Lab Anim Res.  2018 Dec;34(4):257-263. 10.5625/lar.2018.34.4.257.

Disruption of the Tff1 gene in mice using CRISPR/Cas9 promotes body weight reduction and gastric tumorigenesis

• Kim H
• Jeong H
• Cho Y
• Lee J
• Nam KT
• Lee HW

• KMID: 2459303
• DOI: http://doi.org/10.5625/lar.2018.34.4.257

Abstract

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. TFF1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated TFF1-knockout (KO) mice, without a neomycin resistant (NeoR) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our TFF1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established TFF1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that TFF1 expression influences gender differences.

Keyword

Tff1; gastric cancer; CRISPR/Cas9; Tff1-knockout mouse

MeSH Terms

Adenoma
Animals
Body Weight*
Carcinogenesis*
Epithelial Cells
Gastric Mucosa
Genes, Synthetic
Genes, Tumor Suppressor
Humans
Hyperplasia
Lotus
Male
Mice*
Mucous Membrane
Neomycin
Phenotype
Stomach Neoplasms
Neomycin

Figure

• 

  Figure 1 (A) CRISPR/Cas9 targeting strategy to generate Tff1-KO mice. Blue arrows #1–8 indicate the location and orientation of sgRNAs designed to target specific sites. Exons and introns are indicated with boxes and lines, respectively. PCR primers (F, forward; R, reverse) for genotyping are denoted by red arrows. (B) PCR screening results for newborns edited by Cas9 with sgRNAs 1, 2, 5, and 6 (combination 1) or 3, 4, 7, and 8 (combination 2). Mice carrying deletion mutations are highlighted in red. (C) Sequencing results for candidate newborns from combination 1 (upper panel) and 2 (bottom panel). The protospacer adjacent motif (PAM) and target sequences of sgRNAs are shown in red and blue, respectively. The symbol, “-”, indicates a single-nucleotide deletion. (D) PCR genotyping results for heterozygous (HT), homozygous (KO), and wild-type (WT) mice. Each arrow indicates WT-(1181 bp) and KO-specific (325 bp) bands.

• 

  Figure 2 (A–B) Body weight changes in WT (black, n=7) and Tff1-KO (red, n=7) mice. Total body weights of male (A) and female (B) mice were measured once a week from 4 weeks to 36 weeks. Data are presented as mean±SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001). (C) Mean weights (g) of total body, liver, kidney, lung, heart, and spleen in male WT and Tff1-KO mice after sacrifice. Data are presented as mean±SEM (WT, n=2; KO, n=7).

• 

  Figure 3 (A) Representative macroscopic images of WT (upper panel) and Tff1-KO mice (lower panel) at 8 months. The dotted line indicates the boundaries between the fundus and the antrum. (B) Representative H&E images of WT (a, b), 8-month-old (c, d) and 14-month-old Tff1-KO mice (e-h). Higher magnification views of images in the dotted boxes in the left panels are shown on the right. Hyperproliferation of the gastric epithelium and infiltration of immune cells are observed in the antrum of Tff1-KO mice (c, d). Adenoma is observed in the antrum region of Tff1-KO mice (e, f). Yellow asterisk indicates the tumor region (f). Mild lesions are observed in 14-month-old Tff1-KO mice (g, h). Black asterisk indicates cystic dilatation (h). Bars: 200 µm (e); 100 µm (a, c, f, g); 40 µm (b, d, h).

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