[Construction of vector tandem expressing rat ventricular myocyte Kir2.1 shRNA and its effect in vitro].
OBJECTIVE
To construct a recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem and its effect in vitro


METHODS
Ventricular myocytes were collected from newborn Wistar rats and cultured. Five sites targeting on the rat Kir2.1 gene were selected. Accordingly 5 pairs of oligonucleotide fragments were designed, synthesized, and annealed to obtain double-stranded DNAs. The 5 pairs of oligonucleotide were then cloned into the vector pGenesil-1 by repeated excision and ligation successively. The tandem recombinant vector pEGFP6-1Kir2.1 was thus constructed and transfected into the cultured rat myocytes. RT-PCR and Western blotting were used to detect the mRNA and protein expression of Kir2.1 in the myocytes. Sequence not related to Kir2.1 sequence with mismatched bases was designed and used as control.


RESULTS
A recombinant vector that expresses 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem was constructed. 96 hours after the transfection RT-PCR showed that the Kir2.1 mRNA transcription was suppressed by 83, 6%, and Western blotting showed that the Kir2.1 protein transcription was suppressed by 68.1% in comparison with the control.


CONCLUSIONS
The vector that expresses the 5 shRNAs targeting on the rat ventricular myocyte Kir2.1 gene in tandem is able to suppress the expression of Kir2.1 in rat ventricular myocytes. Application of such vector may be a new method to produce a new type of heart biological pacemaker.
Lei
Yin-sheng
YS
Department of Cardiac Surgery, Shandong Provincial Hospital, School of Medicine, Shandong University, Jinan 250021, China.
Zhang
Hai-zhou
HZ
Wang
Lai-cheng
LC
Guo
Lan-min
LM
Fan
Quan-xin
QX
Zou
Cheng-Wei
CW
Li
Hong-xin
HX
Wang
An-biao
AB
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D020868:Gene Silencing; D005822:Genetic Vectors; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D020080:Tandem Repeat Sequences; D014162:Transfection
C489461:Kir2.1 channel; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D034741:RNA, Small Interfering