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- Relander, Thomas, et al.
(författare)
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Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures
- 2000
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Ingår i: Experimental Hematology. - 1873-2399. ; 28:4, s. 373-381
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: We wanted to determine whether the long-term bone marrow culture (LTBMC) transduction system would lead to efficient gene transfer and engraftment of murine repopulating hematopoietic stem cells (HSC), particularly in nonablated recipients. MATERIALS AND METHODS: Congenic mouse strains expressing Ly 5.1 or Ly 5.2 and the GP+E86 cell line producing the MGirL22Y vector carrying the gene for enhanced GFP were used. Murine LTBMCs were established and demi-depopulated on days 7 and 14 with addition of vector supernatant on days 8 and 15. RESULTS: Cell recovery on day 21 was 21.3%+/-3.8% of input cells and CFU-C recovery was 9.7+/-3.4% as compared with CFU-C of input cells. In vitro transduction efficiency determined by CFU-C expressing GFP was 22.2%+/-1.6%. In irradiated (950 cGy) mice transplanted with 2x10(6) LTBMC cells, 94% of nucleated cells in the blood at week 16 were of donor origin. However, GFP was only detected at low level in a few animals at week 4 and not later. Analysis of bone marrow from these mice at week 20 did not show any GFP expression and semiquantitative PCR revealed a transgene level of <1%. When 3.5-20.8x10(6) LTBMC cells (corresponding to 20-100x10(6) fresh cells) were transplanted to nonablated recipients, no engraftment or GFP expression were detected. Competitive repopulation experiments showed that the long-term repopulation ability (LTRA) of the LTMC cells was only 7% of fresh cells. CONCLUSION: These results indicate that LTBMC transduction of murine cells leads to low-level transduction of progenitors, no gene transfer to repopulating stem cells, and reduction in LTRA in ablated and nonablated recipients.
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| 2. |
- Relander, Thomas, et al.
(författare)
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Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes
- 2002
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Ingår i: Journal of Gene Medicine. - : Wiley. - 1521-2254 .- 1099-498X. ; 4:2, s. 122-132
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Tidskriftsartikel (refereegranskat)abstract
- Background The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34(+)/CD38(low) and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes. Methods CB cells were transduced on Retronectin using an MSCV-based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13-MGIN (GALV) 293GPG-MGIN (VSV-G) or AM12-MGIN (amphotropic). Results Sorted CD34(+)/CD38(low) cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP-positive cells was 61.8+/-6.6% (PG13-MGIN), 26.9+/-3.5% (293GPG-MGIN), and 39.3+/-4.8% (AM12-MGIN). For transplantation experiments, CD34(+) cells were prestimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5 x 10(5) cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum-free medium and transduction of CD34(+) cells using VSV-G-pseudotyped vectors under serum-free conditions was very inefficient. In contrast, transduction with PG13-MGIN under serum-free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3+/-6.6%), and efficient gene transfer to SRCs (46.2+/-4.8%). Conclusions The best conditions for transduction and engraftment of CB SRCs were obtained with GALV-pseudo typed vectors using serum-free conditions. Copyright (C) 2002 John Wiley Sons, Ltd.
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| 3. |
- Relander, Thomas, et al.
(författare)
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Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium
- 2001
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Ingår i: Journal of Gene Medicine. - 1521-2254. ; 3:3, s. 207-218
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The objective of the present study was to optimize conditions for retroviral transduction of human cord blood (CB) CD34+ cells and to reveal mechanisms which interfere with efficient gene transfer. METHODS: An MSCV based retroviral vector with the gene for enhanced green fluorescent protein (MGIN) produced by GP+envAM12 (amphotropic envelope), PG13 (gibbon ape leukemia virus envelope) or 293GPG (vesicular stomatitis virus envelope) cell lines was used to transduce cord blood CD34+ cells on Retronectin (fibronectin fragment CH-296) in three different ways: either in vector containing medium (VCM), in fresh medium on Retronectin pre-loaded with vector or in VCM on Retronectin pre-loaded with vector. RESULTS: Paradoxically, the transduction efficiency obtained with pre-load of vector onto Retronectin alone was higher than pre-load plus VCM for PG13-MGIN (67.9 +/- 6.0% vs 24.9 +/- 8.0%) and AM12-MGIN (47.5 +/- 5.8% vs 38.7 +/- 2.2%). Further experiments showed that transduction on Retronectin pre-loaded with PG13-MGIN or AM12-MGIN was inhibited by the presence of the same VCM at high concentrations, but not by the presence of a VCM with a different receptor specificity. If no pre-load of vector was performed, the highest transduction efficiencies were seen when VCMs were diluted 1:10 (MOIs of 3). The inhibitory effect of high titer PG13-MGIN VCM was confirmed in more primitive CD34+CD38low cells and in NOD/SCID repopulating cells, and was also seen in experiments with bone marrow CD34+ cells. CONCLUSIONS: Retroviral transduction of CB CD34+ cells on Retronectin is inhibited by high titer PG13 and GP+envAM12 vector containing medium. Efficient gene transfer to human hematopoietic cells can be obtained by preload alone of the vector onto Retronectin. These findings are of importance for the design of transduction protocols for repopulating hematopoietic cells.
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