Construction and characterization of cDNA library for IRM-2 mice
10.3760/cma.j.issn.0254-5098.2010.03.011
- VernacularTitle:IRM-2小鼠全长cDNA文库的构建及鉴定
- Author:
Qin WANG
;
Jin LI
;
Li SONG
;
Qiang LIU
;
Jingyin YUE
;
Chuanjie MU
;
Weisheng TANG
;
Feiyue FAN
- Publication Type:Journal Article
- Keywords:
IRM-2 mice;
cDNA library;
SMART techique
- From:
Chinese Journal of Radiological Medicine and Protection
2010;30(3):274-278
- CountryChina
- Language:Chinese
-
Abstract:
Objective To screen and isolate the radioresistance related genes of IRM-2 mice.Methods cDNA library of IRM-2 mice was constructed by SMART technique.Total RNA was isolated from spleens of IRM-2 male mice.The first-strand cDNA was synthesized by using PowerScript reverse transeriptase,and double-strand cDNA was synthesized and amplified by long PCR.The PCR products were purified,digested with restriction enzyme Sfi I.The ds-cDNA fragment lessthan 500 bp was fractionated and ligated to the Sfi I-digested pDNR-LIB vector.The ligation mixture was transformed into E.coil DH5α by electroporution transformation to generate the unamplified cDNA library.The quality of cDNA library was identified by PCR technique.130 clones from cDNA library were sequenced and compared with GenBank database.Results The cDNA library contained 2.25 x 106 independent clones with an average insert size of 1.2 kb.The ratio of recombination and full-length was 95% and 55%,respectively.21 pieces of EST sequences from cDNA library were not the same as the known mice genes and registered into GenBank EST database,with registered number DW474856-DW474876.Conclusions cDNA library of IRM-2 mice has been constructed successfully.21 pieces of EST implies that radioresistance correlative genes may be in IRM-2 mice,which will lay a foundation for isolating and identifying radioresistance related genes in further study.
