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[The investigation of the technology of microcell mediated chromosome transfer for functional localization of
Hu Liu1, Sheng-long Ye, Jiong Yang
1Liver Cancer Institute & Zhongshan Hospital, Fudan University, Shanghai, 200032 PR China.
Objective:
In order to seek the functional evidence that there could be metastatsis suppressor gene for liver cancer on human chromosomes, the objective of this study is to establish a method of microcell mediated chromosome transfer (MMCT).
Methods:
Human chromosome 8 randomly marked with neo gene was introduced into highly metastatic rat liver cancer C5F cell line by treating the single human chromosome donor cells with sequential steps of micronucleation, enucleation and microcell fusion. Double selections of G418 and HAT were applied to screen positive microcell hybrids, which were cloned by single cell isolation. Microcell hybrid clones were confirmed by STS-PCR and WCP-FISH.
Results:
Microcell hybrids resistant to HAT and G418 were obtained, from which 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. The human chromosome 8 introduced into C5F was found to have random loss of chromosome fragments by STS-PCR and consistent recombination with rat chromosome by WCP-FISH.
Conclusion:
The successfulls introduction of human chromosome into highly metastatic rat liver cancer cell line has established the technical basis for functional localization of metastasis suppressor gene(s) for liver cancer on human chromosomes.
