Characterization of Neural Stem Cells Using the Nestin Enhancer

Nestin, one of the class VI intermediate filament, is known as a specific marker for neural stem cells and regulated by the enhancer located on the second intron of the gene. The second intron is highly conserved among species and crucial for the nestin expression in central nerve system (CNS) progenitor cells. The transgenic mice that constructed to express green fluorescence protein (GFP) with nestin enhancer have been developed and have great advantage to represent live neural stem cells during research in vivo and vitro. In this study, we characterized GFP expressing the neural stem cells or progenitors in vitro culture using various antibodies. We also investigated that ngn1, which is a basic helix-loop-helix (bHLH) transcription factor that is expressed in neuronal precursors during development of the nervous system, can promote nestin expression by nestin enhancer located on nestin 2nd intron. Two E-boxes (CANNTG) in 637bp of the nestin 2nd intron (nestin enhancer) were thought to be strong candidates for bHLH factors during the early neurogenesis. ngn1 increased the second E-box mediated reporter gene activity. To evaluate the functional mechanism of E-box mediated nestin gene expression in vivo, we generated transgenic (Tg) mice with the 637bp fragment of nestin second intron containing the wild type or a mutated E-boxes. Wild type tg mice successfully exhibited CNS-specific LacZ expression. In contrast, tg mice containing the mutant sequence exhibited very few lacZ-stained cells or a few positive cells in ectopic sites. These results indicate that proneural ngn1 promotes the nestin expression through a E-box located in the nestin second intron. Finally, we also performed microarray to characterize the GFP positive and negative cells in nestin-GFP tg mice neurosphere. The microarray profile may help to understand the meaning of GFP in the tg neural stem cells and may give cue to isolate neural stem cell from progenitors.

Author dc:creator

• 유, 순태

Contributors dc:contributor

• 서, 해영
• 대학원 의학과
• 200524192

• Nestin
• transgenic mouse
• basic Helix-Loop-Helix
• neurogenin1
• microarray
• 신경줄기세포

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000002036
000000002036

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1450