RESUMO
The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.
RESUMO
OBJECTIVE: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance. METHODS: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice. Whole knee joint histological sections were stained using van Gieson or Fast green/Safranin-O to analyze collagen or proteoglycan content, respectively. Microarray analysis was performed to detect gene expression changes within knee joints. RESULTS: Epn-deficient and Epn/Bgn double-deficient mice appeared normal at birth. No significant difference in body weight or femur length was detected in any animal at 1 month of age. However, 9-month Epn/Bgn double-deficient mice were significantly lighter and had shorter femurs than wild type mice, regardless of gender. Male Epn-deficient mice also had significantly shorter femurs than wild type mice at 9 months. Most of the deficient animals developed osteoarthritis (OA) with age; the onset of OA was observed earliest in Epn/Bgn double-deficient mice. Message RNA isolated from Epn/Bgn double-deficient knee joints displayed increased matrix protein expression compared with wild type mice, including other small leucine-rich proteoglycan (SLRP) members such as asporin, fibromodulin and lumican. CONCLUSION: Similar to other previously studied SLRPs, EPN plays an important role in maintaining joint integrity. However, the severity of the OA phenotype in the Epn/Bgn double-deficient mouse suggests a synergy between these two proteins. These data are the first to show a genetic interaction involving class I and class III SLRPs in vivo.
Assuntos
Articulação do Joelho/química , Osteoartrite do Joelho/fisiopatologia , Proteoglicanas/análise , Proteoglicanas/deficiência , Animais , Southern Blotting , Peso Corporal , Colágeno/análise , Fêmur/anatomia & histologia , Imuno-Histoquímica , Articulação do Joelho/patologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Osteoartrite do Joelho/genética , Fenótipo , Reação em Cadeia da Polimerase , Proteoglicanas/genética , RNA Mensageiro/análiseRESUMO
A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.
Assuntos
Rim/anormalidades , Proteínas de Membrana/fisiologia , Proteínas/fisiologia , Proteinúria/etiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Perfilação da Expressão Gênica , Humanos , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas/genéticaRESUMO
Embryonic stem cells have become versatile genetic vehicles. Two-step recombination protocols have been used to modify endogenous mouse genes, and yeast artificial chromosomes containing human genes have been transmitted into the mouse germline. Recently, it has become possible to evaluate homozygous deficiencies in specific developmental compartments either through the use of chimeras or by activating recombination in vivo using potent recombinases.
Assuntos
Biotecnologia/métodos , Expressão Gênica , Células-Tronco , Transfecção/métodos , Animais , Células Cultivadas , Quimera , Cromossomos Artificiais de Levedura , Embrião de Mamíferos , Humanos , Camundongos , Recombinação Genética , Deleção de SequênciaRESUMO
We have constructed a new pair of plasmid vectors for the efficient expression of mammalian genes. The first of the new plasmids, pAVE1, was derived from pCMVcat [Foecking and Hofstetter, Gene 45 (1986) 101-105] by replacing the chloramphenicol acetyltransferase-encoding sequences in the latter for a multiple cloning site. Since it possesses the powerful enhancer-promoter unit of the immediate early gene of human cytomegalovirus, pAVE1 is ideal for the expression of mammalian genes. The second expression vector, pAVE2, resulted when the 3'-end flanking region from the human growth hormone-encoding gene (hGH) was incorporated in pAVE1. This region provides sequences for 3'-end processing and polyadenylation of primary transcripts. Thus, pAVE2 is suitable for expression of cDNAs in cultured cells, where introns have little effect on gene expression. To test our new vectors, we inserted the structural region of the chromosomal hGH gene into pAVE1, and its cDNA into pAVE2. By independently transfecting the resulting recombinant plasmids into COS-7 cells, we have achieved high levels of hGH transient expression with both vectors.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos , Plasmídeos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , Citomegalovirus/genética , Elementos Facilitadores Genéticos , Genes , Hormônio do Crescimento/genética , Humanos , Regiões Promotoras GenéticasRESUMO
Genetic modification of endogenous genes in mice has become possible by applying gene targeting techniques to embryonic stem (ES) cells and using specific clones of cells to generate mice. Despite the experimental opportunities offered by the creation of organisms with specific genetic changes, there are considerable technical obstacles which can confound the routine implementation of this technology. This review addresses some recent advances in the ability to construct mice with a variety of genetic modifications. These include an increased understanding of the basic cell biology and in vitro growth characteristics of ES cells, which has facilitated germ line transmission of manipulated clones on a routine basis. The techniques that are used to isolate "targeted" clones of ES cells have been summarized, and the current status of strategies which have been successfully used to make very specific modifications of the genome are discussed.
Assuntos
Camundongos Transgênicos/genética , Recombinação Genética/genética , Células-Tronco/fisiologia , Transfecção/genética , Alelos , Animais , Linhagem Celular , Células Clonais/fisiologia , CamundongosAssuntos
Blastocisto/fisiologia , Técnicas Genéticas , Camundongos Mutantes , Camundongos Transgênicos , Células-Tronco/fisiologia , Animais , Southern Blotting/métodos , Células Cultivadas , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , DNA/administração & dosagem , DNA/metabolismo , Eletroporação/métodos , Transferência Embrionária/instrumentação , Transferência Embrionária/métodos , Transferência Embrionária/veterinária , Feminino , Marcadores Genéticos , Vetores Genéticos , Hipoxantina Fosforribosiltransferase/genética , Indicadores e Reagentes , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , MicroinjeçõesRESUMO
Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice with null alleles. Current methods of gene inactivation in ES cells introduce a neomycin gene (neo) cassette both as a mutagen and a selection marker for transfected cells. Although null alleles are valuable, changes at the nucleotide level of a gene are very important for functional analysis. One gene family in which subtle mutations would be particularly valuable are the clusters of Hox homeobox genes. Inactivation of gene in a cluster with a neo cassette that includes promoter/enhancer elements may deregulate transcription of neighbouring genes and generate a phenotype which is difficult to interpret. We describe here a highly efficient gene targeting method, termed the 'hit and run' procedure. This generates ES cells with subtle site-specific mutations with no selectable marker and may be useful for most genes. We have developed this procedure at the hypoxanthine phosphoribosyltransferase (hprt) locus and subsequently isolated ES cells with a premature stop codon in the homeobox of Hox-2.6 (ref. 14).
Assuntos
Genes Homeobox , Células-Tronco/metabolismo , Alelos , Animais , Sequência de Bases , Mapeamento Cromossômico , Eletroforese em Gel de Poliacrilamida , Embrião de Mamíferos , Elementos Facilitadores Genéticos , Hipoxantina Fosforribosiltransferase/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Recombinação Genética , Transcrição GênicaRESUMO
Chromosomal rearrangements are the major cause of inherited human disease and fetal loss. Translocations and loss of heterozygosity are important genetic changes causally involved in neoplasia. Chromosomal variants, such as deficiencies, are commonly exploited in genetic screens in organisms such as Drosophila because a small portion of the genome is functionally hemizygous. In the mouse, deficiencies are not generally available, thus genetic screens for recessive mutations are cumbersome. We report here that defined deficiencies, inversions and duplications extending to 3-4 cM can be constructed in embryonic stem cells. This was achieved by consecutive targeting of loxP recombination substrates to the end points of a genetic interval followed by Cre-induced recombination. This reconstructs a positive selectable marker which facilitates direct selection of clones with a chromosome structure specific to the relative orientation of the loxP sites. Duplication and deletion alleles have been transmitted into the mouse germ line. The availability of mice with defined regions of segmental haploidy will allow their use in genetic screens and enable accurate models of human 'chromosomal' diseases to be generated.
Assuntos
Engenharia Genética , Integrases , Proteínas Virais , Animais , Linhagem Celular , Cricetinae , DNA Nucleotidiltransferases , Deleção de Genes , Marcação de Genes , Humanos , Camundongos , Família Multigênica , Recombinação Genética , Células-TroncoRESUMO
Hox genes regulate axial regional specification during animal embryonic development and are grouped into four clusters. The mouse HoxB cluster contains 10 genes, Hoxb1 to Hoxb9 and Hoxb13, which are transcribed in the same direction. We have generated a mouse strain with a targeted 90-kb deletion within the HoxB cluster from Hoxb1 to Hoxb9. Surprisingly, heterozygous mice show no detectable abnormalities. Homozygous mutant embryos survive to term and exhibit an ordered series of one-segment anterior homeotic transformations along the cervical and thoracic vertebral column and defects in sternum morphogenesis. Neurofilament staining indicates abnormalities in the IXth cranial nerve. Notably, simultaneous deletion of Hoxb1 to Hoxb9 resulted in the sum of phenotypes of single HoxB gene mutants. Although a higher penetrance is observed, no synergistic or new phenotypes were observed, except for the loss of ventral curvature at the cervicothoracic boundary of the vertebral column. Although Hoxb13, the most 5' gene, is separated from the rest by 70 kb, it has been suggested to be expressed with temporal and spatial colinearity. Here, we show that the expression pattern of Hoxb13 is not affected by the targeted deletion of the other 9 genes. Thus, Hoxb13 expression seems to be independent of the deleted region, suggesting that its expression pattern could be achieved independent of the colinear pattern of the cluster or by a regulatory element located 5' of Hoxb9.
Assuntos
Deleção de Genes , Genes Homeobox , Animais , Nervos Cranianos/embriologia , Camundongos , Camundongos Mutantes , Família Multigênica , FenótipoRESUMO
Two Hoxb-4 (Hox-2.6) mutations were introduced into the mouse germline. The overt phenotype caused by one of the mutations was assayed on two different genetic backgrounds, an inbred 129SvEv and a hybrid 129SvEv-C57BL/6J. The allele hoxb-4' is a disruption of the first exon and causes two obvious skeletal changes: a partial homeotic transformation of the second cervical vertebra from axis to atlas and a defective morphogenesis of the sternum. Both phenotypes have incomplete penetrance and variable expressivity when assayed in the hybrid genetic background, but the sternum defect is completely penetrant in the inbred background. The mutant allele hoxb-4s has a premature stop codon, introduced by the "hit and run" method in the second exon, that disrupts the third helix of the homeodomain. This allele also causes the partial homeotic transformation of axis to atlas, but it does not affect the sternum.
Assuntos
Vértebras Cervicais/anormalidades , Genes Homeobox , Esterno/anormalidades , Animais , Sequência de Bases , Quimera , Feminino , Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Dados de Sequência Molecular , Morfogênese , Mutagênese Insercional , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Transcrição Gênica , TransfecçãoRESUMO
Gene targeting applied to totipotent embryonic stem (ES) cells is a very powerful means of creating highly specific mutations of genes in the mouse. The successful application of this technology is however constrained by both the types of mutations that can be generated at a target locus and the ability to reconstruct a germline chimera from the manipulated cells. We have developed two cell lines that can be routinely transmitted through the germline of chimeras after cloning and prolonged selection in tissue culture. We have also established a variety of methods for generating non-selected mutations at the X-linked hprt locus in ES cells. Our observations at this locus have enabled us to generate successfully a subtle mutation at the non-selectable Hox-2.6 locus.
Assuntos
Vetores Genéticos/genética , Células-Tronco/fisiologia , Animais , Linhagem Celular , Células Clonais/fisiologia , Células Germinativas/fisiologia , Camundongos , Mutação/genética , Seleção GenéticaRESUMO
We have joined the promoter-less sequences of the three hPL genes (hPL-1, hPL-3 and hPL-4) to strong transcriptional control elements. in vivo 35S-labeled proteins from the culture medium of cells transfected with the genes were resolved on SDS-polyacrylamide gels. The presence of characteristic labeled bands, visualized by autoradiography, determined that hPL-4 and hPL-3, but not hPL-1, contribute to the production of mature hPL. In these experiments hPL-3 expressed more RNA and protein than hPL-4. By exchanging the first two exons among hPL and hGH genes, we determined that the abundance of chimeric proteins depended on the genetic origin of the first two exons. Finally, we found evidence indicating that the splice mutation (G----A) at the beginning of the second intron of hPL-1, is not the only cause of the apparent lack of inactivity of this gene, since its reversion does not restore expression.
Assuntos
Éxons , Lactogênio Placentário/genética , Transfecção , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Genes , Humanos , Mutação , Plasmídeos , Gravidez , Pseudogenes , Splicing de RNA , Transcrição GênicaRESUMO
Many experimental designs require the analysis of genomic DNA from a large number of samples. Although the polymerase chain reaction (PCR) can be used, the Southern blot is preferred for many assays because of its inherent reliability. The rapid acceptance of PCR, despite a significant rate of false positive/negative results, is partly due to the disadvantages of the sample preparation process for Southern blot analysis. We have devised a rapid protocol to extract high-molecular-weight genomic DNA from a large number of samples. It involves the use of a single 96-well tissue culture dish to carry out all the steps of the sample preparation. This, coupled with the use of a multichannel pipette, facilitates the simultaneous analysis of multiple samples. The procedure may be automated since no centrifugation, mixing, or transferring of the samples is necessary. The method has been used to screen embryonic stem cell clones for the presence of targeted mutations at the Hox-2.6 locus and to obtain data from human blood.
Assuntos
Southern Blotting/métodos , DNA/isolamento & purificação , Animais , Células Clonais , DNA/sangue , Genoma , Humanos , Camundongos , Mapeamento por Restrição , Células-Tronco/químicaRESUMO
The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In the mouse, several single mutants for Hox genes show variably penetrant, partial homeotic transformations of vertebrae at their anterior limits of expression, suggesting that compound Hox mutants might show more complete transformations with greater penetrance than the single Hox mutants. Compound mutants for the paralogous group 3 genes, hoxa-3 and hoxd-3, show deletion of a cervical vertebrae, which is not readily interpretable in terms of an alteration in regional identity. Here, we report the skeletal phenotypes of compound mutants in the group 4 Hox genes, hoxa-4, hoxb-4, and hoxd-4. Mice mutant for each of these genes were intercrossed to generate the three possible double mutant combinations and the triple mutant. In contrast to the hoxa-3, hoxd-3 double mutants, group 4 Hox compound mutants displayed clear alterations in regional identity, including a nearly complete transformation of the second cervical vertebrae toward the morphology of the first cervical vertebra in one double mutant combination. In comparing the types of homeotic transformations observed, different double mutant combinations showed different degrees of synergism. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression.
Assuntos
Vértebras Cervicais/anormalidades , Genes Homeobox , Família Multigênica , Mutação , Fatores de Transcrição/genética , Animais , Vértebras Cervicais/embriologia , Vértebras Cervicais/crescimento & desenvolvimento , Cruzamentos Genéticos , Feminino , Expressão Gênica , Genótipo , Heterozigoto , Masculino , Camundongos , Camundongos Mutantes , Fenótipo , Recombinação Genética , Fatores de Transcrição/metabolismoRESUMO
Current efforts to map the human genome are focused on individual chromosomes or smaller regions and frequently rely on the use of somatic cell hybrids. We report the application of the polymerase chain reaction to direct amplification of human DNA from hybrid cells containing regions of the human genome in rodent cell backgrounds using primers directed to the human Alu repeat element. We demonstrate Alu-directed amplification of a fragment of the human HPRT gene from both hybrid cell and cloned DNA and identify through sequence analysis the Alu repeats involved in this amplification. We also demonstrate the application of this technique to identify the chromosomal locations of large fragments of the human X chromosome cloned in a yeast artificial chromosome and the general applicability of the method to the preparation of DNA probes from cloned human sequences. The technique allows rapid gene mapping and provides a simple method for the isolation and analysis of specific chromosomal regions.