[Constructions of Gcn5 shRNAs interfere the histone acetylation modification with stem cell differentiation].
OBJECTIVE
To construct the Gcn5 shRNA plasmids and to explore the Gcn5 shRNA role in histone acetylation modification with the differentiation of stem cells.


METHODS
Seven shRNA fragments were recombined into pGenesil-1 vector to form 7. Gcn5 shRNA constructions. The mesenchymal stem cells (MSCs) induced for two weeks with 5-aza were transfected by the plasmids with lipofectamine2000. Polyclonal antibodies labeled with TRITC were used to identify the acetylation in MSCs with or without Gcn5 shRNA constructions. The efficiencies of transfection and RNAi were calculated based on the ratio of GFP (green fluorescence)/DAPI (blue fluorescence) and TRITC (red fluorescence)/DAPI, respectively.


RESULTS
Seven Gcn5 shRNA plasmids or constructions were identified by restriction endonucleases Pst I/Sal I and DNA sequencing. Acetylation block was observed after Gcn5 shRNA plasmids transfected into cells. Fluorescent intensity of TRITC in nucleuses were decreased remarkably, or even disappeared in MSCs. The efficiencies of transfection and RNAi were 93.7% and 46.6%, respectively.


CONCLUSIONS
The Gcn5 shRNA plasmids constructed in the present study can decrease the histone acetylation during cell differentiation. It sets the basis for further exploring the role of acetylation in the regulation of cell differentiation.
Zhu
Jing
J
Pediatric Institute of Children's Hospital, Chongqing University of Medical Sciences, Chongqing 400014, P.R.China. zhujing310@yahoo.com.cn
Wang
Ying-xiong
YX
Zhang
Xiao-ping
XP
Wang
Jin-ju
JJ
Zhang
Xiao-fei
XF
Tian
Jie
J
D000107:Acetylation; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D051548:Histone Acetyltransferases / Q000235:genetics*; D006657:Histones / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics*; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D018797:Cell Cycle Proteins; D051548:Histone Acetyltransferases; D006657:Histones; D034741:RNA, Small Interfering; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor