scholarly journals Bone-Directed Expression of Col1a1 Promoter-Driven Self-Inactivating Retroviral Vector in Bone Marrow Cells and Transgenic Mice

2001 ◽  
Vol 3 (4) ◽  
pp. 543-550 ◽  
Author(s):  
Mary Louise Stover ◽  
Chi-Kuang Leo Wang ◽  
Monique B. McKinstry ◽  
Ivo Kalajzic ◽  
Gloria Gronowicz ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Marrow Cells

scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

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.

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 CblY371H Transgene Combined with Hematopoietic Deletion of the Endogenous c-Cbl Gene Results in GM-CSF Hypersensitivity and Leukocytosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3672-3672
Author(s):  
Kenneth Lieuw ◽  
Jayasree Krishnamurthy ◽  
Mignon L. Loh
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgenic Mice ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Embryonic Lethality ◽  
Hematopoietic Stem ◽  
Endogenous Gene ◽  
Gm Csf ◽  
Marrow Cells

Abstract Juvenile Myelomonocytic Leukemia (JMML) is a mixed myeloproliferative/myelodysplastic disease that is rapidly fatal with infiltration of myeloid cells into multiple organs. About 15% of JMML patient samples contain a mutation in c-Cbl, and germline mutation results in the predisposition for developing JMML. The c-Cbl gene encodes a multifunctional adaptor protein that contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif that contains E3 ligase activity, and a C-terminal ubiquitin-associated domain. The TKB domain is involved in adaptor functions of the protein, whereas the ubiquitin ligase domain results in mono-ubiquitination of receptors which promotes lysosomal mediated degradation of activated receptors. Interestingly, a hotspot for mutations at residue 371 exists in JMML patients, where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H. This residue belongs in the linker region of the CBL protein, and it was previously observed that Tyr-371 plays key roles in activating the ubiquitin ligase activity of the protein. In vitro, CblY371H mutation does indeed destroy its ligase function, resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene is silenced. How mutant Cbl gives rise to JMML, however, and how it acts in concert with other genes in the pathogenesis of JMML requires further study. To address these questions, we overexpressed the oncogenic CblY371H mutation using transgenic mice. As expected, overexpression of CblY371H by itself in wildtype mice had no apparent phenotype. Therefore, Cbl transgenic mice were bred to Cbl heterozygous knockout mice (Cbl+/-) followed by further breeding in an attempt to generate Cbl transgenic mice with the endogenous Cbl gene inactivated (CblY371H; Cbl-/-). Surprisingly, unlike Cbl null mice, which are viable, overexpression of mutant Cbl allele in Cbl null mice caused embryonic lethality between 11.5 dpc and 12.5 dpc. In order to circumvent the developmental effects of expressing the mutant Cbl protein, we used a conditional Cbl knockout mouse to tissue specifically delete the endogenous Cbl gene. We chose the MMTV-Cre strain, which expresses Cre recombinase in only 10% of hematopoietic stem cells (CD34-; Lin-; Sca-1+; c-Kit+). With subsequent breeding with the CblY371H transgenic mice, we were able to bypass the embryonic lethality and produce mice with the correct genotype (MMTV-Cre;CblY371H;Cblfl/fl). These mice look normal but develop significant leukocytosis and show GM-CSF hypersensitivity even though only 10% of hematopoietic stem cells are affected. These mice, however, appear unaffected by the leukocytosis, and show no obvious difference with its littermates up to one year of age. We conclude that mutant CblY371H by itself is not sufficient for the development of JMML in this model and requires additional cooperating events. Whether further aging of these mice will result in JMML remains to be seen. In conclusion, we have developed a mouse model overexpressing the CblY371H protein ubiquitously, which causes deleterious development when it is the only c-Cbl protein available. This confirms the important role of c-Cbl activity during development. In hematopoietic cells, the overexpression of CblY371H results in leukocytosis and GM-CSF hypersensitivity when the endogenous gene is inactivated. We are currently investigating the cooperating events that are required for the development of JMML in this mouse model. Figure 1. Phenotype of CblY371H Transgenic Mice A and B. Embryonic lethality of Cbl transgenic mice. The embryos look normal on day 10 of development but by day 12.5, no homozygous embryos are found. C and D. There is significant leukocytosis when the CblY371H transgene is combined with inactivation of the endogenous gene only in hematopoietic stem cells using the MMTV Cre. Figure 1. Phenotype of CblY371H Transgenic Mice A and B. Embryonic lethality of Cbl transgenic mice. The embryos look normal on day 10 of development but by day 12.5, no homozygous embryos are found. C and D. There is significant leukocytosis when the CblY371H transgene is combined with inactivation of the endogenous gene only in hematopoietic stem cells using the MMTV Cre. Figure 2. GM-CSF Hypersensitivity of Bone Marrow Cells from Triple Transgenic Mice A. Western Blot of bone marrow cells stimulated with GM-CSF. Panel A shows time course after stimulation of bone marrow cells from conditional Cbl mice (Cblfl/fl) that have the endogenous Cbl gene deleted using the MMTV-Cre transgene. B-D.Quantitation of several blots showing GM-CSF hypersensitivity. When normalized to the nontransgenic mice at time point zero, there is increased activity of downstream signaling pathways with and without GM-CSF. Figure 2. GM-CSF Hypersensitivity of Bone Marrow Cells from Triple Transgenic Mice A. Western Blot of bone marrow cells stimulated with GM-CSF. Panel A shows time course after stimulation of bone marrow cells from conditional Cbl mice (Cblfl/fl) that have the endogenous Cbl gene deleted using the MMTV-Cre transgene. B-D. Quantitation of several blots showing GM-CSF hypersensitivity. When normalized to the nontransgenic mice at time point zero, there is increased activity of downstream signaling pathways with and without GM-CSF. Disclosures No relevant conflicts of interest to declare.

Continued Expression of the NRAS(G12V) Oncogene Is Required for the Maintenance of Acute Myeloid Leukemia Induced in Cooperation with MLL-AF9.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1615-1615
Author(s):  
Won-Il Kim ◽  
Ilze Matise ◽  
Miechaleen Diers ◽  
David Largaespada
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Transgenic Mice ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Scid Mice ◽  
Myeloid Lineage ◽  
Blast Cells ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract To study the role of the NRAS(G12V) oncogene in the context of acute myeloid leukemia (AML) cells developing with in cooperation with MLL fusion oncogene (MLL-AF9), we used a Vav promoter-Tet transactivator (Vav-tTA)-driven repressible system of NRAS(G12V) expression in Mll-AF9 mice. Vav-tTA; TRE-NRAS(G12V); Mll-AF9 triply-transgenic mice were generated by crossing the Vav-tTA; TRE-NRAS(G12V) doubly-transgenic FVB/n and Mll-AF9 knock-in BL6 mice. The triply-transgenic FVB/n × BL6 F1 mice expressing both the NRAS(G12V) and Mll-AF9 transgenes developed AML, which showed a trend of decreased latency compared with those carrying only the Mll-AF9 knock-in transgene. Mast cell disease also occurred accordingly in the Vav-tTA; TRE-NRAS(G12V) co-transgenic mice. Since the mastocytosis disease is not transplantable, we transplanted bone marrow cells from four independent AML mice into recipient SCID mice to determine whether NRAS(G12V) expression is necessary to maintain AML in the recipient mice without mastocytosis. Continuously treating the transplanted SCID mice with doxycycline (Dox) in drinking water, we found the expression of NRAS(G12V) oncogene was required for AML persistence in three out of the four independent primary AML cells. Furthermore, we transplanted the AML bone marrow cells previously xenografted in the recipient SCID mice into other SCID mice to conditionally repress NRAS(G12V) expression only after the transplanted AML was fully established. We found the number of WBC cells was greatly decreased 4–6 days after the Dox treatment and this was correlated with the significant increase of apoptotic cells in bone marrow and peripheral bloods. The transplanted AML blast cells underwent apoptosis and were mostly removed from the circulating blood, bone marrow, and spleen after 8 days post Dox treatment. In 2–3 weeks after beginning Dox treatment and observing AML remission, Dox-resistant leukemia relapse was observed in recipient SCID mice. The relapsed leukemia failed to express NRAS(G12V) and showed significantly reduced aggressiveness along with less myelosuppression and more differentiated myeloid lineage cells than AML prior to repression of NRAS(G12V) expression. The NRAS(G12V)-independent relapsed disease histopathologically resembles an aggressive myeloproliferative disease (MPD) rather than AML, because the proportion of AML blast cells was less than 20% of myeloid lineage cells. The NRAS(G12V)-independent MPD could be transplanted into recipient SCID mice, but the subsequent anemia was greatly attenuated compared to transplant of the same AML clone expressing NRAS(G12V). We conclude that NRAS(G12V) can be a good molecular target to treat AML, because NRAS(G12V) expression is required for persistence and specific malignant features in AML induced in cooperation with MLL-AF9. Targeting NRAS(G12V) can strongly disturb the maintenance of AML blast cells and myelosuppression, although leukemia cells can relapse without NRAS(G12V) expression.

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.

Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10

Blood ◽  
2001 ◽  
Vol 98 (12) ◽  
pp. 3301-3308 ◽  
Author(s):  
Jon Mar Björnsson ◽  
Elisabet Andersson ◽  
Patrik Lundström ◽  
Nina Larsson ◽  
Xiufeng Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitors ◽  
Tetracycline Transactivator ◽  
Human Cd34 ◽  
Marrow Cells ◽  
Myeloid Progenitors

Abstract Recent studies show that several Hox transcription factors are important for regulation of proliferation and differentiation in hematopoiesis. Among these is H0XA10, which is selectively expressed at high levels in the most primitive subpopulation of human CD34+ bone marrow cells. When overexpressed, H0XA10 increases the proliferation of early progenitor cells and can lead to the development of myeloid leukemia. To study the effects of H0XA10 on primitive hematopoietic progenitors in more detail, transgenic mice were generated with regulatable H0XA10 expression. The transgenic mouse model, referred to as tetO-HOXA10, contains theH0XA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of H0XA10 is inducible and reversible depending on the absence or presence of tetracycline or its analog, doxycycline. A retroviral vector containing the tetracycline transactivator gene (tTA) was used to induce expression of the H0XA10 gene in bone marrow cells from the transgenic mice. Reverse transcription–polymerase chain reaction analysis confirmed regulatable H0XA10 expression in several transgenic lines. H0XA10 induction led to the formation of hematopoietic colonies containing blastlike cells and megakaryocytes. Moreover, the induction of H0XA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFU-S12]), which was reversible on withdrawal of induction. Activation of H0XA10 expression intet0-H0XA10 mice will therefore govern proliferation of primitive myeloid progenitors in a regulated fashion. This novel animal model can be used to identify the target genes of HOXA10 and better clarify the specific role of HOXA10 in normal and malignant hematopoiesis.

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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