Shi HB, Lou JL, Shi HL, Ren F, Chen Y, Duan ZP. Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells. World J Gastroenterol 2017; 23(2): 224-231 [PMID: 28127196 DOI: 10.3748/wjg.v23.i2.224]
Corresponding Author of This Article
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. duan2517@163.com
Research Domain of This Article
Methodology
Article-Type of This Article
Basic Study
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Shi HB, Lou JL, Shi HL, Ren F, Chen Y, Duan ZP. Construction of Gpm6a/ReelinGFPCreERT2 by BAC recombination using a specific gene in hepatic mesothelial or stellate cells. World J Gastroenterol 2017; 23(2): 224-231 [PMID: 28127196 DOI: 10.3748/wjg.v23.i2.224]
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 byNational 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 duan2517@163.com. Participants provided informed consent for data sharing. No additional data are available.
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. duan2517@163.com
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 Processing time: 162 Days and 20.1 Hours
Abstract
AIM
To prepare a Gpm6a/ReelinGFPCreERT2 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 Gpm6aGFPCreERT2 or ReelinGFPCreERT2 was prepared successfully, which will establish a foundation for tracing the hepatic stellate cell lineage and studying its function.
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.
