Cotransfection of glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells / 中国组织工程研究
Chinese Journal of Tissue Engineering Research
;
(53): 9779-9782, 2009.
Artigo
em Chinês
| WPRIM
| ID: wpr-404490
ABSTRACT
BACKGROUND:
Deletion of glial cell-derived neurotrophic factor and endothelin receptor type B gene will induce abnormal development of enteric nervous system. Neural stem cell transplantation can repair nervous system from anatomy and function,and be considered as a vector of gene transfection.OBJECTIVE:
To transfect recombinant adenovirus carrying glial cell-derived neurotrophic factor and endothelin receptor type B gene into mouse neural stem cells, and to observe expression of target gene.DESIGN:
A cell-gene study.MATERIALS New-born Kunming mice were provided by the Animal Center of Tongji Medical College, Huazhong University of Science and Technology, China. jetPEI reagent was purchased from PolyPlus Co, France. The pAdTrack-CMV-GE with green fluorescent protein (GFP) was gifted by Doctor Sun Nianfeng and Zhang Jinghui in our laboratory.METHODS:
Neonatal mouse brain tissues were sterilely obtained to prepare monoplast suspension. Adenovirus expressing glial cell-derived neurotrophic factor and endothelin receptor type B gene with GFP was dissolved in NaCI to prepare JetPEI/DNA complex. Subcultured neural stem cells in DMEM/F12 were regulated to 5×10~8/L, and 400 μL cell suspension and 100 μL JetPEI/DNA complex were seeded on a 24-well plate at 37 ℃ in 5% CO_2 incubator. Neural stem cells were harvested at 24, 48 and 72 hours following transfection.MAIN OUTCOMEMEASURES:
The efficiency of transfection was detected using fluorescence microscope and flow cytometry.Target gene expression in neural stem cells was determined using RT-PCR.RESULTS:
Bright green fluorescence of the transfected cells could be observed under fluorescence microscope after 24 hours of transfection. The positive rate of GFP was 15.36%, 24.67%, 25.73% at 24, 48 and 72 hours following transfection respectively.Neural stem cells expressed glial cell-derived neurotrophic factor and endothelin receptor type B gene at various time points.Strap brightness was low at 24 hours, and exogenous gene expression was great at 72 hours.CONCLUSION:
The target genes were successfully transfected into neural stem cells by using jetPEI reagent. Moreover, glial cell-derived neurotrophic factor and endothelin receptor type B gene effectively transcribed and expressed in target cells.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Idioma:
Chinês
Revista:
Chinese Journal of Tissue Engineering Research
Ano de publicação:
2009
Tipo de documento:
Artigo
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