis and one received cells from the spleen, only, respectively. Altogether the study comprised 15 groups divided into 6 female and 9 male groups. Of these, 4 male and 3 female groups received 3 HSC-mobilization courses with G-CSF at intervals of 2 months starting 3 month after transplantation. Hematopoiesis in the other fraction remained unperturbed. During the observation period of 11–14 months in these tertiary recipients, repeated FACS analyses as well as linear amplification mediated (LAM) PCRs were carried out to track the clonal contributions. A decrease in the percentage of eGFP expressing marked hematopoiesis was observed in most cases. However, eGFP expression never disappeared altogether and could still be detected in the different hematopoietic lineages and successfully sorted for further analyses by MoFlo (Dako-Cytomation). Assessment of the clonal status of the Bri44 by LAM-PCR displayed interesting results. In some mice a decline in clone numbers was observed, whereas clone numbers remained stable in others. Tertiary transplantation with long-term follow-up indicates that this observation may be related to the transplantation of limited long-term repopulating clone numbers and progenitor cell exhaustion over time.

Presence of bone marrow–derived circulating progenitor endothelial cells in the newly formed lymphatic vessels

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4184-4190 ◽  
Author(s):  
Piotr Religa ◽  
Renhai Cao ◽  
Meit Bjorndahl ◽  
Zhongjun Zhou ◽  
Zhenping Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Lymphatic Vessels ◽  
Growth Factor Receptor ◽  
Donor Bone Marrow ◽  
Green Fluorescent ◽  
Corneal Lymphangiogenesis ◽  
Marrow Cells

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPCs) have been reported to incorporate into newly formed blood vessels under physiologic and pathologic conditions. However, it is unknown if CEPCs contribute to lymphangiogenesis. Here we show that in a corneal lymphangiogenesis model of irradiated mice reconstituted with enhanced green fluorescent protein (EGFP)-positive donor bone marrow cells, CEPCs are present in the newly formed lymphatic vessels. Depletion of bone marrow cells by irradiation remarkably suppressed lymphangiogenesis in corneas implanted with fibroblast growth factor-2 (FGF-2). Further, transplantation of isolated EGFP-positive/vascular endothelial growth factor receptor-3-positive (EGFP+/VEGFR-3+) or EGFP+/VEGFR-2+ cell populations resulted in incorporation of EGFP+ cells into the newly formed lymphatic vessels. EGFP+/CEPCs were also present in peritumoral lymphatic vessels of a fibrosarcoma. These data suggest that BM-derived CEPCs may play a role in “lymphvasculogenesis.”

scholarly journals Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 310-317 ◽  
Author(s):  
JF Apperley ◽  
BD Luskey ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Bone Marrow Cells ◽  
Selectable Marker ◽  
Retroviral Vector ◽  
G418 Selection ◽  
Marrow Cells

Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

scholarly journals Retroviral gene transfer of human adenosine deaminase in murine hematopoietic cells: effect of selectable marker sequences on long-term expression

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 310-317 ◽  
Author(s):  
JF Apperley ◽  
BD Luskey ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Bone Marrow Cells ◽  
Selectable Marker ◽  
Retroviral Vector ◽  
G418 Selection ◽  
Marrow Cells

Abstract Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (less than 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK- ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

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