An method for small hairpin RNA expression vector reconstruction for easy single restriction endonuclease identification.
- Author:
Zhi-xin SHAN
1
;
Qiu-xiong LIN
;
Yong-heng FU
;
Chun-yu DENG
;
Xi-yong YU
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Gene Expression; Genetic Engineering; methods; Genetic Vectors; genetics; Green Fluorescent Proteins; genetics; Inverted Repeat Sequences; Molecular Sequence Data; Plasmids; genetics; RNA, Small Interfering; genetics; Restriction Mapping; methods; Sequence Analysis, DNA; Time Factors
- From: Journal of Southern Medical University 2007;27(9):1341-1344
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo develop an effective method for screening recombinant hairpin RNA expression plasmids using single restriction endonuclease analysis.
METHODSThe 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.
RESULT AND CONCLUSIONDNA 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.
