Construction of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

OBJECTIVE:To detect the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential using the suppression subtractive hybridization (SSH) method in order to make the molecular mechanism of lymphatic metastasis clearly. METHODS: cDNA of Hca-F cells was used as tester and cDNA of Hca-P cells was used as driver. The cDNAs which were highly expressed in Hca-F cells were isolated by using SSH method. The isolated cDNA were cloned into T/A cloning vector. The ligation products were transformed into DH5α competent cells. Individual 160 clones were selected randomly and then used for clone PCR amplification. The vector DNA from positive clones were isolated for sequencing. RESULTS: There were 800 positive bacteria clones in the amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95 percent clones contained 100-700 bp inserts. Analysis of 14 sequenced cDNA clones randomly picked from the SSH library revealed 3 known genes (mouse heat shock protein 80 KD, ribosomal protein S13, ethanol induced 6)and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent unknown genes. CONCLUSIONS: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for searching new lymphatic metastasis related genes.