The microcell mediated transfer of human chromosome 8 into highly metastatic rat liver cancer cell line C5F

Abstract

Aim: Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer. In order to seek the functional evidence that there could be a metastatsis suppressor gene(s) for liver cancer on human chromosome 8, we tried to transfer normal human chromosome 8 into rat liver cancer cell line C5F, which had high metastatic potential to lung.

Methods: Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selections of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. Finally, STS-PCR and WCP-FISH were used to confirm the introduction.

Results: Microcell hybrids resistant to HAT and G418 were obtained and 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. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome.

Conclusion: The successful introduction of human chromosome 8 into highly metastatic rat liver cancer cell line builds the basis for seeking functional evidence of a metastasis suppressor gene for liver cancer harboring on human chromosome 8 and its subsequent cloning.

Figure 1

Double selection of microcell hybrids in HAT and G418 (10 × 25 magnification).

Figure 2

Single-cell isolation cloning in 96-well cell culture plate (10 × 25 magnification).

Figure 3

STS-PCR Amplification of D8S277. 1: chromosome donor cell line A9(neo8); 2: recipient cell line C5F; 3-7: microcell hybrids neo8/C5F-1-5; M: 100bp ladders.

Figure 4

WCP-FISH analysis of neo8/C5F microcell hybrids.

Figure 5

WCP-FISH of neo8/C5F microcell hybrids DAPI counterstain.