1. Volume 131, Issue 5, Pages 1553-1560 (November 2006)
Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 Over Expression in Transgenic Mice Suppresses Intestinal Neoplasia 
Seung Joon Baek, Ryuji Okazaki, Seong-Ho Lee, Jeanelle Martinez, Jong-Sik Kim, Kiyoshi Yamaguchi, Yuji Mishina, David W. Martin, Ahmed Shoieb, Michael F. Mcentee, Thomas E. Eling 
Gastroenterology 
Volume 131, Issue 5, Pages (November 2006)
DOI: /j.gastro
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2. Figure 1
Generation and characterization of NAG-1 transgenic mice. (A) Schematic diagram of pCAG-LoxP/CAT/LoxP-NAG-1. The human NAG-1 cDNA34 was cloned into the plasmid pCAG-loxP/CAT/loxP35 to generate a transgene vector pCAG-LoxP/CAT/LoxP-NAG-1. CAG is a strong promoter that ubiquitously drives expression in diverse tissues. The flanking sequences of the CAT reporter gene are loxP sites that would be removed in the presence of Cre recombinase. The fragments of SalI and SphI were used for pronuclear microinjection. Transgenic mice on a C57BL/6 (Jackson Laboratories) background were generated by pronuclear microinjection. Three transgenic founder lines were identified and characterized for CAG-directed CAT expression. Two lines, identified as 1377 and 1398, were chosen for further analysis based on CAT activity. Mice from lines 1377 and 1398 were bred to protamine-Cre transgenic mice (129/Sv background).36 Male Protamine Cre:CAG-LoxP/CAT/LoxP-NAG-1 compound transgenic mice were bred to female C57BL/6 mice to generate offspring that had a deleted floxed CAT reporter, turning on the expression of the NAG-1 gene. (B) Western blot analysis of NAG-1 in different tissue. Two founders (1377 and 1398) were selected based on the CAT activity, and Tg mice were bred with protamine Cre mice that express Cre in all the tissues. Thirty micrograms of total cell lysates were loaded into 15% SDS-PAGE and detected with the NAG-1 antibody. Equal loadings were measured by Actin expression. STD represents control cell lysates from sulindac sulfide-treated HCT-116 cells.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Body weights (g) of male and female NAG-Tg and control littermates. A mixed-model growth curve analysis48 was used, followed by 2-sample t tests at each week to determine which differences were significant. The P values are all 2-sided. The overall pattern of growth differed between NAG-Tg+/− and WT for females (P = .030) and for males (P = .032). Differences were significant beginning in week 7 for females and in week 8 for males. Data are expressed as mean ± SE. *P < .05, **P < .01 for difference between NAG-Tg and control littermates, n = 4–14.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Reduced numbers of AOM-induced aberrant crypt foci in NAGTg+ mice. (A and B) Azoxymethane (AOM)-induced aberrant crypt foci (ACF) and tumor formation in the colon of NAGTg+ (4 males and 10 females) and control littermates (5 males and 13 females). The bottom box represents the incidence of tumor-bearing mice. Values are expressed as mean ± SD. (A) The data represent mean number of ACF per mouse, and (B) mean number of aberrant crypts per focus. (C) Immunohistochemical analysis of NAG-1 expression in colon tissue from control and NAGTg+ mice (original magnification, 200×).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
NAGTg+ mice carrying the ApcMin mutation had a significant reduction in the number of adenomas and overall tumor load in comparison to nontransgenic littermates, but mean tumor size did not differ. Figure 4 shows a representative Western blot of human NAG-1 in the small intestine (A), average tumor diameter in millimeters (B), comparison of the total numbers of polyps of different sizes (C), and tumor load (= number of tumors × average diameter) (D) between control littermates ApcMin+/NAGTg- and ApcMin+/NAGTg+ mice (n = 4 for ApcMin+/NAGTg- and n = 9 for ApcMin+/NAGTg+ mice). The data are represented as mean ± SE using nonparametric Mann–Whitney tests to compare NAG-Tg with control littermates.49 *Significantly lower compared with ApcMin+/NAGTg- mice, at P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions