The Collaborative Cross at Oak Ridge National Laboratory: developing a powerful resource for systems genetics.

Complex traits and disease comorbidity in humans and in model organisms are the result of naturally occurring polymorphisms that interact with each other and with the environment. To ensure the availability of resources needed to investigate biomolecular networks and systems-level phenotypes underlying complex traits, we have initiated breeding of a new genetic reference population of mice, the Collaborative Cross. This population has been designed to optimally support systems genetics analysis. Its novel and important features include a high level of genetic diversity, a large population size to ensure sufficient power in high-dimensional studies, and high mapping precision through accumulation of independent recombination events. Implementation of the Collaborative Cross has been ongoing at the Oak Ridge National Laboratory (ORNL) since May 2005. Production has been systematically managed using a software-assisted breeding program with fully traceable lineages, performed in a controlled environment. Currently, there are 650 lines in production, and close to 200 lines are now beyond their seventh generation of inbreeding. Retired breeders enter a high-throughput phenotyping protocol and DNA samples are banked for analyses of recombination history, allele drift and loss, and population structure. Herein we present a progress report of the Collaborative Cross breeding program at ORNL and a description of the kinds of investigations that this resource will support.

Cdc14B depletion leads to centriole amplification, and its overexpression prevents unscheduled centriole duplication.

Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S phase-arrested cells and proteasome inhibitor Z-L(3)VS-treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.

Nucleophosmin/B23 negatively regulates GCN5-dependent histone acetylation and transactivation.

Nucleophosmin/B23 is a multifunctional phosphoprotein that is overexpressed in cancer cells and has been shown to be involved in both positive and negative regulation of transcription. In this study, we first identified GCN5 acetyltransferase as a B23-interacting protein by mass spectrometry, which was then confirmed by in vivo co-immunoprecipitation. An in vitro assay demonstrated that B23 bound the PCAF-N domain of GCN5 and inhibited GCN5-mediated acetylation of both free and mononucleosomal histones, probably through interfering with GCN5 and masking histones from being acetylated. Mitotic B23 exhibited higher inhibitory activity on GCN5-mediated histone acetylation than interphase B23. Immunodepletion experiments of mitotic extracts revealed that phosphorylation of B23 at Thr 199 enhanced the inhibition of GCN5-mediated histone acetylation. Moreover, luciferase reporter and microarray analyses suggested that B23 attenuated GCN5-mediated transactivation in vivo. Taken together, our studies suggest a molecular mechanism of B23 in the mitotic inhibition of GCN5-mediated histone acetylation and transactivation.

Neurobehavioral mutants identified in an ENU-mutagenesis project.

We report on a battery of behavioral screening tests that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU-mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and used a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open-field mutants (one displaying hyperlocomotion, the other hypolocomotion), four tail-suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning-and-memory mutant (displaying reduced response to the conditioned stimulus). These findings highlight the utility of a set of behavioral tasks used in a high-throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

Detection of mouse parvovirus in Mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction assay.

We used primary and nested polymerase chain reaction (PCR) assays to determine the presence of mouse parvovirus (MPV) in mouse sperm, oocytes, preimplantation embryos, and ovarian tissues collected from MPV-infected mice. The primary PCR assay detected MPV in 56% of the sperm samples. MPV was not eliminated by passing sperm samples through a Percoll gradient. After Percoll treatment, MPV was still present in 50% of the samples according to primary PCR assay. Oocyte samples that did not undergo extensive washing procedures had detectable MPV in 7% of the samples based on the primary PCR assay, but nested PCR assay detected higher (28%) infection rate. However, MPV was not detected in oocytes that underwent extensive washing procedures, as assessed by either primary or nested PCR assay. Although primary PCR did not detect MPV in embryos, a nested PCR assay determined that 50% of the embryos were positive for the virus. In addition, ovarian tissues were collected from 3 different mouse colonies with enzootic MPV infection. Ovarian tissue collected from 129CT, 101/R1, and Sencar mice had high incidence (38%, 63%, and 65%, respectively) of MPV infection on the basis of nested PCR amplification. These results demonstrate that mouse gametes, embryos, and ovarian tissues may be contaminated with MPV and therefore caution is necessary when infected germplasm is used for assisted reproductive technologies such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization, ovary transplantation, and intracytoplasmic sperm injection.

Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects.

The mammalian Nell1 gene encodes a protein kinase C-beta1 (PKC-beta1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell1(6R), a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell1(6R) has a T-->A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell1(6R) mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell1(6R) mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice.

BACKGROUND: Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. RESULTS: We produced the Cryopreserved Mutant Mouse Bank (CMMB), which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE) was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41%) result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI) of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. CONCLUSIONS: The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

Identification of mutations from phenotype-driven ENU mutagenesis in mouse chromosome 7.

We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.

Lack of Pwcr1/MBII-85 snoRNA is critical for neonatal lethality in Prader-Willi syndrome mouse models.

Prader-Willi syndrome (PWS) is a neurobehavioral disorder caused by the lack of paternal expression of imprinted genes in the human chromosome region 15q11-13. Recent studies of rare human translocation patients narrowed the PWS critical genes to a 121-kb region containing PWCR1/HBII-85 and HBII-438 snoRNA genes. The existing mouse models of PWS that lack the expression of multiple genes, including Snrpn, Ube3a, and many intronic snoRNA genes, are characterized by 80%-100% neonatal lethality. To define the candidate region for PWS-like phenotypes in mice, we analyzed the expression of several genetic elements in mice carrying the large radiation-induced p(30PUb) deletion that includes the p locus. Mice having inherited this deletion from either parent develop normally into adulthood. By Northern blot and RT-PCR assays of brain tissue, we found that Pwcr1/MBII-85 snoRNAs are expressed normally, while MBII-52 snoRNAs are not expressed when the deletion is paternally inherited. Mapping of the distal deletion breakpoint indicated that the p30PUb deletion includes the entire MBII-52 snoRNA gene cluster and three previously unmapped EST sequences. The lack of expression of these elements in mice with a paternal p30PUb deletion indicates that they are not critical for the neonatal lethality observed in PWS mouse models. In addition, we identified MBII-436, the mouse homolog of the HBII-436 snoRNA, confirmed its imprinting status, and mapped it outside of the p30PUb deletion. Taking together all available data, we conclude that the lack of Pwcr1/MBII-85 snoRNA expression is the most likely cause for the neonatal lethality in PWS model mice.

Phenotype screening for genetically determined age-onset disorders and increased longevity in ENU-mutagenized mice.

With the goal of discovering genes that contribute to late-onset neurological and ocular disorders and also genes that extend the healthy life span in mammals, we are phenotyping mice carrying new mutations induced by the chemical N-ethyl-N-nitrosourea (ENU). The phenotyping plan includes basic behavioral, neurohistological, and vision testing in sibling cohorts of mice aged to 18 months, and then evaluation for markers of growth trajectory and stress response in these same cohorts aged up to 28 months. Statistical outliers are identified by comparison to test results of similar aged cohorts, and potential mutants are recovered for re-aging to confirm heritability of the phenotype.

Physical mapping of the pink-eyed dilution complex in mouse chromosome 7 shows that Atp10c is the only transcript between Gabrb3 and Ube3a.

Phenotypic analyses of a set of homozygous-lethal deletion mutants at the pink-eyed dilution (p) locus has resulted in the identification of p-linked obesity locus 1 (plo 1), distal to the p locus, as a locus involved in the modulation of body fat and/or affecting lipid metabolism in these mice. The plo 1 region maps to mouse chromosome 7 (MMU 7) between two genes, Gabrb3 and Ube3a, which have been used as anchor points to generate an integrated deletion and physical map of plo 1 that encompasses about 1.2-1.3 Mb. A deletion/physical map was constructed and the genomic DNA between the two loci was sequenced to identify genes mapping to this region. Data show that Atp10c, a novel type IV ATPase a putative phospholipid transporter, is the only coding unit in this region of the chromosome.

The Collaborative Cross, a community resource for the genetic analysis of complex traits.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

Mice heterozygous for Atp10c, a putative amphipath, represent a novel model of obesity and type 2 diabetes.

Atp10c is a novel type IV P-type ATPase and is a putative phospholipid transporter. The purpose of this study was to assess the overall effect of the heterozygous deletion of Atp10c on obesity-related phenotypes and metabolic abnormalities in mice fed a high-fat diet. Heterozygous mice with maternal inheritance of Atp10c were compared with heterozygous mice with paternal inheritance of Atp10c and wild-type controls. Body weight, adiposity index, and plasma insulin, leptin and triglyceride concentrations were significantly greater in the mutants inheriting the deletion maternally compared with their sex- and age-matched control male mice fed a 10% fat (% energy) diet and female mice fed a 45% fat (% energy) diet. Glucose and insulin tolerance tests were performed after mice consumed the diets for 4 and 8 wk. Mutants had altered glucose tolerance and insulin response compared with controls, suggesting insulin resistance in both sexes. Mice were killed at 12 wk and routine gross and histological evaluations of the liver, pancreas, adipose tissue, and heart were performed. Histological evaluation showed micro- and macrovesicular lipid deposition within the hepatocytes that was more severe in the mutant mice than in age-matched controls. Although sex differences were observed, our data suggest that heterozygous deletion along with an unusual pattern of maternal inheritance of the chromosomal region containing the single gene, Atp10c, causes obesity, type 2 diabetes, and nonalcoholic fatty liver disease in these mice.

Phenotype- and gene-driven approaches to discovering the functions of mammalian genes.

All of us are involved in discovery science as we pursue the genes, networks, cellular processes and biophysical principles that govern our chosen biological question. For those of us who choose to proceed using plant or animal models to dissect the elements of our favorite biological system, there are many classical and newer approaches available for our use, including two complementary strategies by which the discovery process is proceeding at the Oak Ridge National Laboratory (ORNL). The ORNL has been known for six decades for its investigations of the effects of radiation and chemicals in inducing heritable mutations in mouse germ cells, and for using mouse mutations as tools for the cloning and characterization of mammalian genes. Our history and experience in making mouse models are being applied via these two complementary strategies: 1), a phenotype-driven approach, in which mice carrying random chemically-induced mutations are screened for abnormal phenotypes; and 2) a gene-driven approach in which heritable single nucleotide polymorphisms (SNP) in preselected genes already thought likely to influence a biological system of choice can be recovered in live mice. The SNP-carrying mice can then be phenotyped for alterations in one's target biology. Both approaches have value and are necessary; while we can use mutations in genes that we already know to be of interest in our favorite biology to discover gene function, we also know that biology is full of surprise genes whose effects on our favorite biology would not be predicted and which will be identified only through phenotype screening.

Cloning, functional study and comparative mapping of Luzp2 to mouse chromosome 7 and human chromosome 11p13-11p14.

A novel leucine-zipper gene, leucine zipper protein 2 (Luzp2), has been cloned as part of an aberrant deletion-fusion transcript in the chromosomal interval between Gas2 and Herc2 on mouse Chromosome 7 (Chr 7). Luzp2 is normally expressed only in brain and spinal cord. The human homolog of Luzp2 maps to Chr 11p13-11p14 by radiation-hybrid mapping and is deleted in some patients with Wilms tumor-Aniridia-Genitourinary anomalies-mental Retardation (WAGR) syndrome. Disruption of Luzp2 by gene targeting in mice did not result in any obvious abnormal phenotypes.

Functional annotation of mammalian genomic DNA sequence by chemical mutagenesis: a fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15.

Eleven independent, recessive, N-ethyl-N-nitrosourea-induced mutations that map to a approximately 1- to 2-cM region of mouse chromosome (Chr) 7 homologous to human Chr 11p14-p15 were recovered from a screen of 1,218 gametes. These mutations were initially identified in a hemizygous state opposite a large p-locus deletion and subsequently were mapped to finer genomic intervals by crosses to a panel of smaller p deletions. The 11 mutations also were classified into seven complementation groups by pairwise crosses. Four complementation groups were defined by seven prenatally lethal mutations, including a group (l7R3) comprised of two alleles of obvious differing severity. Two allelic mutations (at the psrt locus) result in a severe seizure and runting syndrome, but one mutation (at the fit2 locus) results in a more benign runting phenotype. This experiment has added seven loci, defined by phenotypes of presumed point mutations, to the genetic map of a small (1-2 cM) region of mouse Chr 7 and will facilitate the task of functional annotation of DNA sequence and transcription maps both in the mouse and the corresponding human 11p14-p15 homology region.