[Construction of "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference].
OBJECTIVE
To clone the "pEGFP-C1-pU6-dsRNA" recombinant for human DNA polymerase beta RNA interference, to provide research tool for the study on the function of DNA polymerase beta in repairing of human DNA damaged by environmental chemical pollutants (ECPs).


METHODS
According to the gene sequence of polymerase beta cDNA published in Genbank, double strand RNA(dsRNA) sequence which was used in RNA interference was designed by dsRNA oligonucleotide designer and synthesized by chemical methods. DNA recombination technology was used to insert the up related dsRNA sequence into the vector of pSIREN-RetroQ, and then the "pSIREN-RetroQ-dsRNA" recombinant was obtained. After E. coli DH5alpha was transformed with the "pSIREN-RetroQ-dsRNA" recombinant and screened with ampicillin for positive clones, plasmid was extracted and digested by EcoR I and Bgl II , the fragment of"pU6-dsRNA"was purified. And then the "pU6-dsRNA"fragment was cloned into the vector of pEGFP-C1 by recombination technology, the recombinant of "pEGFP-C1-pU6-dsRNA" was obtained and identified by restriction endonuclease analysis and sequencing.


RESULTS
The "pEGFP-C1-pU6-dsRNA" recombinant lied in the predicted band, and the sequence of insert was identical to the designed target fragment.


CONCLUSIONS
The "pEGFP-C1-pU6-dsRNA" recombinant was successfully cloned for human DNA polymerase beta RNA interference, it was an important research tool for the further study.
Hu
Da-Lin
DL
School of Public Health, Sun Yat-sen University, Guangzhou 510080, China.
Zhuang
Zhi-Xiong
ZX
He
Yun
Y
Ji
Wei-Dong
WD
Tang
Dongsheng
D
Yuan
Jianhui
J
Fang
Daokui
D
Sha
Yan
Y
Yang
Jianping
J
Tu
Xiaozhi
X
Hu
Gonghua
G
D003001:Cloning, Molecular; D019951:DNA Polymerase beta / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004785:Environmental Pollutants / Q000009:adverse effects; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000096:biosynthesis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004785:Environmental Pollutants; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; C405486:U6 small nuclear RNA; D049452:Green Fluorescent Proteins; D019951:DNA Polymerase beta