[Knocking down rat Mecp2 expression by RNAi].
OBJECTIVE
To find the valid siRNA (small interference RNA) sequence to knock down rat Mecp2 expression for the analysis of Mecp2 function by RNA interference (RNAi).


METHODS
A plasmid (pMecp2-RFP) expressing rat Mecp2 and marker gene red fluorescent protein (RFP) as a fusion gene was constracted. We first selected a candidate valid sequence by cotransfecting the pMecp2-RFP with 4 candidate siRNAs targeting rat Mecp2 to HEK293T cells respectively, and then identified the RNAi efficiency of the candidate valid siRNA by detecting its effect on suppressing the expression of endogenous rat Mecp2 in PC12 cells.


RESULTS
siRNA sequence (5'-GCUGUGAAGGAAUCUUCUA-3') targeting rat Mecp2 mRNA 918-936nt was the valid sequence to knock down rat Mecp2 expression by RNA interference. As the target sequence was located in exon 4 of rat Mecp2 mRNA, we assumed that it could suppress the expression of Mecp2alpha and Mecp2beta at the same time. And as the target sequence was located in the coding region of rat Mecp2, we assumed that it could suppress the expression of both 1.9 kb and 10 kb of rat Mecp2 transcript.


CONCLUSIONS
This study has laid the foundation for constructing rat Mecp2 knocked-down neuronal cell model to study the gene function of Mecp2 in brain development.
Zhang
Yu-zhi
YZ
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Wang
Han-sen
HS
Pan
Hong
H
Li
Mei-rong
MR
Bao
Xin-hua
XH
Jin
Jing
J
Wu
Xi-ru
XR
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016716:PC12 Cells; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D051783:Methyl-CpG-Binding Protein 2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C497394:Mecp2 protein, rat