Gene marking of human neural stem/precursor cells using green fluorescent proteins.
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 hNSCs, and is highly useful in vivo. The nature, properties, and possible side effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.
Navarro-Galve
Beatriz
B
Center of Molecular Biology Severo Ochoa, Autonomous University of Madrid, Campus Cantoblanco, 28049 Madrid, Spain.
Villa
Ana
A
Bueno
Carlos
C
Thompson
Lachlan
L
Johansen
Jens
J
Martínez-Serrano
Alberto
A
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D017690:Cell Transplantation; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000166:cytology / Q000378:metabolism; D004027:Diencephalon / Q000166:cytology; D017930:Genes, Reporter; D005819:Genetic Markers; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014162:Transfection
D005819:Genetic Markers; D049452:Green Fluorescent Proteins