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Gene marking of human neural stem/precursor cells using green fluorescent proteins

Navarro-Galve, B; Villa, A; Bueno, C; Thompson, Lachlan LU ; Johansen, J and Martinez-Serrano, A (2005) In Journal of Gene Medicine 7(1). p.18-29
Abstract
Background Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. Methods hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting,... (More)
Background Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. Methods hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. Results Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. Conclusions Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of and is highly useful in vivo. The nature, properties, and possible side hNSCs, effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
neural precursor, transplantation, neural progenitor, protein, green fluorescent, reporter gene, cell therapy, GFP, gene therapy
in
Journal of Gene Medicine
volume
7
issue
1
pages
18 - 29
publisher
John Wiley & Sons
external identifiers
  • wos:000226620500002
  • pmid:15508144
  • scopus:12744253636
ISSN
1521-2254
DOI
10.1002/jgm.639
language
English
LU publication?
yes
id
1f8d2572-3d45-4538-a4e1-bd336f504119 (old id 254820)
date added to LUP
2007-08-14 10:34:40
date last changed
2017-01-01 04:30:30
@article{1f8d2572-3d45-4538-a4e1-bd336f504119,
  abstract     = {Background Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. Methods hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. Results Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. Conclusions Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of and is highly useful in vivo. The nature, properties, and possible side hNSCs, effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.},
  author       = {Navarro-Galve, B and Villa, A and Bueno, C and Thompson, Lachlan and Johansen, J and Martinez-Serrano, A},
  issn         = {1521-2254},
  keyword      = {neural precursor,transplantation,neural progenitor,protein,green fluorescent,reporter gene,cell therapy,GFP,gene therapy},
  language     = {eng},
  number       = {1},
  pages        = {18--29},
  publisher    = {John Wiley & Sons},
  series       = {Journal of Gene Medicine},
  title        = {Gene marking of human neural stem/precursor cells using green fluorescent proteins},
  url          = {http://dx.doi.org/10.1002/jgm.639},
  volume       = {7},
  year         = {2005},
}