Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector - PubMed (original) (raw)
. 1999 Nov 15;94(10):3349-57.
Affiliations
- PMID: 10552944
- PMCID: PMC9071851
Improved expression in hematopoietic and lymphoid cells in mice after transplantation of bone marrow transduced with a modified retroviral vector
S Halene et al. Blood. 1999.
Abstract
Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.
Figures
Fig 1.
Vector constructs. Vectors were constructed so that the LTR drives expression of the reporter gene for eGFP and the SV40 promoter drives expression of the gene for neomycin resistance. The 5′LTR of LeGFPSN is the MoMuLV LTR. The MND LTR is based on the MoMuLV LTR, but was modified by replacing the MoMuLV enhancer by the MPSV enhancer (M), deleting the negative control region (N) and replacing the primer binding site (PBS) of the MoMuLV by the PBS of the endogenous murine retrovirus dl587rev (D). Vectors are otherwise identical.
Fig 2.
FACS analysis of eGFP expression. FACS dot blots and histogram blotsfrom a representative pair of mice are shown. (A and B) Cells from bone marrow, spleen, thymus, and blood from recipients of LeGFPSN-transduced, MNDeGFPSN-transduced, and LN-transduced (data not shown) marrow were harvested and analyzed by FACS for eGFP expression (x axis) in granulocytes (Gr-1), monocytes/macrophages (MAC-1), B cells (B220), T cells (CD4, CD8), and RBC (Ter-119). Lineage-specific staining is shown on the y-axis. Quadrant statistics give the percentage of gated events. (C) Distribution of fluorescence intensity is shown in histogram plots for LeGFPSN (dark line) and MNDeGFPSN (solid area).
Fig 2.
FACS analysis of eGFP expression. FACS dot blots and histogram blotsfrom a representative pair of mice are shown. (A and B) Cells from bone marrow, spleen, thymus, and blood from recipients of LeGFPSN-transduced, MNDeGFPSN-transduced, and LN-transduced (data not shown) marrow were harvested and analyzed by FACS for eGFP expression (x axis) in granulocytes (Gr-1), monocytes/macrophages (MAC-1), B cells (B220), T cells (CD4, CD8), and RBC (Ter-119). Lineage-specific staining is shown on the y-axis. Quadrant statistics give the percentage of gated events. (C) Distribution of fluorescence intensity is shown in histogram plots for LeGFPSN (dark line) and MNDeGFPSN (solid area).
Fig 3.
eGFP expression in tissues. eGFP expression was assessed in bone marrow, spleen, blood (A), and thymus (B) from recipients of LeGFPSN-transduced, MNDeGFPSN-transduced, and LN-transduced (data not shown) bone marrow 2 weeks, 8 to 12 weeks, 4 to 5 months, and 6 to 8 months after BMT. Values are given as the mean ± standard error of the mean. n is the number of animals analyzed for each time point and vector. * and ** mark differences in the percentage of expression between MNDeGFPSN and LeGFPSN that are statistically significant (*P < .05; **P < .005).
Fig 4.
eGFP expression in lineages. eGFP expression is shown for B lymphocytes (A) and T lymphocytes (B) in bone marrow, spleen, blood, and thymus from recipients of MNDeGFPSN-transduced and LeGFPSN-transduced (data for negative control LN not shown) bone marrow. Animals were killed and analyzed 2 weeks, 8 to 12 weeks, 4 to 5 months, and 6 to 8 months after BMT. Values for the percentage of cells showing expression were calculated by dividing the number of expressing lin+ cells by the total number of lin+ cells and are given as the mean ± standard error of the mean. n is the number of animals analyzed for each time point and vector. * and ** mark differences in the percentage of cells showing expression between MNDeGFPSN and LeGFPSN that are statistically significant (*P < .05 and **P < .005).
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