[An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification].
OBJECTIVE
To develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.


METHODS
The double-strand DNA fragment containing a ClaI site (the flanking sequences of which were not complementary) was annealed and ligated into small hairpin RNA (shRNA) expression vector pSilencer-4.1 that did not contain ClaI site to construct the circular pSilencer-4.1-ClaI vector. With BamHI and HindIII, the pSilencer-4.1-ClaIwas digested and ligated with the DNA template of green fluorescence protein (GFP) shRNA that did not include a ClaI site. The plasmid DNA of the positive clones was extracted and digested with ClaI, and the inserted DNA sequence of the non-linearized plasmid was identified by sequence analysis.


CONCLUSIONS
DNA sequencing showed that pSilencer-4.1-ClaI was correctly constructed and the plasmids resistant to ClaI digestion were all recombinant vectors encoding GFP shRNA. The constructed pSilencer-4.1-ClaI can be used as a universal vector to construct the shRNA expression plasmid, and the incorporated ClaI sites may allow efficient screening of recombinant shRNA expression vectors.
Shan
Zhi-xin
ZX
Research Center of Medical Sciences, People's Hospital of Guangdong Province, Guangzhou 510080, China. zhixinshan@yahoo.com.cn
Lin
Qiu-xiong
QX
Fu
Yong-heng
YH
Deng
Chun-yu
CY
Yu
Xi-yong
XY
D001483:Base Sequence; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D015183:Restriction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA; D013997:Time Factors
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins