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AIM:In order to elucidate the molecular mechanism oflymphatic metastasis of hepatocarcinoma,we detectedthe difference of gene expression between mousehepatocarcinoma cell lines Hca-F and Hca-P with differentlymphatic metastasis potential.METHODS:cDNA of Hca-F cells was used as a tester andcDNA of Hca-P cells was used as a driver,cDNAs highlyexpressed in Hca-F cells were isolated by the suppressionsubtractive hybridization (SSH) method.The isolated cDNAwas cloned into T/A cloning vector.The ligation productswere transformed into DH5 α competent cells.Individual doneswere randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing.RESULTS:There were 800 positive clones in amplifiedsubtracted cDNA library.Random analysis of 160 clones withPCR showed that 95% of the clones contained 100-700 bpinserts.Analysis of 20 sequenced cDNA clones randomlypicked from the SSH library revealed 4 known genes(mouse heat shock protein 84 ku,DNA helicase,ribosomalprotein S13 ,ethanol induced 6 gene) and 3 expressedsequence tags (ESTs).Four cDNAs showed no homologyand presumably represent novel genes.CONCLUSION:A subtracted cDNA library of differentiallyexpressed genes in mouse heptocarcinoma cell lines withdifferent lymphatic metastasis potential was successfullyconstructed with SSH and T/A cloning techniques.Thelibrary is efficient and lays a solid foundation for searchingnew lymphatic metastasis related genes.The expressionof mouse heat shock protein gene,DNA helicase and other4 novel gene may be different between mouse heptocarcinomacell lines with different lymphatic metastasis potential.
AIM: In order to elucidate the molecular mechanism oflymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mousehepatocarcinoma cell lines Hca-F and Hca-P with differentlymphatic 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 suppressionsubtractive hybridization (SSH) method. The isolated cDNA was cloned into T / A cloning vector.The ligation productswere transformed into DH5a competent cells. Individual doneswere randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing.RESULTS: There were 800 positive clones in amplifiedsubtracted cDNA library.Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bpinserts . Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes (mouse heat shock protein 84 ku, DNA helicase, ri bosomal 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 searchingnew lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other4 novel genes may be different between mouse heptocarcinomacell lines with different lymphatic metastasis potential.