A step towards annotating the mouse genome is to use forward genetics in phenotype-driven screens to saturate the genome with mutations. The purpose of this article is to highlight the new projects in North America that are focused on isolating mouse mutations after ENU mutagenesis and phenotype screening.
balancer chromosomes; Human Genome Project; Mouse Genome Project; mouse mutagenesis; phenotype screens
Obesity is the result of excess energy intake relative to expenditure, however little is known about why some individuals are more prone to weight gain than others. Inbred strains of mice also vary in their susceptibility to obesity and therefore represent a valuable model to study the genetics and physiology of weight gain and its comorbidities such as type 2 diabetes. C57BL/6J mice are susceptible to obesity and insulin resistance when fed an obesogenic diet, whereas A/J mice are resistant despite increased caloric intake. Analysis of B6- and A/J-derived chromosome substitution strains and congenic strains revealed a complex genetic and physiological basis for this phenotype. To improve our understanding of the molecular mechanisms underlying susceptibility to metabolic disease we analyzed global gene expression patterns in 6C1 and 6C2 congenic strains. 6C1 is susceptible whereas 6C2 is resistant to diet-induced obesity. In addition, we demonstrate that 6C1 is glucose intolerant and insulin resistant relative to 6C2. Pathway analysis of global gene expression patterns in muscle, adipose, and liver identified expression level differences between 6C1 and 6C2 in pathways related to basal transcription factors, endocytosis, and mitochondrial oxidative phosphorylation (OxPhos). The OxPhos expression differences were subtle but evident in each complex of the electron transport chain and were associated with a marked increase in mitochondrial oxidative capacity in the livers of the obese strain 6C1 relative to the obesity-resistant strain 6C2. These data suggests the importance of hepatic mitochondrial function in the development of obesity and insulin resistance.
Prepulse inhibition (PPI) is a measure of sensorimotor gating, a pre-attentional inhibitory brain mechanism that filters extraneous stimuli. PPI is correlated with measures of cognition and executive functioning, and is considered an endophenotype of schizophrenia and other psychiatric illnesses in which patients demonstrate PPI impairments. As a first step towards identifying genes that regulate PPI, we performed a quantitative trait locus (QTL) screen of PPI phenotypes in a panel of mouse chromosome substitution strains (CSS). We identified five CSSs with altered PPI compared to the host C57BL/6J strain: CSS-4 exhibited decreased PPI, whereas CSS-10, -11, -16, and -Y exhibited higher PPI compared to C57BL/6J. These data indicate that A/J chromosomes 4, 10, 11, 16, and Y harbor at least one QTL region that modulates PPI in these CSSs. QTLs for the acoustic startle response were identified on seven chromosomes. Like PPI, habituation of the startle response is also disrupted in schizophrenia, and in the present study CSS-7 and -8 exhibited deficits in startle habituation. Linkage analysis of an F2 intercross identified a highly significant QTL for PPI on chromosome 11 between positions 101.5Mb – 114.4Mb (peak LOD = 4.54). Future studies will map the specific genes contributing to these QTLs using congenic strains and other genomic approaches. Identification of genes that modulate PPI will provide insight into the neural mechanisms underlying sensorimotor gating, as well as the psychopathology of disorders characterized by gating deficits.
Sensorimotor gating; Quantitative trait locus; Linkage; Startle; Cognition; Psychopathology; Psychiatric disorders; Behavior; Mouse genetics
Since Mendel, studies of phenotypic variation and disease risk have emphasized associations between genotype and phenotype among affected individuals in families and populations. Although this paradigm has led to important insights into the molecular basis for many traits and diseases, most of the genetic variants that control the inheritance of these conditions continue to elude detection. Recent studies suggest an alternative mode of inheritance where genetic variants that are present in one generation affect phenotypes in subsequent generations, thereby decoupling the conventional relations between genotype and phenotype, and perhaps, contributing to ‘missing heritability’. Under some conditions, these transgenerational genetic effects can be as frequent and strong as conventional inheritance, and can persist for multiple generations. Growing evidence suggests that RNA mediates these heritable epigenetic changes. The primary challenge now is to identify the molecular basis for these effects, characterize mechanisms and determine whether transgenerational genetic effects occur in humans.
epigenetics; heritability; transgenerational
Testicular germ cell tumors (TGCT) are sex limited, occurring only in males with a Y chromosome. Recently, the gr/gr deletion on the human Y chromosome was associated with increased risk of TGCTs. In addition, the presence of Y chromosome sequences is associated with TGCTs in cases of gonadal dysgenesis. TGCTs in strain 129 males recapitulate many aspects of testicular cancer in human infants and can be used to evaluate the role of the Y chromosome in TGCT risk. We used chromosome substitution strains and a sex-reversing mutant to test the role of the Y chromosome on TGCT susceptibility. Our results show that a Y-linked gene that does not differ among the tested strains is essential for tumorigenesis.
Certain mutations in the Deadend1 (Dnd1) gene are the most potent modifiers of testicular germ cell tumor (TGCT) susceptibility in mice and rats. In the 129 family of mice, the Dnd1Ter mutation significantly increases occurrence of TGCT-affected males. To test the hypothesis that he Dnd1Ter allele is a loss-of-function mutation; we characterized the consequences of a genetically-engineered loss-of-function mutation in mice, and compared these results with those for Dnd1Ter.
We found that intercrossing Dnd1+/KO heterozygotes to generate a complete loss-of-function led to absence of Dnd1KO/KO homozygotes and significantly reduced numbers of Dnd1+/KO heterozygotes. Further crosses showed that Dnd1Ter partially rescues loss of Dnd1KO mice. We also found that loss of a single copy of Dnd1 in Dnd1KO/+ heterozygotes did not affect baseline occurrence of TGCT-affected males and that Dnd1Ter increased TGCT risk regardless whether the alternative allele was loss-of-function (Dnd1KO) or wild-type (Dnd1+). Finally, we found that the action of Dnd1Ter was not limited to testicular cancer, but also significantly increased polyp number and burden in the Apc+/Min model of intestinal polyposis.
These results show that Dnd1 is essential for normal allelic inheritance and that Dnd1Ter has a novel combination of functions that significantly increase risk for both testicular and intestinal cancer.
Testicular cancer; Allelic segregation; Intestinal neoplasia; DND1
Disruption of cellular processes affected by multiple genes and accumulation of numerous insults throughout life dictate the progression of age-related disorders, but their complex etiology is poorly understood. Postmitotic neurons, such as photoreceptor cells in the retina and epithelial cells in the adjacent retinal pigmented epithelium, are especially susceptible to cellular senescence, which contributes to age-related retinal degeneration (ARD). The multigenic and complex etiology of ARD in humans is reflected by the relative paucity of effective compounds for its early prevention and treatment. To understand the genetic differences that drive ARD pathogenesis, we studied A/J mice, which develop ARD more pronounced than that in other inbred mouse models. Although our investigation of consomic strains failed to identify a chromosome associated with the observed retinal deterioration, pathway analysis of RNA-Seq data from young mice prior to retinal pathological changes revealed that increased vulnerability to ARD in A/J mice was due to initially high levels of inflammatory factors and low levels of homeostatic neuroprotective factors. The genetic signatures of an uncompensated preinflammatory state and ARD progression identified here aid in understanding the susceptible genetic loci that underlie pathogenic mechanisms of age-associated disorders, including several human blinding diseases.
Despite strong heritability, little is known about the genetic control of susceptibility to testicular germ cell tumors (TGCTs) in humans or mice. Although the mouse model of spontaneous TGCTs has been extensively studied, conventional linkage analysis has failed to locate the factors that control both teratocarcinogenesis in the susceptible 129 family of inbred strains. As an alternative approach, we used both chromosome substitution strains (CSSs) to identify individual chromosomes that harbor susceptibility genes, and a panel of congenic strains derived from a selected CSS to determine the number and location of susceptibility variants on the substituted chromosome. We showed that 129-Chr 18MOLF males are resistant to spontaneous TGCTs and that at least four genetic variants control susceptibility in males with this substituted chromosome. In addition, early embryonic cells from this strain fail to establish embryonic stem (ES) cell lines as efficiently as those from the parental 129/Sv strain. For the first time, 129-derived genetic variants that control TGCT susceptibility and fundamental aspects of ES cell biology have been localized in a genetic context where the genes can be identified and functionally characterized.
The objective of this work was to identify strain-specific characteristics from real-time measurements of circadian rhythms of two inbred mouse strains. In particular, heart rate, temperature, and activity data collected from A/J and C57BL/6J (B6) mice using telemetry are analyzed. The influence of activity on heart rate and temperature is minimized by correlation analysis followed by regression analysis. The correlation analysis is used to determine the length of the activity data filter that results in the best correlation between activity data and heart rate or temperature. After the activity data are filtered, they are used in regression analysis. The temperature and heart rate rhythms obtained as the intercepts of the regression analysis are interpreted as the zero-activity rhythms and consequently are good estimates of the circadian rhythms. The circadian temperature rhythms for the B6 mice follow a smoother cosine-like time waveform, whereas those for the A/J mice follow a more square-wave-like waveform. To quantify the difference between these two temperature rhythms, a feature based on Fourier analysis of the time-series data is used. Detrended fluctuation analysis is used to identify features in the heart rate rhythms. The results of this work show that the features for the circadian temperature and heart rate rhythms can be used as distinguishing characteristics of the A/J and B6 strains. This work provides the foundation for future studies directed at investigating the influence of chromosomal substitutions on the regulation of circadian rhythms in these two strains.
Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The pre-implantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.
epiblast stem cells; pluripotency; embryonic stem cells; blastocyst
Crooked tail (Cd) mice bear a gain-of-function mutation in Lrp6, a co-receptor for canonical WNT signaling, and are a model of neural tube defects (NTDs), preventable with dietary folic acid (FA) supplementation. Whether the FA response reflects a direct influence of FA on LRP6 function was tested with prenatal supplementation in LRP6-deficient embryos. The enriched FA (10 ppm) diet reduced the occurrence of birth defects among all litters compared with the control (2 ppm FA) diet, but did so by increasing early lethality of Lrp6−/− embryos while actually increasing NTDs among nulls alive at embryonic days 10–13 (E10–13). Proliferation in cranial neural folds was reduced in homozygous Lrp6−/− mutants versus wild-type embryos at E10, and FA supplementation increased proliferation in wild-type but not mutant neuroepithelia. Canonical WNT activity was reduced in LRP6-deficient midbrain–hindbrain at E9.5, demonstrated in vivo by a TCF/LEF-reporter transgene. FA levels in media modulated the canonical WNT response in NIH3T3 cells, suggesting that although FA was required for optimal WNT signaling, even modest FA elevations attenuated LRP5/6-dependent canonical WNT responses. Gene expression analysis in embryos and adults showed striking interactions between targeted Lrp6 deficiency and FA supplementation, especially for mitochondrial function, folate and methionine metabolism, WNT signaling and cytoskeletal regulation that together implicate relevant signaling and metabolic pathways supporting cell proliferation, morphology and differentiation. We propose that FA supplementation rescues Lrp6Cd/Cd fetuses by normalizing hyperactive WNT activity, whereas in LRP6-deficient embryos, added FA further attenuates reduced WNT activity, thereby compromising development.
Current treatments have largely failed to slow the rapidly increasing world-wide prevalence of obesity and its co-morbidities. Despite a strong genetic contribution to obesity (40–70%), only a small percentage of heritability is explained with current knowledge of monogenic abnormalities, common sequence variants and conventional modes of inheritance. Epigenetic effects are rarely tested in humans because of difficulties arranging studies that distinguish conventional and transgenerational inheritance while simultaneously controlling environmental factors and learned behaviors. However, growing evidence from model organisms implicates genetic and environmental factors in one generation that affect phenotypes in subsequent generations. In this report, we provide the first evidence for paternal transgenerational genetic effects on body weight and food intake. This test focused on the obesity-resistant 6C2d congenic strain, which carries the Obrq2aA/J allele on an otherwise C57BL/6J background. Various crosses between 6C2d and the control C57BL/6J strain showed that the Obrq2aA/J allele in the paternal or grandpaternal generation was sufficient to inhibit diet-induced obesity and reduce food intake in the normally obesity-susceptible, high food intake C57BL/6J strain. These obesity-resistant and reduced food intake phenotypes were transmitted through the paternal lineage but not the maternal lineage with equal strength for at least two generations. Eliminating social interaction between the father and both his offspring and the pregnant dam did not significantly affect food intake levels, demonstrating that the phenotype is transmitted through the male germline rather than through social interactions. Persistence of these phenotypes across multiple generations raises the possibility that transgenerational genetic effects contribute to current metabolic conditions.
Testicular germ cell tumors (TGCTs) originate from germ cells. The 129-Ter and M19 (129.MOLF-Chr19 consomic) mouse strains have extremely high incidences of TGCTs. We found that the expression levels of Sf1 encoded Splicing factor 1 (SF1) can modulate the incidence of TGCTs. We generated mice with inactivated Sf1. Sf1 null mice (Sf1-/-) died before birth. Mice with one intact allele of Sf1 (Sf1+/-) were viable but expressed reduced levels of Sf1. When Sf1 deficient mice (Sf1+/-) were crossed to the 129-Ter and M19 strains, we observed decreased incidence of TGCTs in Sf1+/-;Ter and Sf1+/-;M19/+ mice compared to that in control cohorts. Therefore, Sf1 deficiency protects against TGCT development in both strains. Sf1 is expressed in the testes. We found that Sf1 levels vary significantly in the testes of inbred strains such as 129 and MOLF and as such Sf1 is an oncogenic tumor susceptibility factor from 129. Our results also highlight the complications involved in evaluating Sf1 levels and TGCT incidences. When a large number of tumor promoting factors are present in a strain, the protective effect of lower Sf1 levels is masked. However, when the dosage of tumor promoting factors is reduced, the protective effect of lower Sf1 levels becomes apparent. SF1 is involved in splicing of specific pre-mRNAs in cells. Alternate splicing generates the complex proteosome in eukaryotic cells. Our data indicates that Sf1 levels in mouse strains correlate with their incidences of TGCTs and implicate the importance of splicing mechanisms in germ cell tumorigenesis.
To identify genes affecting bone strength, we studied how genetic variants regulate components of a phenotypic covariation network that was previously shown to accurately characterize the compensatory trait interactions involved in functional adaptation during growth. Quantitative trait loci (QTLs) regulating femoral robustness, morphologic compensation, and mineralization (tissue quality) were mapped at three ages during growth using AXB/BXA Recombinant Inbred (RI) mouse strains and adult B6-iA Chromosome Substitution Strains (CSS). QTLs for robustness were identified on chromosomes 8, 12, 18, and 19 and confirmed at all three ages, indicating that genetic variants established robustness postnatally without further modification. A QTL for morphologic compensation, which was measured as the relationship between cortical area and body weight, was identified on chromosome 8. This QTL limited the amount of bone formed during growth and thus acted as a setpoint for diaphyseal bone mass. Additional QTLs were identified from the CSS analysis. QTLs for robustness and morphologic compensation regulated bone structure independently (ie, in a nonpleiotropic manner), indicating that each trait may be targeted separately to individualize treatments aiming to improve strength. Multiple regression analyses showed that variation in morphologic compensation and tissue quality, not bone size, determined femoral strength relative to body weight. Thus an individual inheriting slender bones will not necessarily inherit weak bones unless the individual also inherits a gene that impairs compensation. This systems genetic analysis showed that genetically determined phenotype covariation networks control bone strength, suggesting that incorporating functional adaptation into genetic analyses will advance our understanding of the genetic basis of bone strength. © 2010 American Society for Bone and Mineral Research.
systems genetics; recombinant inbred mouse strains; bone; morphology; biomechanics; growth; phenotypic covariation; QTL; strength; chromosome substitution strains
Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#A/J strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6A/J mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A→ T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A→G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.
Traditionally, we understand that individual phenotypes result primarily from inherited genetic variants together with environmental exposures. However, many studies showed that a remarkable variety of factors including environmental agents, parental behaviors, maternal physiology, xenobiotics, nutritional supplements and others lead to epigenetic changes that can be transmitted to subsequent generations without continued exposure. Recent discoveries show transgenerational epistasis and transgenerational genetic effects where genetic factors in one generation affect phenotypes in subsequent generation without inheritance of the genetic variant in the parents. Together these discoveries implicate a key signaling pathway, chromatin remodeling, methylation, RNA editing and microRNA biology. This exceptional mode of inheritance complicates the search for disease genes and represents perhaps an adaptation to transmit useful gene expression profiles from one generation to the next. In this review, I present evidence for these transgenerational genetic effects, identify their common features, propose a heuristic model to guide the search for mechanisms, discuss the implications, and pose questions whose answers will begin to reveal the underlying mechanisms.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with ∼70% of cases resulting from hepatitis B and C viral infections, aflatoxin exposure, chronic alcohol use or genetic liver diseases. The remaining ∼30% of cases are associated with obesity, type 2 diabetes and related metabolic diseases, although a direct link between these pathologies and HCCs has not been established. We tested the long-term effects of high-fat and low-fat diets on males of two inbred strains of mice and discovered that C57BL/6J but not A/J males were susceptible to non-alcoholic steatohepatitis (NASH) and HCC on a high-fat but not low-fat diet. This strain–diet interaction represents an important model for genetically controlled, diet-induced HCC. Susceptible mice showed morphological characteristics of NASH (steatosis, hepatitis, fibrosis and cirrhosis), dysplasia and HCC. mRNA profiles of HCCs versus tumor-free liver showed involvement of two signaling networks, one centered on Myc and the other on NFκB, similar to signaling described for the two major classes of HCC in humans. miRNA profiles revealed dramatically increased expression of a cluster of miRNAs on the X chromosome without amplification of the chromosomal segment. A switch from high-fat to low-fat diet reversed these outcomes, with switched C57BL/6J males being lean rather than obese and without evidence for NASH or HCCs at the end of the study. A similar diet modification may have important implications for prevention of HCCs in humans.
Two overlapping quantitative trait loci (QTLs) for clot stability, Hmtb8 and Hmtb9, were identified on mouse chromosome 17 in an F2 intercross derived from C57BL/6J (B6) and B6-Chr17A/J (B6-Chr17) mouse strains. The intervals were in synteny with a QTL for thrombotic susceptibility on chromosome 18 in a human study, and there were 23 homologs between mouse and human. The objective of this study was to determine whether any of these genes in the syntenic region are likely candidates as modifiers for clot stability. Seven genes, Twsg1, Zfp161, Dlgap1, Ralbp1, Myom1, Rab31, and Emilin2, of the 23 genes with single nucleotide polymorphisms (SNPs) in the mRNA-UTR had differential expression in B6 and A/J mice. Dlgap1, Ralbp1, Myom1, and Emilin2 also had nonsynonymous SNPs. In addition, two other genes had nonsynonymous SNPs, Lama1 and Ndc80. Of these nine candidate genes, Emilin2 was selected for further analysis since other EMILIN (Elastin Microfibril Interface Located Protein) proteins have known functions in vascular structure and coagulation. Differences were found between B6 and A/J mice in vessel wall architecture and EMILIN2 protein in plasma, carotid vessel wall, and thrombi formed after ferric chloride injury. In B6-Chr17A/J mice both clot stability and Emilin2 mRNA expression were higher compared to those in B6 and A/J mice, suggesting the exposure of epistatic interactions. Although other homologous genes in the QTL region cannot be ruled out as causative genes, further investigation of Emilin2 as a candidate gene for thrombosis susceptibility is warranted.
Electronic supplementary material
The online version of this article (doi:10.1007/s00335-010-9274-6) contains supplementary material, which is available to authorized users.
A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.
Dnd1; Ter; testicular teratoma; teratocarcinoma; testicular germ cell tumor
The agouti-yellow (Ay) deletion is the only genetic modifier known to suppress testicular germ cell tumor (TGCT) susceptibility in mice or humans. The Ay mutation deletes Raly and Eif2s2, and induces the ectopic expression of agouti, all of which are potential TGCT-modifying mutations. Here we report that the reduced TGCT incidence of heterozygous Ay males and the recessive embryonic lethality of Ay are caused by the deletion of Eif2s2, the beta subunit of translation initiation factor eIF2. We found that the incidence of affected males was reduced 2-fold in mice that were partially deficient for Eif2s2 and that embryonic lethality occurred near the time of implantation in mice that were fully deficient for Eif2s2. In contrast, neither reduced expression of Raly in gene-trap mice nor ectopic expression of agouti in transgenic or viable-yellow (Avy) mutants affected TGCT incidence or embryonic viability. In addition, we provide evidence that partial deficiency of Eif2s2 attenuated germ cell proliferation and differentiation, both of which are important to TGCT formation. These results show that germ cell development and TGCT pathogenesis are sensitive to the availability of the eIF2 translation initiation complex and to changes in the rate of translation.
Congenic strains continue to be a fundamental resource for dissecting the genetic basis of complex traits. Traditionally, genetic variants (QTLs) that account for phenotypic variation in a panel of congenic strains are sought first by comparing phenotypes for each strain to the host (reference) strain, and then by examining the results to identify a common chromosome segment that provides the best match between genotype and phenotype across the panel. However, this ‘‘common-segment’’ method has significant limitations, including the subjective nature of the genetic model and an inability to deal formally with strain phenotypes that do not fit the model. We propose an alternative that we call ‘‘sequential’’ analysis and that is based on a unique principle of QTL analysis where each strain, corresponding to a single genotype, is tested individually for QTL effects rather than testing the congenic panel collectively for common effects across heterogeneous backgrounds. A minimum spanning tree, based on principles of graph theory, is used to determine the optimal sequence of strain comparisons. For two traits in two panels of congenic strains in mice, we compared results for the sequential method with the common-segment method as well as with two standard methods of QTL analysis, namely, interval mapping and multiple linear regression. The general utility of the sequential method was demonstrated with analysis of five additional traits in congenic panels from mice and rats. Sequential analysis rigorously resolved phenotypic heterogeneity among strains in the congenic panels and found QTLs that other methods failed to detect.
Several genetic variants act as modifiers of testicular germ cell tumor (TGCT) susceptibility in the 129/Sv mouse model of human pediatric TGCTs. One such modifier, the Steel locus, encodes the transmembrane-bound and soluble ligand of the kit receptor. Some (Sl and SlJ) but not all (Sld) mutations of the Steel locus increase TGCT incidence in heterozygous mutant mice. Because Sl and SlJ are large deletions that affect multiple transcripts and Sld is an intragenic deletion of the kit ligand (Kitl) from which only the soluble protein is produced, it was uncertain whether Kitl or a neighboring gene is a modifier of TGCT susceptibility. We tested the effect of the small Steel grizzle-belly (Slgb) deletion on TGCT susceptibility to determine whether Kitl is a TGCT modifier gene. An increase in TGCT incidence was observed in Slgb/+ heterozygotes and fine-mapping of the deletion breakpoints revealed that Kitl is the only conventional gene deleted by the mutation, suggesting that Kitl is the TGCT modifier gene at the Steel locus. Additionally, we propose that soluble KITL in Sld/+ heterozygous mutant mice complements a dosage effect of transmembrane-associated KITL on TGCT susceptibility and that the kit receptor (Kit) is haplosufficient for primordial germ cell (PGC) development.
Acute lung injury (ALI) is a devastating condition resulting from diverse causes. Genetic studies of human populations indicate that ALI is a complex disease with substantial phenotypic variance, incomplete penetrance, and gene–environment interactions. To identify genes controlling ALI mortality, we previously investigated mean survival time (MST) differences between sensitive A/J (A) and resistant C57BL/6J (B) mice in ozone using quantitative trait locus (QTL) analysis. MST was significantly linked to QTLs (Aliq1-3) on chromosomes 11, 13, and 17, respectively. Additional QTL analyses of separate and combined backcross and F2 populations supported linkage to Aliq1 and Aliq2, and established significance for previously suggestive QTLs on chromosomes 7 and 12 (named Aliq5 and Aliq6, respectively). Decreased MSTs of corresponding chromosome substitution strains (CSSs) verified the contribution of most QTL-containing chromosomes to ALI survival. Multilocus models demonstrated that three QTLs could explain the MST difference between progenitor strains, agreeing with calculated estimates for number of genes involved. Based on results of QTL genotype analysis, a double CSS (B.A-6,11) was generated that contained Aliq1 and Aliq4 chromosomes. Surprisingly, MST and pulmonary edema after exposure of B.A-6,11 mice were comparable to B mice, revealing an unpredicted loss of sensitivity compared with separate CSSs. Reciprocal congenic lines for Aliq1 captured the corresponding phenotype in both background strains and further refined the QTL interval. Together, these findings support most of the previously identified QTLs linked to ALI survival and established lines of mice to further resolve Aliq1.
acute respiratory distress syndrome; chromosome substitution strain; congenic; mean survival; pulmonary edema
The Ter mutation causes primordial germ cell (PGC) loss on all mouse genetic backgrounds (Fig. 1a) with deficiency of PGCs1 starting at embryonic day 8. Ter is also a potent modifier of spontaneous testicular germ cell tumour (TGCT) susceptibility in the 129 family of inbred strains and increases TGCT incidence from a baseline rate of 5% in 129 to 94% in 129-Ter/Ter males2-4 (Figs. 1b & c). In 129, some of the remaining PGCs transform into undifferentiated pluripotent embryonal carcinoma (EC) cells2-6 and after birth they differentiate into various cells and tissues that compose TGCTs. Positional cloning of Ter revealed a point mutation that introduces a termination codon in the mouse ortholog (Dnd1) of the zebrafish dead-end (dnd) gene. PGC deficiency is corrected both with BACs that contain Dnd1 and with a Dnd1 encoding transgene. Dnd1 is expressed in fetal gonads during the critical period when TGCTs originate. DND1 has an RNA recognition motif (RRM) and is most similar to the apobec complementation factor (Acf), a component of the cytidine to uridine RNA editing complex. These results suggest that Ter may adversely affect essential aspects of RNA biology during PGC development. DND1 is the first protein with an RRM that is directly implicated as a heritable cause of spontaneous tumourigenesis. TGCT development of the 129-Ter strain models pediatric TGCT in humans. This work will have important implications for our understanding of the genetic control of TGCT pathogenesis and PGC biology.
We examined femora from adult AXB/BXA recombinant inbred (RI) mouse strains to identify skeletal traits that are functionally related and to determine how functional interactions among these traits contribute to genetic variability in whole-bone stiffness, strength, and toughness. Randomization of A/J and C57BL/6J genomic regions resulted in each adult male and female RI strain building mechanically functional femora by assembling unique sets of morphologic and tissue-quality traits. A correlation analysis was conducted using the mean trait values for each RI strain. A third of the 66 correlations examined were significant, indicating that many bone traits covaried or were functionally related. Path analysis revealed important functional interactions among bone slenderness, cortical thickness, and tissue mineral density. The path coefficients describing these functional relations were similar for both sexes. The causal relationship among these three traits suggested that cellular processes during growth simultaneously regulate bone slenderness, cortical thickness, and tissue mineral density so that the combination of traits is sufficiently stiff and strong to satisfy daily loading demands. A disadvantage of these functional interactions was that increases in tissue mineral density also deleteriously affected tissue ductility. Consequently, slender bones with high mineral density may be stiff and strong but they are also brittle. Thus, genetically randomized mouse strains revealed a basic biological paradigm that allows for flexibility in building bones that are functional for daily activities but that creates preferred sets of traits under extreme loading conditions. Genetic or environmental perturbations that alter these functional interactions during growth would be expected to lead to loss of function and suboptimal adult bone quality.