Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization.
OBJECTIVE
To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.


METHODS
Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH) was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the blue-white screening system to establish cDNA library.


RESULTS
After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.


CONCLUSIONS
A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.
Liang
Li
L
Department of Pathology, the First Military Medical University, Guangzhou 510515, Guangdong Province, China.
Ding
Yan-Qing
YQ
Li
Xin
X
Yang
Guang-Zhi
GZ
Xiao
Jun
J
Lu
Li-Chun
LC
Zhang
Jin-Hua
JH
D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D006801:Humans; D009362:Neoplasm Metastasis; D009693:Nucleic Acid Hybridization / Q000379:methods*