Construction of Gpm6a/Reelin(GFPCreERT2) by BAC recombination using a specific gene in hepatic mesothelial or stellate cells.

AIM: To prepare a Gpm6a/Reelin(GFPCreERT2) construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC).

METHODS: Gpm6a and Reelin BACs were purified and transformed into SW102 E. coli by electroporation. The GFPCreERT2 fragment was prepared from a shuttle vector and transformed into SW102 E. coli carrying a BAC. Homologous recombination was induced in SW102 E. coli. Recombinant clones were screened and confirmed by PCR and restriction enzyme digestion. Recombinant clones were transformed into SW102 E. coli to remove the kanamycin unit.

RESULTS: A complete BAC was successfully transformed into SW102 E. coli by electroporation because BAC purified from SW102 E. coli showed the same pattern as the original BAC with BamHI digestion. The GFPCreERT2 fragment was deemed to have been prepared successfully because we obtained the same size fragment as expected. Homologous recombination was induced, and GFPCreERT2 was deemed to have been inserted into the correct site of the BAC because we found the band change was the same as the expected pattern after restriction enzyme digestion. The kanamycin unit was deemed to have been removed successfully because we obtained different sizes of bands that were consistent with the results expected by PCR with different primers.

CONCLUSION: The construct of Gpm6a(GFPCreERT2) or Reelin(GFPCreERT2) was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.