Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 14, 2017; 23(2): 224-231
Published online Jan 14, 2017. doi: 10.3748/wjg.v23.i2.224
Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells
Hong-Bo Shi, Jin-Li Lou, Hong-Lin Shi, Feng Ren, Yu Chen, Zhong-Ping Duan
Hong-Bo Shi, Hong-Lin Shi, Feng Ren, Zhong-Ping Duan, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
Jin-Li Lou, Clinical Laboratory Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
Yu Chen, Zhong-Ping Duan, Artificial Liver Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
Author contributions: Shi HB and Lou JL contributed equally to this work; Shi HB and Shi HL carried out most of the experiments; Ren F purified the strain and performed PCR; Chen Y analyzed the pattern of restriction enzyme digestion; Shi HB drafted the manuscript, and Lou JL analyzed the experimental data; Duan ZP and Lou JL conceived and supervised the study; Duan ZP was involved in editing the manuscript; all authors read and approved the final manuscript.
Supported by National Natural Science Foundation of China, No. 81300349 and No. 81270532; the Beijing Natural Science Foundation, No. 7144216; the Beijing Nova Program, No. Z131107000413016; the Project of Science and Technology Activities of Preferred Overseas Personnel of Beijing (2014); the Project of Cultivation of High Level Medical Technical Personnel in the Health System of Beijing, No. 2014-3-090 and No. 2013-3-071; Beijing Municipal Institute of public medical research development and reform pilot project, No. 2016-2.
Conflict-of-interest statement: Shi HB, Lou JL, Shi HL, Ren F, Chen Y and Duan ZP are employees of Beijing Youan Hospital, Capital Medical University. The authors declare no conflicts of interest.
Data sharing statement: Technical appendices, statistical codes and datasets are available from the corresponding author at Participants provided informed consent for data sharing. No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, 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. See:
Correspondence to: Zhong-Ping Duan, MD, PhD, Professor, Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, 8 Xitoutiao, Youwai Street, Fengtai District, Beijing 100069, China.
Telephone: +86-10-63291007 Fax: +86-10-63295258
Received: August 2, 2016
Peer-review started: August 3, 2016
First decision: September 21, 2016
Revised: October 5, 2016
Accepted: November 2, 2016
Article in press: November 2, 2016
Published online: January 14, 2017

To prepare a Gpm6a/ReelinGFPCreERT2 construct with a rapid and reliable strategy using a bacterial artificial chromosome (BAC).


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.


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.


The construct of Gpm6aGFPCreERT2 or ReelinGFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.

Keywords: 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/ReelinGFPCreERT2 construct for the first time, which is the first step for the preparation of a Gpm6aGFPCreERT2 or ReelinGFPCreERT2 mouse line.