Two genes abrogate the inhibition of murine hepatocarcinogenesis by ovarian hormones.

Hormonal and genetic factors strongly influence the susceptibility of inbred mice to hepatocarcinogenesis. Female C57BR/cdJ (BR) mice are extremely susceptible to liver tumor induction relative to other strains because they are genetically insensitive to the inhibition of hepatocarcinogenesis by ovarian hormones. To determine the genetic basis for the sensitivity of BR mice relative to resistant C57BL/6J (B6) mice, we treated 12-day-old B6BRF1 x B6 and B6BRF1 x B6BRF1 (F2) animals with N,N-diethylnitrosamine (0.1 micromol/g of body weight) and enumerated liver tumors at 32 weeks of age in males and at 50 weeks in females. Genomic DNA samples from backcross and F2 mice were analyzed for 70 informative simple sequence length polymorphism markers. Genetic markers on chromosome 17 (D17Mit21) and chromosome 1 (D1Mit33) cosegregated with high tumor multiplicity in both sexes. Together, these loci [designated Hcf1 and Hcf2 (Hepatocarcinogenesis in females), respectively] account for virtually all of the difference in sensitivity between BR and B6 mice. The Hcf1 locus accounts for a majority of the higher susceptibility of BR mice of both sexes. Backcross female mice heterozygous at both loci (33 +/- 23 tumors per mouse) and at Hcf1 only (17 +/- 18) were 15- and 8-fold more sensitive, respectively, than mice homozygous for the B6 alleles at Hcf1 and Hcf2 (2.2 +/- 3.9). In backcross male mice, the double heterozygotes (35 +/- 22) and Hcf1 heterozygotes (28 +/- 12) were 5.4- and 4.3-fold more sensitive than mice homozygous for B6 alleles at both loci (6.5 +/- 5.4).

Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice.

The prevalence of cholesterol gallstones differs among inbred strains of mice fed a diet containing 15% (wt/wt) dairy fat, 1% (wt/wt) cholesterol, and 0.5% (wt/wt) cholic acid. Strains C57L, SWR, and A were notable for a high prevalence of cholelithiasis; strains C57BL/6, C3H, and SJL had an intermediate prevalence; and strains SM, AKR, and DBA/2 exhibited no cholelithiasis after consuming the diet for 18 weeks. Genetic analysis of the difference in gallstone prevalence rates between strains AKR and C57L was carried out by using the AKXL recombinant inbred strain set and (AKR x C57L)F1 x AKR backcross mice. Susceptibility to gallstone formation was found to be a dominant trait determined by at least two genes. A major gene, named Lith1, mapped to mouse chromosome 2. When examined after 6 weeks on the lithogenic diet, the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.88) was downregulated as expected in the gallstone-resistant strains, AKR and SJL, but this enzyme failed to downregulate in C57L and SWR, the gallstone-susceptible strains. This suggests that regulation of the rate-limiting enzyme in cholesterol biosynthesis may be pivotal in determining the occurrence and severity of cholesterol hypersecretion and hence lithogenicity of gallbladder bile. These studies indicate that genetic factors are critical in determining gallstone formation and that the genetic resources of the mouse model may permit these factors to be identified.

Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent protein kinase C (PKC) substrate in brain that is expressed highly in hippocampal granule cells and their axons, the mossy fibers. Here, we examined hippocampal infrapyramidal mossy fiber (IP-MF) limb length and spatial learning in heterozygous Macs mutant mice that exhibit an ≈50% reduction in MARCKS expression relative to wild-type controls. On a 129B6(N3) background, the Macs mutation produced IP-MF hyperplasia, a significant increase in hippocampal PKCɛ expression, and proficient spatial learning relative to wild-type controls. However, wild-type 129B6(N3) mice exhibited phenotypic characteristics resembling inbred 129Sv mice, including IP-MF hypoplasia relative to inbred C57BL/6J mice and impaired spatial-reversal learning, suggesting a significant contribution of 129Sv background genes to wild-type and possibly mutant phenotypes. Indeed, when these mice were backcrossed with inbred C57BL/6J mice for nine generations to reduce 129Sv background genes, the Macs mutation did not effect IP-MF length or hippocampal PKCɛ expression and impaired spatial learning relative to wild-type controls, which now showed proficient spatial learning. Moreover, in a different strain (B6SJL(N1), the Macs mutation also produced a significant impairment in spatial learning that was reversed by transgenic expression of MARCKS. Collectively, these data indicate that the heterozygous Macs mutation modifies the expression of linked 129Sv gene(s), affecting hippocampal mossy fiber development and spatial learning performance, and that MARCKS plays a significant role in spatial learning processes.

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F1 genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F1 ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F1 ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F1 ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F1 ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F1 ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.

Identification of multiple quantitative trait loci linked to prion disease incubation period in mice

Polymorphisms in the prion protein gene are known to affect prion disease incubation times and susceptibility in humans and mice. However, studies with inbred lines of mice show that large differences in incubation times occur even with the same amino acid sequence of the prion protein, suggesting that other genes may contribute to the observed variation. To identify these loci we analyzed 1,009 animals from an F2 intercross between two strains of mice, CAST/Ei and NZW/OlaHSd, with significantly different incubation periods when challenged with RML scrapie prions. Interval mapping identified three highly significantly linked regions on chromosomes 2, 11, and 12; composite interval mapping suggests that each of these regions includes multiple linked quantitative trait loci. Suggestive evidence for linkage was obtained on chromosomes 6 and 7. The sequence conservation between the mouse and human genome suggests that identification of mouse prion susceptibility alleles may have direct relevance to understanding human susceptibility to bovine spongiform encephalopathy (BSE) infection, as well as identifying key factors in the molecular pathways of prion pathogenesis. However, the demonstration of other major genetic effects on incubation period suggests the need for extreme caution in interpreting estimates of variant Creutzfeldt–Jakob disease epidemic size utilizing existing epidemiological models.

Genetic and environmental factors modify bovine spongiform encephalopathy incubation period in mice

The incubation period (IP) and the neuropathology of transmissible spongiform encephalopathies (TSEs) have been extensively used to distinguish prion isolates (or strains) inoculated into panels of inbred mouse strains. Such studies have shown that the bovine spongiform encephalopathy (BSE) agent is indistinguishable from the agent causing variant Creutzfeldt–Jakob disease (vCJD), but differs from isolates of sporadic CJD, reinforcing the idea that the vCJD epidemic in Britain results from consumption of contaminated beef products. We present a mouse model for genetic and environmental factors that modify the incubation period of BSE cross-species transmission. We have used two mouse strains that carry the same prion protein (PrP) allele, but display a 100-day difference in their mean IP following intracerebral inoculation with primary BSE isolate. We report genetic effects on IP that map to four chromosomal regions, and in addition we find significant factors of host environment, namely the age of the host's mother, the age of the host at infection, and an X-cytoplasm interaction in the host.

A locus on chromosome 7 determines myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

Dystrophic cardiac calcinosis, an age-related cardiomyopathy that occurs among certain inbred strains of mice, involves myocardial injury, necrosis, and calcification. Using a complete linkage map approach and quantitative trait locus analysis, we sought to identify genetic loci determining dystrophic cardiac calcinosis in an F2 intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains. We identified a single major locus, designated Dyscalc, located on proximal chromosome 7 in a region syntenic with human chromosomes 19q13 and 11p15. The statistical significance of Dyscalc (logarithm of odds score 14.6) was tested by analysis of permuted trait data. Analysis of BxH recombinant inbred strains confirmed the mapping position. The inheritance pattern indicated that this locus influences susceptibility of cells both to enter necrosis and to subsequently undergo calcification.

A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.

Previously, a hypomorphic mutation in CD18 was generated by gene targeting, with homozygous mice displaying increased circulating neutrophil counts, defects in the response to chemically induced peritonitis, and delays in transplantation rejection. When this mutation was backcrossed onto the PL/J inbred strain, virtually all homozygous mice developed a chronic inflammatory skin disease with a mean age of onset of 11 weeks after birth. The disease was characterized by erythema, hair loss, and the development of scales and crusts. The histopathology revealed hyperplasia of the epidermis, subcorneal microabscesses, orthohyperkeratosis, parakeratosis, and lymphocyte exocytosis, which are features in common with human psoriasis and other hyperproliferative inflammatory skin disorders. Repetitive cultures failed to demonstrate bacterial or fungal organisms potentially involved in the pathogenesis of this disease, and the dermatitis resolved rapidly after subcutaneous administration of dexamethasone. Homozygous mutant mice on a (PL/J x C57BL/6J)F1 background did not develop the disease and backcross experiments suggest that a small number of genes (perhaps as few as one), in addition to CD18, determine susceptibility to the disorder. This phenotype provides a model for inflammatory skin disorders, may have general relevance to polygenic human inflammatory diseases, and should help to identify genes that interact with the beta2 integrins in inflammatory processes.

Isolation and characterization of a pseudoautosomal region-specific genetic marker in C57BL/6 mice using genomic representational difference analysis.

Representational difference analysis was used to identify strain-specific differences in the pseudoautosomal region (PAR) of mouse X and Y chromosomes. One second generation (C57BL/6 x Mus spretus) x Mus spretus interspecific backcross male carrying the C57BL/6 (B6) PAR was used for tester DNA. DNA from five backcross males from the same generation that were M. spretus-type for the PAR was pooled for the driver. A cloned probe designated B6-38 was recovered that is B6-specific in Southern analysis. Analysis of genomic DNA from several inbred strains of laboratory mice and diverse Mus species and subspecies identified a characteristic Pst I pattern of fragment sizes that is present only in the C57BL family of strains. Hybridization was observed with sequences in DBA/2J and to a limited extent with Mus musculus (PWK strain) and Mus castaneus DNA. No hybridization was observed in DNA of different Mus species, M. spretus, M. hortulanus, and M. caroli. Genetic analyses of B6-38 was conducted using C57BL congenic males that carry M. spretus alleles for distal X chromosome loci and the PAR and outcrosses of heterozygous congenic females with M. spretus. These analyses demonstrated that the B6-38 sequences were inherited with both the X and Y chromosome. B6-38 sequences were genetically mapped as a locus within the PAR using two interspecific backcrosses. The locus defined by B6-38 is designated DXYRp1. Preliminary analyses of recombination between the distal X chromosome gene amelogenin (Amg) and the PAR loci for either TelXY or sex chromosome association (Sxa) suggest that the locus DXYRp1 maps to the distal portion of the PAR.