ABSTRACT
Germ line gene transposition technology has been used to generate "libraries" of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular "disease" phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes.
Subject(s)
DNA Transposable Elements , Germ Cells , Mice, Mutant Strains/genetics , Mice, Transgenic/genetics , Mutagenesis , Transposases/genetics , Animals , Cardiovascular Diseases/embryology , Cardiovascular Diseases/genetics , Gene Knock-In Techniques , Mice , Mice, Inbred C57BL , Spermatogenesis/geneticsABSTRACT
Gata1 is a transcription factor essential for erythropoiesis. Erythroid cells lacking Gata1 undergo apoptosis, while overexpression of Gata1 results in a block in erythroid differentiation. However, erythroid cells overexpressing Gata1 differentiate normally in vivo when in the presence of wild-type cells. We have proposed a model, whereby a signal generated by wild-type cells (red cell differentiation signal; REDS) overcomes the intrinsic defect in Gata1-overexpressing erythroid cells. The simplest interpretation of this model is that wild-type erythroid cells generate REDS. To substantiate this notion, we have exploited a tissue specific Cre/loxP system and the process of X-inactivation to generate mice that overexpress Gata1 in half the erythroid cells and are Gata1 null in the other half. The results show that the cells supplying REDS are erythroid cells. This study demonstrates the importance of intercellular signalling in regulating Gata1 activity and that this homotypic signalling between erythroid cells is crucial to normal differentiation.
Subject(s)
DNA-Binding Proteins/metabolism , Erythrocytes/cytology , Transcription Factors/metabolism , Animals , Apoptosis , Blotting, Western , Cell Differentiation , Cell Lineage , Cell Separation , Crosses, Genetic , Erythroid-Specific DNA-Binding Factors , Female , Flow Cytometry , GATA1 Transcription Factor , Genotype , Hematopoietic Stem Cells/metabolism , Heterozygote , Liver/embryology , Male , Mice , Mice, Knockout , Mice, Mutant Strains , Mice, Transgenic , Models, Genetic , Mutation , Organ Culture Techniques , Phenotype , Recombination, Genetic , Signal Transduction , Time Factors , TransgenesABSTRACT
The human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the beta-globin gene in the maintenance of the ACH. When the human beta-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the beta-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the beta-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the beta-globin gene are required to maintain the appropriate spatial configuration in vivo.
Subject(s)
Chromatin/ultrastructure , Globins/genetics , Locus Control Region/genetics , Animals , Humans , Mice , Mice, Transgenic , Nucleic Acid Conformation , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid/genetics , Sequence Deletion , Transcription, GeneticABSTRACT
Deletions at the 3' end of the human beta-globin locus are associated with the hereditary persistence of fetal hemoglobin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal gamma-globin genes. We have tested how sequences at the HPFH-2, HPFH-3, and HPFH-6 breakpoints, which act as enhancers in vitro, affect the silencing of a locus control region A gamma (LCRA gamma) transgene in the adult stage of mice. We found persistent A gamma expression in the adult blood of most of the multicopy HPFH-2, HPFH-3, or HPFH-6 lines, in contrast to the control LCRA gamma lines which were silenced. Cre-mediated generation of single copy lines showed persistent gamma gene expression maintained in some of the HPFH-2 and HPFH-6 lines, but not in any of the HPFH-3 or LCRA gamma lines. In the HPFH-2 and HPFH-6 lines, persistent gamma gene expression correlated with euchromatic transgene integrations. Thus, our observations provide support for a model whereby HPFH conditions arise from the juxtaposition of enhancers as well as permissive chromatin subdomains in the vicinity of the gamma-globin genes.
Subject(s)
Fetal Hemoglobin/genetics , Gene Expression Regulation, Developmental , Globins/genetics , Sequence Deletion/physiology , Adult , Amino Acid Sequence , Animals , Enhancer Elements, Genetic , Gene Dosage , Gene Silencing , Genes, Switch , Humans , Locus Control Region/genetics , Mice , Mice, TransgenicABSTRACT
We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.
Subject(s)
DNA Transposable Elements , Drosophila/genetics , Animals , Blotting, Southern , In Situ Hybridization, Fluorescence , MiceABSTRACT
To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing alpha- and beta-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only alpha-globin genes (alpha-locus), whereas the other also contains a beta-globin gene (alpha beta-locus). This is the first poikilothermic species analyzed in which the physical linkage of the alpha- and beta-globin genes has been uncoupled, supporting a model in which the separation of the alpha- and beta-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu alpha beta-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian beta-globin clusters, may not be present in the Fugu alpha beta-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.
