Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis

Abstract

Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines.

Figure 1

Comparison of polyp number and location between Apc^Min/+ mice and mice from the Cav1^−/−x Apc^Min/+ colony at 120 days of age. (A) Average polyp number detected in the small intestine and colon combined. (B) Average polyp numbers detected in the proximal, middle and distal portion of the small intestine and colon. (*p < 0.05. **p < 0.005. ***p < 0.001. n = 147 for Apc^Min/+ mice and n = 104 for mice from the Cav1^−/−x Apc^Min/+ colony. Error bars represent standard deviation).

Figure 2

Cav1 genotype does not correlate with intestinal polyp number in Apc^Min/+ mice. (A) Distal small intestine samples were taken from B6 Cav1^−/− mice and transgenic mice expressing Myc-tagged Cav1 (B6 Cav1Tg). Cav1 expression was analyzed by western blot. Actin was used as a protein loading control. (B) Average polyp numbers detected in the intestine and colon of Apc^Min/+ mice with Cav1^+/+, Cav1^−/+ or Cav1^−/− genotypes. (n = 39 for Cav1^+/+ mice, n = 33 for Cav1^−/+ mice and n = 32 for Cav1^−/− mice. Error bars represent standard deviation).

Figure 3

Female mice from the Cav1^−/−x Apc^Min/+ colony develop more polyps than male mice at 120 days of age. Average polyp number detected in the small intestine and colon combined. (*p < 0.05; **p < 0.005; ***p < 0.001; n = 46 for female mice and n = 58 for male mice from the Cav1^−/−x Apc^Min/+ colony; n = 50 for female Apc^Min/+ mice and n = 97 for male Apc^Min/+ mice. Error bars represent standard deviation).

Figure 4

Map of residual 129X1/SvJ genomic DNA in the congenic region of the Cav1 knockout allele. Tail DNA was tested for SSLP markers across the length of chromosome 6. Marker locations are given in Mb from the centromere. Gray boxes indicate the presence of 129X1 genomic DNA. Black boxes indicate the absence of 129X1 genomic DNA. The maximum congenic region is a 51 Mb area between the D6Mit138 and D6Mit316 markers.

Figure 5

Polyp multiplicity for mice in the Cav1^−/−x Apc^Min/+ colony based on sex and D6Mit33 genotype. Polyp number data includes both small intestine and colon. Female mice were separated into (A) B6/B6 (n = 25) and (B) 129/B6 (n = 18) genotypes. High polyp multiplicity in B6/B6 female mice was defined as >150 polyps. Male mice were also separated into (C) B6/B6 (n = 35) and (D) 129/B6 (n = 14) genotypes. High polyp multiplicity in B6/B6 male mice was defined as >110 polyps.

Figure 6

Representation of the effects of Mom12 and Mom13 on intestinal tumorigenesis. the effect of Mom12 on polyp number is represented by the dashed gray curve. the effect of Mom13 is represented by the black curve. Both modifiers individually have an apparent bimodal distribution. Mice with resistant alleles of both modifiers develop the same number of polyps as B6 Apc^Min/+ controls. Mice carrying a susceptible allele at either Mom12 or Mom13 have increased polyp numbers. Mice carrying susceptible alleles at both modifier loci have the Mom12 phenotype. Expected genotypes for each modifier locus are listed above the phenotypic groups. (R, resistant allele; S, susceptible allele).