Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 7, 2019; 25(5): 584-599
Published online Feb 7, 2019. doi: 10.3748/wjg.v25.i5.584
Claudin-7 gene knockout causes destruction of intestinal structure and animal death in mice
Chang Xu, Kun Wang, Yu-Han Ding, Wen-Jing Li, Lei Ding
Chang Xu, Kun Wang, Yu-Han Ding, Wen-Jing Li, Lei Ding, Department of Oncology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China
Author contributions: Ding L designed the study; Xu C performed the research and wrote the paper; Wang K and Ding YH fed the mice; Li WJ analysed the data.
Supported by the National Natural Science Foundation of China, No. 81372585 and No. 81772557; and Beijing Health System High Level Training Plan of Health Technical Personnel, No. 2014-3-048.
Institutional review board statement: The study was reviewed and approved by the Medical Ethics Committee of the Capital Medical University Affiliated Beijing Shijitan Hospital Institutional Review Board.
Institutional animal care and use committee statement: All protocols were carried out in accordance with relevant guidelines and regulations.
Conflict-of-interest statement: The authors declare no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
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 noncommercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Lei Ding, MD, PhD, Associate Professor, Chief Doctor, Surgeon, Surgical Oncologist, Department of Oncology, Beijing Shijitan Hospital, Capital Medical University, No. 10, Tieyi Road, Haidian District, Beijing 100038, China.
Telephone: +86-10-63926296 Fax: +86-10-63926296
Received: October 24, 2018
Peer-review started: October 25, 2018
First decision: November 29, 2018
Revised: January 10, 2019
Accepted: January 18, 2019
Article in press: January 18, 2019
Published online: February 7, 2019
Research background

Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanisms. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system to provide a basis for further studying the role of Claudin-7 in intestinal tumors.

Research motivation

Claudin-7 is currently considered to play an inhibitory role in colorectal inflammation and colorectal cancer. The most effective way to study inhibitors in vivo is to knock out the gene in an animal and observe its overall phenotype. Therefore, we constructed conventional Claudin-7 gene knockout (CKO) mice and conditional Claudin-7 gene knockout (cKO) mice using Cre/LoxP system, and we also generated inducible conditional Claudin-7 gene knockout (ICKO) mice and induced Cre expression by injecting tamoxifen. The successful construction of these mouse lines as intestinal inflammation and intestinal adenoma models could provide a basis for further studying the role of Claudin-7 in intestinal tumors.

Research objectives

The main objective was to construct three lines of Claudin-7 gene knockout mice to achieve space- and time-specific knockout of Claudin-7 and prolong the survival time of mice. Due to the prolonged growth time of mice, the organs matured when Claudin-7 was knocked out. So this animal model can provide a basis for further study of Claudin-7.

Research methods

We constructed three lines of Claudin-7 knockout mice using the Cre/LoxP system. First, chimeric mice were constructed by transfecting the target vector into embryonic stem cells, screening the hybrid clones and injecting them into the female mouse blastocysts, and transplanting the blastocysts into the female mouse uterus. Chimeric mice were then purified to obtain Claudin-7-floxed mice. Second, Claudin-7-floxed mice were crossed with CMV-Cre mice, vil1-Cre mice, and villin-CreERT2 mice ot obtain Claudin-7 CKO mice, Claudin-7 cKO mice, and Claudin-7 ICKO mice, respectively. ICKO mice were induced by intraperitoneal injection of tamoxifen to knockout Claudin-7 in intestinal tissue. Finally, Western blot was used to verify the knockout efficiency of Claudin-7. Hematoxylin-eosin (HE) staining was used to confirm the structural changes and pathological changes of the intestinal tract in Claudin-7 knockout mice. Immunohistochemical staining was used to observe the proliferation markers. The construction of cKO mice prolonged the lifespan of CKO mice, and the ICKO mouse was the first animal model to specifically knock out Claudin-7 in a spatial and temporal manner.

Research results

We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice successfully. Claudin-7 CKO mice and Claudin-7 cKO mice were stunted and died during the perinatal period, and intestinal HE staining revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. Claudin-7 ICKO mice had a normal phenotype at birth, but after the induction with tamoxifen, the mice exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were also observed. Intestinal immunohistochemistry analysis showed abnormal expression and distribution of Ki67, and the normal intestinal proliferation balance was disrupted.

Research conclusions

This study innovatively constructed three lines of Claudin-7 gene knockout mice, which clarified that Claudin-7 plays an inhibitory role in colon inflammation and colon adenoma, and initially found that Claudin-7 may promote the development of colon adenomas by affecting proliferation. This study successfully simulated intestinal inflammation and intestinal adenoma, and proposed new animal models. This study clarified the role of Claudin-7 in colonic inflammation and tumors, laying the groundwork for finding early clinical diagnosis and potential therapeutic targets.

Research perspectives

This article describes the construction of Claudin-7-floxed mice and the process of crossing with three Cre mice. Based on this experience, we can construct ICKO mice in which Claudin-7 is ablated in the kidney, skin, and some other organs, and then observe changes in mice before and after Claudin-7 deletion. In the next step of the study, the dose of tamoxifen should be clarified, and the tumor should be induced in the intestinal tract of ICKO mice. The direction of the future research is to clarify the specific mechanism of Claudin-7 in inflammatory bowel disease and intestinal tumorigenesis, invasion, and metastasis; and to explore the relationship between Claudin-7 and stem cells as well as its role in intestinal development.