AIM:In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma,we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential.METHODS:cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver.cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method.The isolated cDNA was cloned into T/A cloning vector.The ligation products were transformed into DH5 α competent cells.Individual clones were randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing. RESULTS:There were 800 positive clones in amplified subtracted cDNA library.Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts.Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku,DNA helicase,ribosomal protein S13,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs).Four cDNAs showed no homology and presumably represent novel genes.CONCLUSION:A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques.The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes.The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.