Biomedical subjects
Publications and source records attributed to Neal G Copeland.
Hair graying is an obvious sign of human aging, yet little was known about its causes. Two recent papers provide compelling evidence that hair graying is due to incomplete melanocyte stem cell maintenance and identify Pax3 and Mitf as key molecules that help regulate the balance between melanocyte stem cell maintenance and differentiation.
Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (lambda) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA. Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection. This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site. All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive lambda prophage that makes recombineering possible. SW105 and SW106 cells in addition carry l-arabinose-inducible Cre or Flp genes, respectively. The galK function can be selected both for and against. This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published. We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors.
Dense genetic maps of mammalian genomes facilitate a variety of biological studies including the mapping of polygenic traits, positional cloning of monogenic traits, mapping of quantitative or qualitative trait loci, marker association, allelic imbalance, speed congenic construction, and evolutionary or phylogenetic comparison. In particular, single nucleotide polymorphisms (SNPs) have proved useful because of their abundance and compatibility with multiple high-throughput technology platforms. SNP genotyping is especially suited for the genetic analysis of model organisms such as the mouse because biallelic markers remain fully informative when used to characterize crosses between inbred strains. Here we report the mapping and genotyping of 673 SNPs (including 519 novel SNPs) in 55 of the most commonly used mouse strains. These data have allowed us to construct a phylogenetic tree that correlates and expands known genealogical relationships and clarifies the origin of strains previously having an uncertain ancestry. All 55 inbred strains are distinguishable genetically using this SNP panel. Our data reveal an uneven SNP distribution consistent with a mosaic pattern of inheritance and provide some insight into the changing dynamics of the physical architecture of the genome. Furthermore, these data represent a valuable resource for the selection of markers and the design of experiments that require the genetic distinction of any pair of mouse inbred strains such as the generation of congenic mice, positional cloning, and the mapping of quantitative or qualitative trait loci.
Recent consolidation of the whole-genome sequence with genome-wide transcriptome profiling revealed the existence of functional units within the genome in specific chromosomal regions, as seen in the coordinated expression of gene clusters and colocalization of functionally related genes. An efficient region-specific mutagenesis screen would greatly facilitate research in addressing the importance of these clusters. Here we use the 'local hopping' phenomenon of a DNA-type transposon, Sleeping Beauty (SB), for region-specific saturation mutagenesis. A transgenic mouse containing both transposon (acts as a mutagen) and transposase (recognizes and mobilizes the transposon) was bred for germ-cell transposition events, allowing us to generate many mutant mice. All genes within a 4-Mb region of the original donor site were mutated by SB, indicating the potential of this system for functional genomic studies within a specific chromosomal region.
PURPOSE: To establish a transgenic mouse line that expresses Cre-recombinase in retinal rod bipolar cells for the generation of rod bipolar cell-specific knockout mutants. METHODS: The IRES-Cre-cDNA fragment was inserted into a 173-kb bacterial artificial chromosome (BAC) carrying the intact Pcp2 gene, by using red-mediated recombineering. Transgenic mice were generated with the modified BAC and identified. The Cre-transgenic mice were crossed with ROSA26 and Z/EG reporter mice to detect Cre-recombinase activity. RESULTS: X-gal staining showed that strong Cre-recombinase activities were present in retinal inner nuclear layers and cerebellar Purkinje cells. Double staining with an anti-GFP antibody and an anti-PKCalpha antibody (specific for retinal rod bipolar cells) revealed that Cre-recombinase activity localized exclusively to the rod bipolar cells in the retina. CONCLUSIONS: A mouse BAC-Pcp2-IRES-Cre transgenic line that expresses Cre-recombinase in retinal rod bipolar neurons has been established. Because mutations in some ubiquitously expressed genes may result in retinal degenerative diseases, the mouse strain BAC-Pcp2-IRES-Cre will be a useful new tool for investigating the effects of retinal rod bipolar cell-specific gene inactivation.
MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.
Machado-Joseph disease (MJD) is an inherited neurodegenerative disorder caused by ataxin-3 with a polyglutamine expansion. It is proposed that a toxic cleavage fragment of mutant ataxin-3 alternatively spliced isoform mjd1a triggers neurodegeneration, although this fragment has not yet been detected in the brains of MJD patients or in animal models. We have now generated transgenic mice expressing human mutant (Q71) or normal (Q20) ataxin-3 mjd1a under the control of the mouse prion promoter. Q71 transgenic mice expressing mutant ataxin-3 mjd1a above a critical level developed a phenotype similar to MJD including progressive postural instability, gait and limb ataxia, weight loss, premature death, neuronal intranuclear inclusions, and decreased tyrosine hydroxylase-positive neurons in the substantia nigra (determined by unbiased stereology). Q20 transgenic mice had normal behavior and pathology. Brains from sick Q71 transgenic mice contained an abundant mutant ataxin-3 mjd1a putative-cleavage fragment (Fragment), which was scarce in normal Q71 transgenic mice. Reactivity of the Fragment with a panel of antibodies and comigration with truncations of mutant ataxin-3 revealed that it contained residues C terminal to amino acid 221 to include the polyglutamine expansion. A similar portion of mutant ataxin-3 mjd1a expressed in transfected neuroblastoma cells was toxic above a critical concentration. The Fragment was more abundant in two affected brain regions of MJD patients. Thus, we have developed a murine model for mutant ataxin-3 mjd1a toxicity and identified a putative-cleavage fragment of the disease protein in the brains of these transgenic mice and MJD patients that is cytotoxic above a critical concentration.
The chromosomal translocation t(7; 11)(p15;p15), observed in human myeloid leukemia, results in a NUP98 and HOXA9 gene fusion. We generated a transgenic mouse line that specifically expressed the chimeric NUP98-HOXA9 gene in the myeloid lineage. While only 20% of the transgenic mice progressed to leukemia after a latency period, myeloid progenitor cells from nonleukemic transgenic mice still exhibited increased proliferative potential. This suggested that the NUP98-HOXA9 fusion induced a preleukemic phase, and other factors were required for complete leukemogenesis. NUP98-HOXA9 expression promoted the onset of retrovirus-induced BXH2 myeloid leukemia. This phenomenon was used to identify cooperative disease genes as common integration sites (CISs). Meis1, a known HOX cofactor, was identified as a CIS with a higher integration frequency in transgenic than in wild-type BXH2 mice. By the same means we identified further 4 candidate cooperative genes, Dnalc4, Fcgr2b, Fcrl, and Con1. These genes cooperated with NUP98-HOXA9 in transforming NIH 3T3 cells. The system described here is a powerful tool to identify cooperative oncogenes and will assist in the clarification of the multistep process of carcinogenesis.
Huntington's disease (HD) results from the expansion of a glutamine repeat near the N-terminus of huntingtin (htt). At post-mortem, neurons in the central nervous system of patients have been found to accumulate N-terminal fragments of mutant htt in nuclear and cytoplasmic inclusions. This pathology has been reproduced in transgenic mice expressing the first 171 amino acids of htt with 82 glutamines along with losses of motoric function, hypoactivity and abbreviated life-span. The relative contributions of nuclear versus cytoplasmic mutant htt to the pathogenesis of disease have not been clarified. To examine whether pathogenic processes in the nucleus disproportionately contribute to disease features in vivo, we fused a nuclear localization signal (NLS) derived from atrophin-1 to the N-terminus of an N171-82Q construct. Two lines of mice (lines 8A and 61) that were identified expressed NLS-N171-82Q at comparable levels and developed phenotypes identical to our previously described HD-N171-82Q mice. Western blot and immunohistochemical analyses revealed that NLS-N171-82Q fragments accumulate in nuclear, but not cytoplasmic, compartments. These data suggest that disruption of nuclear processes may account for many of the disease phenotypes displayed in the mouse models generated by expressing mutant N-terminal fragments of htt.
The early B-cell factor (EBF)-associated zinc-finger protein (EBFAZ) binds to and negatively regulates EBF, a basic helix-loop-helix transcription factor required for B-cell lineage commitment and development of the olfactory epithelium. It also binds to SMA- and MAD-related protein 1 (SMAD1) and SMAD4 in response to bone morphogenic protein 2 (BMP2) signaling. It is highly related to ecotropic viral integration site 3 (EVI3), a protein that, like EBFAZ, contains 30 Krüppel-like zinc-finger repeats. In previous studies, we showed that Evi3 is a frequent target of retroviral integration in AKXD27 B-cell lymphomas. Here, we show that EBFAZ is also a frequent target. Integrations at Ebfaz and Evi3 are mutually exclusive, suggesting that they function in the same tumor pathway. Lymphomas with integrations at Ebfaz or Evi3 express the pre-B-cell-specific marker immunoglobulin lambda chain 5, and contain immunoglobulin heavy-chain rearrangements, suggesting that they are blocked at an early B-cell stage. Unlike Evi3, which is expressed at low levels in normal B cells, or Ebfaz, which is not expressed in B cells, both genes are highly expressed following viral integration. Collectively, our results suggest that ectopic expression of Ebfaz can substitute for the upregulated expression of Evi3 in B-cell disease and highlight the importance of this gene family in hematopoietic cancer.
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Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.
Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a potent cancer gene discovery tool in the post-genome-sequence era. To manage multiple high-throughput insertional mutagenesis screening projects, we developed the Retroviral Tagged Cancer Gene Database (RTCGD; http://RTCGD.ncifcrf.gov). A sequence analysis pipeline determines the genomic position of each retroviral integration site cloned from a mouse tumor, the distance between it and the nearest candidate disease gene(s) and its orientation with respect to the candidate gene(s). The pipeline also identifies genomic regions that are targets of retroviral integration in more than one tumor (common integration sites, CISs) and are thus likely to encode a disease gene. Users can search the database using a specified gene symbol, chromosome number or tumor model to identify both CIS genes and unique viral integration sites or compare the integration sites cloned by different laboratories using different models. As a default setting, users first review the CIS Lists and then Clone Lists. CIS Lists describe CISs and their candidate disease genes along with links to other public databases and clone lists. Clone Lists describe the viral integration site clones along with the tumor model and tumor type from which they were cloned, candidate disease gene(s), genomic position and orientation of the integrated provirus with respect to the candidate gene(s). It also provides a pictorial view of the genomic location of each integration site relative to neighboring genes and markers. Researchers can identify integrations of interest and compare their results with those for multiple tumor models and tumor types using RTCGD.
The Purkinje neuron, one of the most fascinating components of the cerebellar cortex, is involved in motor learning, motor coordination, and cognitive function. Purkinje cell protein 2 (Pcp2/L7) expression is highly restricted to Purkinje and retinal bipolar cells, where it has been exploited to enable highly specific, Cre recombinase-mediated, site-specific recombination. Previous studies showed that mice carrying a Cre transgene produced by insertion of Cre cDNA into a small 2.88-kb Pcp2 DNA fragment expressed Cre in Purkinje cells; however, some Cre activity was also observed outside the target tissues. Here, we used Red-mediated recombineering to insert Cre cDNA into a 173-kb BAC carrying the entire intact Pcp2 gene, and characterize the resultant BAC/Cre transgenic mice for Cre expression. We show that BAC/Cre transgenic mice have exclusive Cre expression in Purkinje and bipolar cells and nowhere else. These mice will facilitate Purkinje cell and retinal bipolar cell-specific genetic manipulation.
More than 70 different mutations in presenilin 1 (PS1) have been associated with inherited early onset Alzheimer's disease (AD). How all these different mutations cause disease has not been clearly delineated. Our laboratory has previously shown that co-expression of mutant PS1 in mice transgenic for amyloid precursor protein (APPswe) dramatically accelerates the rate of amyloid deposition in the brain. In our original animals mutant PS1 was substantially over-expressed, and the stabilized pool of mouse PS1 fragments was largely replaced by the human protein. In this setting the accelerated amyloid pathology in the double transgenic mice could have been due, in part, to decreased endogenous PS1 activity. To investigate this possibility, we generated APP transgenic mice with reduced levels of endogenous PS1. We find that mice harboring only one functional PS1 allele and co-expressing Mo/HuAPPswe do not develop amyloid deposits at ages comparable to mice expressing mutant PS1. We next tested whether hypo-expression of mutant PS1 could accelerate the rate of amyloid deposition using an unusual line of transgenic mice expressing PS1dE9 at low levels, finding no significant acceleration. Our findings demonstrate that the accelerated amyloid pathology, caused by so many different mutations in PS1, is clearly not a result of haplo-insufficiency that might result from inactivating mutations. Instead, our data are consistent with a gain of property mechanism.
The first mouse microphthalmia transcription factor (Mitf ) mutation was discovered over 60 years ago, and since then over 24 spontaneous and induced mutations have been identified at the locus. Mitf encodes a member of the Myc supergene family of basic helix-loop-helix zipper (bHLH-Zip) transcription factors. Like Myc, Mitf regulates gene expression by binding to DNA as a homodimer or as a heterodimer with another related family member, in the case of Mitf the Tfe3, Tfeb, and Tfec proteins. The study of Mitf has provided many insights into the biology of melanocytes and helped to explain how melanocyte-specific gene expression and signaling is regulated. The human homologue of MITF is mutated in patients with the pigmentary and deafness disorder Waardenburg Syndrome Type 2A (WS2A). The mouse Mitf mutations therefore serve as a model for the study of this human disease. Mutations and/or aberrant expression of several MITF family member genes have also been reported in human cancer, including melanoma (MITF), papillary renal cell carcinoma (TFE3, TFEB), and alveolar soft part sarcoma (TFE3). Genes in the MITF/TFE pathway may therefore also represent valuable therapeutic targets for the treatment of human cancer. Here we review recent developments in the analysis of Mitf function in vivo and in vitro and show how traditional genetics, modern forward genetics and in vitro biochemical analyses have combined to produce an intriguing story on the role and actions of a gene family in a living organism.
Amyloid precursor protein (APP) is endoproteolytically processed by BACE1 and gamma-secretase to release amyloid peptides (Abeta40 and 42) that aggregate to form senile plaques in the brains of patients with Alzheimer's disease (AD). The C-terminus of Abeta40/42 is generated by gamma-secretase, whose activity is dependent upon presenilin (PS 1 or 2). Missense mutations in PS1 (and PS2) occur in patients with early-onset familial AD (FAD), and previous studies in transgenic mice and cultured cell models demonstrated that FAD-PS1 variants shift the ratio of Abeta40 : 42 to favor Abeta42. One hypothesis to explain this outcome is that mutant PS alters the specificity of gamma-secretase to favor production of Abeta42 at the expense of Abeta40. To test this hypothesis in vivo, we studied Abeta40 and 42 levels in a series of transgenic mice that co-express the Swedish mutation of APP (APPswe) with two FAD-PS1 variants that differentially accelerate amyloid pathology in the brain. We demonstrate a direct correlation between the concentration of Abeta42 and the rate of amyloid deposition. We further show that the shift in Abeta42 : 40 ratios associated with the expression of FAD-PS1 variants is due to a specific elevation in the steady-state levels of Abeta42, while maintaining a constant level of Abeta40. These data suggest that PS1 variants do not simply alter the preferred cleavage site for gamma-secretase, but rather that they have more complex effects on the regulation of gamma-secretase and its access to substrates.
While studying the unique Nramp1 (Slc11a1)-independent susceptibility to Mycobacterium bovis (BCG) infection of BXH-2 mice, we noted that these mice develop important splenomegaly and enlargement of lymph nodes. Segregation analyses in several F2 crosses showed that splenomegaly segregates as a single recessive trait caused by a novel mutation in BXH-2, independent of the infection. Histologic and fluorescence-activated cell sorter (FACS) analyses indicated that splenomegaly is associated with a large increase in Mac1+/GR1+ (macrophage antigen-1+/granulocyte differentiation antigen 1+) granulocyte precursors in spleen, lymph nodes, and bone marrow, resembling a myeloproliferative syndrome. This is concomitant to extramedullary erythropoiesis in the spleen, as measured by proportion of Ter119+ erythroid cells. The locus controlling this myeloproliferative syndrome and splenomegaly was designated Myls and maps to an 18 centimorgan (cM) region of chromosome 8, which also contains an integrated copy of an N-ecotropic murine leukemia virus (MuLV) provirus (Emv2). The relationship between Myls, expansion of Mac1+/GR1+ cells, and Emv2 was investigated. Homozygosity at Myls is necessary but not sufficient for B-ecotropic virus replication in splenocytes, the extent of which appears to be under separate genetic control. Our results suggest a model in which Myls-dependent myeloproliferation in BXH-2 acts as a predisposing factor for the subsequent development of virally induced myeloid leukemia characteristic of this strain.