Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes
Relander, Thomas LU ; Karlsson, Stefan LU
and Richter, Johan
LU
(2002)
In Journal of Gene Medicine
4(2).
p.122-132
- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/339927
- author
- Relander, Thomas
LU
; Karlsson, Stefan
LU
and Richter, Johan
LU
- organization
- publishing date
- 2002
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- VSV-G, GALV, gene transfer, CD34+, NOD/SCID, amphotropic
- in
- Journal of Gene Medicine
- volume
- 4
- issue
- 2
- pages
- 122 - 132
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- wos:000175071000002
- scopus:0036518338
- ISSN
- 1521-2254
- DOI
- 10.1002/jgm.246
- language
- English
- LU publication?
- yes
- id
- 23fee20f-2f42-4257-bac3-0ae21e2d8ef3 (old id 339927)
- date added to LUP
- 2016-04-01 12:17:06
- date last changed
- 2022-01-27 01:28:35
@article{23fee20f-2f42-4257-bac3-0ae21e2d8ef3,
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.}},
author = {{Relander, Thomas and Karlsson, Stefan and Richter, Johan}},
issn = {{1521-2254}},
keywords = {{VSV-G; GALV; gene transfer; CD34+; NOD/SCID; amphotropic}},
language = {{eng}},
number = {{2}},
pages = {{122--132}},
publisher = {{John Wiley & Sons Inc.}},
series = {{Journal of Gene Medicine}},
title = {{Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes}},
url = {{http://dx.doi.org/10.1002/jgm.246}},
doi = {{10.1002/jgm.246}},
volume = {{4}},
year = {{2002}},
}