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

abstract: ...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 BamH I 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.

...World J Gastroenterol World J. Gastroenterol WJG World Journal of Gastroenterology 1007-9327 2219-2840 Baishideng Publishing Group Inc 28127196 5236502 jWJG.v23.i2.pg224 10.3748/wjg.v23.i2.224 Basic Study Construction of Gpm6a/Reelin GFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells Shi Hong-Bo Lou Jin-Li Shi Hong-Lin Ren Feng Chen Yu Duan Zhong-Ping Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan

..., build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. 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

... Gpm6a GFPCreERT2 or Reelin GFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function. Bacterial artificial chromosome Homologous recombination Glycoprotein M6a Reelin Core tip: Until now, there have been few specific mouse lines that allowed recombination for tracing hepatic mesothelial cells or hepatic stellate cells. Here, we describe a rapid and reliable strategy for construct preparation using a bacterial artificial chromosome. This study prepared a Gpm6a/Reelin GFPCreERT2 construct for the first time, which is the first step for the preparation of a Gpm6a GFPCreERT2 or Reelin GFPCreERT2 mouse line. INTRODUCTION Excessive extracellular matrix (ECM) of the liver results in cirrhosis, which is an end stage liver disease with high mortality and for which efficacious medical treatments are not currently available, except for liver transplantation. Hepatic stellate cell (HSC) activation is considered a major

... cells or HSCs. The Wt1 CreERT2 mice are useful to trace hepatic mesothelial cells, but the labeling efficiency and specificity is low[ 4 - 6 ]. Specific genes have been identified in hepatic mesothelial cells and HSCs by microarray[ 7 , 8 ]. We plan to develop Cre mouse lines with specific markers for the study of HSCs or hepatic mesothelial cells. Based on a previous study, glycoprotein M6a (Gpm6a) has been identified as a specific surface marker of hepatic mesothelial cells. It covers the surface of the liver and migrates from the surface into the center[ 4 ]. Reelin is an extracellular matrix glycoprotein, which is a specific HSC marker in the mouse liver and has similar amounts in resting and activated HSCs[ 9 ]. The Gpm6a GFPCreERT2 or Reelin GFPCreERT2 mouse line will express a fusion protein of green fluorescent protein (GFP), Cre, and estrogen receptor induced by tamoxifen (ERT2) under the control of the Gpm6a or Reelin promoter. GFP is used to track the labeled protein as a marker. Cre recombinase is used to delete a segment of DNA flanked by LoxP sites (flox). The ERT2 system is used to activate Cre activity by tamoxifen treatment[ 10 , 11 ]. Cre mouse lines are very useful tools that can generate knockout mice through the cross breeding of Cre and flox mouse lines. A mouse line is needed to trace HSC lineages and study their function through the knockout of specific genes in specific cells. It is, therefore, necessary to prepare a Gpm6a GFPCreERT2 or Reelin GFPCreERT2

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Gene: GPM6A - glycoprotein M6A - (NCBI)

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