Alexandra Shedlovsky

Identification of Mom7, a Novel Modifier of ApcMin/+ on Mouse Chromosome 18

The ApcMin mouse model of colorectal cancer provides a discrete, quantitative measurement of tumor multiplicity, allowing for robust quantitative trait locus analysis. This advantage has previously been used to uncover polymorphic modifiers of the Min phenotype: Mom1, which is partly explained by Pla2g2a; Mom2, a spontaneous mutant modifier; and Mom3, which was discovered in an outbred cross. Here, we describe the localization of a novel modifier, Mom7, to the pericentromeric region of chromosome 18. Mom7 was mapped in crosses involving four inbred strains: C57BL/6J (B6), BTBR/Pas (BTBR), AKR/J (AKR), and A/J. There are at least two distinct alleles of Mom7: the recessive, enhancing BTBR, AKR, and A/J alleles and the dominant, suppressive B6 allele. Homozygosity for the enhancing alleles increases tumor number by approximately threefold in the small intestine on both inbred and F1 backgrounds. Congenic line analysis has narrowed the Mom7 region to within 7.4 Mb of the centromere, 28 Mb proximal to Apc. Analysis of SNP data from various genotyping projects suggests that the region could be as small as 4.4 Mb and that there may be five or more alleles of Mom7 segregating among the many strains of inbred mice. This has implications for experiments involving ApcMin and comparisons between different or mixed genetic backgrounds.

The genetics of teratocarcinoma transplantation. Tumor formation in allogeneic hosts by the embryonal carcinoma cell lines f9 and pcc3.

Two cultured lines of murine embryonal carcinoma, F9 and PCC3, have been grafted to a variety of allogeneic hosts. The host strains have been classified by their resistance or sensitivity to these carcinomas. Resistance seems to be immunological in nature.Allograft rejection does not correlate withH-2 haplotype, and seems to be controlled by a limited number of recessive factors, presumably histocompatibility genes. We infer that these factors have limited polymorphism in the mouse species. Recombinational analysis of strain A/He has revealed the presence of a recessive factor linked to theH-2 locus. Tumor resistance of strains C57BL/6 and AKR appears to result from the interaction of dominant or semi-dominant factors in theH-2 region with other recessive elements in the genetic background.Though F1 hybrids between resistant mouse strains and the syngeneic strain 129 are largely tumor-sensitive, a low level of hybrid resistance to F9 has been observed and shown to be eliminated by X-irradiation.

Sex disparity in colonic adenomagenesis involves promotion by male hormones, not protection by female hormones

Significance The age-adjusted incidence of colonic adenomas and colorectal cancer is higher in men than in women. In a careful analysis of two established animal models, we found that castration reduced, and testosterone supplementation restored, the number of adenomas in the male rat and mouse colon, whereas ovariectomy and replacement of female hormones had no measureable effect on colonic adenomagenesis. In Min mice, in which most of the tumors arise in the small intestine, this testosterone-dependent sexual dimorphism in mice was specific to the colon. Our results support a paradigm shift: Testosterone promotes early adenomagenesis through an indirect mechanism, explaining the enhanced susceptibility of males to colonic adenomagenesis in the human, rat, and mouse. It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the ApcPirc/+ (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the ApcMin/+ mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.

Near-microscopic magnetic resonance imaging of the brains of phenylalanine hydroxylase-deficient mice, normal littermates, and of normal BALB/c mice at 9.4 Tesla.

The near-microscopic resolution of the mouse brain, by magnetic resonance imaging (MRI) at 9.4 T, permits in situ examination of the entire brain and longitudinal studies of neural development. MRI can be utilized to reveal brain structure at a resolution of 100 microns in the X, Y, and Z planes of brain, to differentiate the gray from white (myelin-rich) matter, and to reveal the ventricular compartments. The present report describes the structure of normal BALB/c mouse brain as revealed by imaging at 9.4 T and by histological stains; the structure of normal brain is compared with that from a phenylalanine hydroxylase-deficient mouse mutant line (Pah(enu2)) and those from normal littermates. The brains of patients with phenylketonuria (PKU) were reported to have demyelination and other structural abnormalities revealed by magnetic resonance imaging (MRI). Therefore, high-resolution MRI was used to examine the brain of this mutant, an animal model for the study of human phenylketonuria. Our study revealed no evidence of demyelination or other abnormalities in the brains of Pah(enu2) mice. Histologically, the mutant and normal mouse brains appear similar. This is consistent with a recent study from our laboratory which demonstrated that the histology of the brain of an untreated male patient, who died with PKU at the age of 29, was similar to control brain with the exception of changes directly related to visual blindness and seizures experienced by the patient.

Strong teratocarcinoma transplantation loci, Gt-1 and Gt-2, Flank H-2

Evidence is presented for the existence of two strong murine teratocarcinoma transplantation antigens (Gt) on the cell line PCC3. It is shown that the loci governing expression of these antigens are linked to the H-2 complex. These loci have been further mapped with respect to the brachyury marker (T) and H-2: Gt-1 lies 5±2 crossover units proximal to H-2 and 12±2 crossover units distal to T, Gt-2 lies 21±4 crossover units distal to H-2. It is possible that these strong transplantation antigens provide an embryonic analogue to the adult major histocompatibility system.

A candidate mouse model for Hartnup Disorder deficient in neutral amino acid transport

The mutant mouse strain HPH2 (hyperphenylalaninemia) was isolated after N-ethyl-N-nitrosourea (ENU) mutagenesis on the basis of delayed plasma clearance of an injected load of phenylalanine. Animals homozygous for the recessive hph2 mutation excrete elevated concentrations of many of the neutral amino acids in the urine, while plasma concentrations of these amino acids are normal. In contrast, mutant homozygotes excrete normal levels of glucose and phosphorus. These data suggest an amino acid transport defect in the mutant, confirmed in a small reduction in normalized values of 14C-labeled glutamine uptake by kidney cortex brush border membrane vesicles (BBMV). The hyperaminoaciduria pattern is very similar to that of Hartnup Disorder, a human amino acid transport defect. A subset of Hartnup Disorder cases also show niacin deficiency symptoms, which are thought to be multifactorially determined. Similarly, the HPH2 mouse exhibits a niacin-reversible syndrome that is modified by diet and by genetic background. Thus, HPH2 provides a candidate mouse model for the study of Hartnup Disorder, an amino acid transport deficiency and a multifactorial disease in the human.

Genetic mapping of hph2, a mutation affecting amino acid transport in the mouse

We describe the genetic mapping of hyperphenylalaninemia 2 (hph2), a recessive mutation in the mouse that causes deficient amino acid transport similar to Hartnup Disorder, a human genetic amino acid transport disorder. The hph2 locus was mapped in three separate crosses to identify candidate genes for hph2 and a region of homology in the human genome where we propose the Hartnup Disorder gene might lie. The mutation maps to mouse Chromosome (Chr) 7 distal of the simple sequence length polymorphism (SSLP) marker D7Mit140 and does not recombine with D7Nds4, an SSLP marker in the fibroblast growth factor 3 (Fgf3) gene. Unexpectedly, the mutant chromosome affects recombination frequency in the D7Mit12 to D7Nds4 interval.

Meiotic mapping of murine chromosome 17: The string of loci around l(17)-2Pas

We describe a genetic analysis of l(17)-2Pas, an embryonic lethal mutation on murine chromosome 17. Males transmitted the l(17)-2 allele to only 38% of their offspring, whereas females transmitted this allele at 50%. Two-point crosses revealed tight linkage between l(17)-2 and brachyury (T), and deletion mapping placed l(17)-2 outside of the hairpin-tail deletion (Thp). To map this mutation more precisely, we intercrossed hybrid mice that carry distinct alleles at many classical and DNA loci on chromosome 17 and obtained 172 animals recombinant in the T to H-2 region. Strong positive interference was observed over the 14 cM interval from T to H-2K. Thus, a single recombinant can be informative; one such recombinant places l(17)-2 distal of the molecular marker D17Leh66D. Robust genetic maps can be constructed with multilocus crosses that share anchor loci. DNA markers can be interpolated onto these maps retrospectively.

Detailed physical and genetic mapping in the region of plasminogen, D17Rp17e, and quaking

We present here a detailed physical map encompassing over 600 kb of mouse Chromosome (Chr) 17 in the region of plasminogen, D17Rp17e, and quaking. This region is cloned in yeast artificial chromosomes (YACs). We have identified several CpG islands within this region from pulsed field gel mapping of mouse genomic DNA and YAC DNA. Five new DNA probes have been generated. One, D17Leh514, is a minimum of about 90 kb distal to plasminogen. Four, D17Leh513, D17Leh512, D17Leh511, and D17Leh510, are proximal to D17Rp17e, the closest previously described genetic marker to quakingviable and quakinglethal-1 mutations. We have genetically mapped D17Leh511 to within 0.15 cM of these mutations. The genetic distance to D17Rp17e from D17Leh511 is also 0.15 cM; the physical distance of less than 360 kb (minimum 200 kb) is consistent with an approximation of 2 Mbp per cM.

H-2 class I and Gt (H-2) antigens are identical: Evidence from H-2 mutant mice

Target (Gt) antigens involved in the rejection of embryonal carcinoma (EC) transplants map to both the K and D regions of H-2 and elsewhere (Johnson et al. 1983a, b). Paradoxically, EC cells do not appear to express H-2 class I antigens (Artzt and Jacob 1974, Morello et al. 1978, 1982, Croce et al. 1981, Maher and Dove 1984). It has recently been reported that mice coisogenically immunized against H-2 class I antigens on transfected L cells reject transplanted EC cells (D6mant and Oudshoorn-Snoek 1985, Moser et al. 1985). From these results, we have concluded that either the EC cells express H-2 class I antigens or the Gt (H-2) antigens share cross-reactive determinants with the H-2 molecules encoded by the transfected genes. We now report experiments performed to distinguish between these possibilities. We find that mice bearing mutations in the H 2 K b gene reject implants of the EC line PCC3/A/1 (PCC3, genotypically H-2 b) after immunization with C57BL/6J (H-2 b) spleen cells. Thus, we conclude that PCC3 cells express H-2 b determinants that are missing, or altered, in at least two different K b mutations. This strongly suggests that the Gt antigens reside on H-2Kb-encoded molecules themselves, rather than simply sharing an antigenic determinant. The experimental strategy for the present experiments depends on the following previously reported observations (Tyan and Cole 1963, Johnson et al. 1983a, b, Moser et al. 1985). Mice immunized against various transplantation antigens and then sublethally irradiated reject subsequently administered grafts which express those antigens. In contrast, irradiated control mice pre-immunized against extraneous antigens do not reject such grafts, or do so only belatedly. Thus, the capacity of irradiated recipients to reject these grafts is an assay for the expression of antigens on a challenge