RESUMO
Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.
Assuntos
Desoxirribonucleases/genética , Técnicas de Inativação de Genes/métodos , Genoma/genética , Ratos/embriologia , Ratos/genética , Transfecção/métodos , Dedos de Zinco/genética , Animais , Engenharia Genética/métodos , Ratos TransgênicosRESUMO
The rabbit is a preferred model system for diverse areas of human disease research, such as hypertension and atherosclerosis, for its close resemblance to human physiology. Its larger size than that of rodents allows for more convenient physiological and surgical manipulations as well as imaging. The rapid development of nuclease technologies enables the rabbit genome to be engineered as readily as that of rats and mice, offering rabbit models a chance to make their due impact on medical research. Here, we report the efficient creation of an APOE knockout rabbit by using zinc finger nucleases. The knockout rabbits had drastically elevated cholesterol and moderately increased triglyceride levels, mimicking symptoms in human heart disease. So far the rabbit genome has been successfully modified with three nuclease technologies. With a gestation period only days longer than those of rodents, we hope additional reports on their creation and characterization will help encourage the use of rabbit models where they are most relevant to human conditions.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/genética , Dedos de Zinco/genética , Animais , Aterosclerose/fisiopatologia , Colesterol/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Genoma , Humanos , Coelhos , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. RESULTS: After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαß+ as well as CD8+/CD3+/TCRαß+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. CONCLUSION: The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.
Assuntos
Endonucleases/metabolismo , Marcação de Genes , Proteínas de Homeodomínio/genética , Dedos de Zinco , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Embrião de Mamíferos/metabolismo , Citometria de Fluxo , Mutação da Fase de Leitura/genética , Genótipo , Células Germinativas , Proteínas de Homeodomínio/química , Humanos , Depleção Linfocítica , Tecido Linfoide/patologia , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Ratos MutantesRESUMO
"Animal Models of Neural Disease" was the focus of General Session 5 at a 2010 scientific symposium that was sponsored jointly by the Society of Toxicologic Pathology (STP) and the International Federation of Societies of Toxicologic Pathologists (IFSTP). The objective was to consider issues that dictate the choice of animal models for neuropathology-based studies used to investigate neurological diseases and novel therapeutic agents to treat them. In some cases, no animal model exists that recapitulates the attributes of the human disease (e.g., fibromyalgia syndrome). Alternatively, numerous animal models are available for other conditions, so an essential consideration is selecting the most appropriate experimental system (e.g., Alzheimer's disease). New technologies (e.g., genetically engineered rodent models) promise the opportunity to generate suitable animal models for syndromes that currently lack any in vivo animal model, while in vitro models offer the opportunity to evaluate xenobiotic effects in specific neural cell populations. The complex nature of neurological disease requires regular reassessment of available and potential options to ensure that animal-derived data sets support translational medicine efforts to improve public health.
Assuntos
Modelos Animais de Doenças , Doenças do Sistema Nervoso/patologia , Síndromes Neurotóxicas/patologia , Doença de Alzheimer/terapia , Animais , Animais Geneticamente Modificados , Congressos como Assunto , Fibromialgia/patologia , Humanos , Doenças do Sistema Nervoso/induzido quimicamente , Síndromes Neurotóxicas/terapia , Neurotoxinas , Sociedades CientíficasRESUMO
Osteosarcoma is the most common primary malignant bone tumor affecting children and adolescents. The majority of patients are treated by surgery and chemotherapy but have limited alternative therapeutic options. Kinases play an important role in the growth and survival of tumor cells. We aim to identify specific kinases to be vital in the survival of osteosarcoma cells and thus may be a key target in creating novel anticancer therapies. A lentiviral short hairpin RNA kinase library, screened osteosarcoma cells, identified kinase minibrain-related kinase (Mirk) (Dyrk1B) as a potential target. Knockdown Mirk expression could inhibit cell growth and induce apoptosis. Chemically synthetic small interfering RNA knockdown and complementary DNA rescue assay further confirmed the results from the decrease of Mirk gene expression. The relationship between Mirk gene expression and the clinical characteristics of patients with osteosarcoma was investigated using tissue microarray and immunohistochemistry analysis. The data indicate that the overall survival rate of patients with Mirk high staining (high levels of Mirk protein expression) is significantly shorter than those with Mirk low staining and moderate staining. This highlights Mirk's potential to serve as a promising target for molecular therapy in the treatment of osteosarcoma.
Assuntos
Neoplasias Ósseas/patologia , Osteossarcoma/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Apoptose , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Humanos , Osteossarcoma/terapia , Prognóstico , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Interferente Pequeno/genética , Quinases DyrkRESUMO
Published reports implicate a variety of mechanisms that may contribute to drug resistance in ovarian cancer. The chief aim of this study is to understand the relationship between overexpression of drug resistance associated genes and multidrug resistance in ovarian cancer. Using lentiviral short hairpin RNA collections targeting 132 genes identified from transcriptional profiling of drug-resistant cancer cell lines, individual knockdown experiments were done in the presence of sublethal doses of paclitaxel. Specific genes whose knockdown was found to be associated with cellular toxicity included MDR1 (ABCB1), survivin, and pre-mRNA processing factor-4 (PRP-4). These genes, when repressed, can reverse paclitaxel resistance in the multidrug-resistant cell line SKOV-3(TR) and OVCAR8(TR). Both MDR1 and survivin have been reported previously to play a role in multidrug resistance and chemotherapy-induced apoptosis; however, the effect of PRP-4 expression on drug sensitivity is currently unrecognized. PRP-4 belongs to the serine/threonine protein kinase family, plays a role in pre-mRNA splicing and cell mitosis, and interacts with CLK1. Northern analysis shows that PRP-4 is overexpressed in several paclitaxel-resistant cell lines and confirms that PRP-4 expression could be significantly repressed by PRP-4 lentiviral short hairpin RNA. Both clonogenic and MTT assays confirm that transcriptional repression of PRP-4 could reverse paclitaxel resistance 5-10-fold in SKOV-3(TR). Finally, overexpression of PRP-4 in drug-sensitive cells could induce a modest level of drug resistance to paclitaxel, doxorubicin, and vincristine.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Resistência a Múltiplos Medicamentos/fisiologia , Lentivirus/genética , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/uso terapêutico , RNA Viral/genética , Ribonucleoproteína Nuclear Pequena U4-U6/fisiologia , Antineoplásicos Fitogênicos/farmacologia , Sequência de Bases , Northern Blotting , Primers do DNA , Feminino , Humanos , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
An shRNA tumor suppressor panel was screened using reverse infection of an A549 tumorigenic cell line and exposing it to a predetermined concentration of paclitaxel, an anticancer drug. The shRNAs targeting a positive control gene, MDR1, were found to effectively decrease mRNA levels and cause cells to become more sensitive to the chemotherapeutic drug. A set of genes were identified in the screen of a panel of tumor suppressors which, when down-regulated, were found to increase or decrease cell sensitivity in regards to treatment with paclitaxel. In many cases, there were multiple clones to a single gene that provided a positive result. The shRNAs targeting SMAD4, LZTS2, ST14 and VHL all increased the cell's sensitivity to paclitaxel. The loss of other tumor suppressors such as GLTSCR2, LATS1, NF1, PTEN, TP53 and WT1 induced a protective effect in the cell, making it more resistant to the effect of the drug. Further investigation of VHL mRNA levels after down-regulation with shRNA show a direct correlation between gene expression levels and paclitaxel sensitivity. This study credits the identified genes with the potential to act as prognostic biomarkers for use in genetic profiling, or even as targets in pathways of tumorigenesis yet to be fully understood.
Assuntos
Genes Supressores de Tumor , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adenocarcinoma , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Rim , Neoplasias Pulmonares , Dados de Sequência Molecular , Plasmídeos , TransfecçãoRESUMO
Extensive efforts are under way to identify antiangiogenic therapies for the treatment of human cancers. Many proposed therapeutics target vascular endothelial growth factor (VEGF) or the kinase insert domain receptor (KDR/VEGF receptor-2/FLK-1), the mitogenic VEGF receptor tyrosine kinase expressed by endothelial cells. Inhibition of KDR catalytic activity blocks tumor neoangiogenesis, reduces vascular permeability, and, in animal models, inhibits tumor growth and metastasis. Using a gene expression profiling strategy in rat tumor models, we identified a set of six genes that are selectively overexpressed in tumor endothelial cells relative to tumor cells and whose pattern of expression correlates with the rate of tumor endothelial cell proliferation. In addition to being potential targets for antiangiogenesis tumor therapy, the expression patterns of these genes or their protein products may aid the development of pharmacodynamic assays for small molecule inhibitors of the KDR kinase in human tumors.
Assuntos
Biomarcadores Tumorais , Perfilação da Expressão Gênica/métodos , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Primers do DNA/química , Células Endoteliais/citologia , Endotélio Vascular/citologia , Humanos , Imuno-Histoquímica , Microcirculação/citologia , Microscopia de Fluorescência , Metástase Neoplásica , Transplante de Neoplasias , Neovascularização Patológica , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful â¼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/genética , Marcação de Genes/métodos , Fatores de Troca do Nucleotídeo Guanina/genética , RNA Guia de Cinetoplastídeos/genética , Alelos , Animais , Sequência de Bases , Reparo do DNA por Junção de Extremidades , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Endonucleases/metabolismo , Éxons , Feminino , Proteínas Ativadoras de GTPase , Edição de Genes , Fatores de Troca do Nucleotídeo Guanina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microinjeções , Mutagênese Insercional , RNA Guia de Cinetoplastídeos/metabolismo , Zigoto/citologia , Zigoto/metabolismoRESUMO
Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications.
Assuntos
Edição de Genes , Genoma , Imunodeficiência Combinada Severa/genética , Envelhecimento/patologia , Animais , Animais Recém-Nascidos , Blastômeros/metabolismo , Cruzamento , Callithrix , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Efeito Fundador , Técnicas de Inativação de Genes , Humanos , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Masculino , Mosaicismo , Fenótipo , Reprodutibilidade dos Testes , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/parasitologia , Espermatozoides/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Dedos de ZincoRESUMO
In a concerted effort to identify biomarkers for lung and colon carcinomas by genome-wide transcriptional profiling, we describe the identification and cloning of one such gene as well as two additional closely related genes. Due to the strong sequence homology to the C. elegans UNC-112 we call this gene URP1, for UNC-112 related protein. We have also isolated the full-length clones for another novel related gene, URP2 and the previously discovered MIG-2 gene. Collectively, these proteins, together with two from Drosophila, appear to form a novel membrane-associated FERM and PH domain-containing protein family. Transcriptional analysis shows that only URP1 is significantly differentially regulated, being over-expressed in 70% of the colon carcinomas and 60% of the lung carcinomas tested. Quantification of URP1 expression by qRT-PCR showed up-regulation of the gene by 60-fold in lung tumors and up to nearly 6-fold in colon tumors. Northern blot analysis of URP1 indicates that normal expression is restricted to neuromuscular tissues. In contrast, the expression of URP2 appears to be confined primarily to tissues of the immune system. SNP analysis of URP1 reveals that it is highly polymorphic, containing seven sites, four of which are in the coding region and one position that results in the interchangeable substitution of glutamic acid and lysine. Finally, we have shown that the genomic structure for all three genes is nearly identical with all encoded by 15 exons although URP1 gene localized to chromosome 20p13, URP2 to 11q12 and MIG-2 to 14q22. This conserved exon structure suggests that all three members probably arose by gene duplication from one ancestral gene. The presence of multiple FERM domains characteristic of cytoplasmic plasma membrane to cytoskeleton linkers and a PH domain typical of membrane-anchored proteins involved in signal transduction suggest an important role for URP1 in tumorigenesis.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Sequência de Aminoácidos , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Clonagem Molecular , Neoplasias do Colo/genética , DNA Complementar/biossíntese , DNA Complementar/química , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Análise Serial de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de SequênciaRESUMO
The molecular mechanisms involved in the progression of colon carcinomas from a primary to a metastatic tumor have been only partially elucidated and poorly understood. This study combines suppression subtractive hybridization and cDNA array hybridization to identify genes with expression differences between a primary human colon tumor cell line (HT29) and three isogenic lung tumor metastases. The positive clones isolated in this screen were further validated and quantitated with real-time reverse transcription polymerase chain reactions. HES-6 was identified as up-regulated in each of the individual tumor metastases, as well as in a panel of primary human tumors derived from the lung, breast and kidney. These findings demonstrate that it is possible to utilize longitudinal samples from an in vivo model of colon carcinoma to identify genes up-regulated in metastases and that HES-6 may be an important marker of a range of primary cancers as well as metastatic colon carcinoma.
Assuntos
Aberrações Cromossômicas , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Repressoras , Fatores de Transcrição/genética , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias da Mama/genética , Neoplasias do Colo/patologia , Primers do DNA , Feminino , Biblioteca Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Metástase Neoplásica , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The tumor suppressor TP53 plays a crucial role in cancer biology, and the TP53 gene is the most mutated gene in human cancer. Trp53 knockout mouse models have been widely used in cancer etiology studies and in search for a cure of cancer with some limitations that other model organisms might help overcome. Via pronuclear microinjection of zinc finger nucleases (ZFNs), we created a Tp53 knockout rat that contains an 11-bp deletion in exon 3, resulting in a frameshift and premature terminations in the open reading frame. In cohorts of 25 homozygous (Tp53(Δ11/Δ11)), 37 heterozygous (Tp53(Δ11/+)) and 30 wild-type rats, the Tp53(Δ11/Δ11) rats lived an average of 126 days before death or removal from study because of clinical signs of abnormality or formation of tumors. Half of Tp53(Δ11/+) were removed from study by 1 year of age because of tumor formation. Both Tp53(Δ11/+) and Tp53(Δ11/Δ11) rats developed a wide spectrum of tumors, most commonly sarcomas. Interestingly, there was a strikingly high incidence of brain lesions, especially in Tp53(Δ11/Δ11) animals. We believe that this mutant rat line will be useful in studying cancer types rarely observed in mice and in carcinogenicity assays for drug development.
Assuntos
Técnicas de Inativação de Genes/métodos , Genes p53 , Neoplasias Experimentais/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , DNA/genética , Modelos Animais de Doenças , Feminino , Fertilidade/genética , Mutação da Fase de Leitura , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Deleção de SequênciaRESUMO
Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses.
Assuntos
Peso Corporal/genética , Ingestão de Alimentos/genética , Leptina/genética , Obesidade/genética , Ratos Transgênicos , Animais , Densidade Óssea/genética , Colesterol/sangue , Metabolismo Energético/genética , Fêmur/metabolismo , Insulina/sangue , Leptina/metabolismo , Obesidade/metabolismo , Fenótipo , Ratos , Triglicerídeos/sangue , Dedos de ZincoRESUMO
Gene targeting is indispensible for reverse genetics and the generation of animal models of disease. The mouse has become the most commonly used animal model system owing to the success of embryonic stem cell-based targeting technology, whereas other mammalian species lack convenient tools for genome modification. Recently, microinjection of engineered zinc-finger nucleases (ZFNs) in embryos was used to generate gene knockouts in the rat and the mouse by introducing nonhomologous end joining (NHEJ)-mediated deletions or insertions at the target site. Here we use ZFN technology in embryos to introduce sequence-specific modifications (knock-ins) by means of homologous recombination in Sprague Dawley and Long-Evans hooded rats and FVB mice. This approach enables precise genome engineering to generate modifications such as point mutations, accurate insertions and deletions, and conditional knockouts and knock-ins. The same strategy can potentially be applied to many other species for which genetic engineering tools are needed.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Embrião de Mamíferos/metabolismo , Marcação de Genes , Engenharia Genética/métodos , Recombinação Genética , Dedos de Zinco/genética , Animais , Sequência de Bases , Células-Tronco Embrionárias , Técnicas de Inativação de Genes/métodos , Camundongos , Microinjeções , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We describe an optimized systematic screen of known kinases using osteosarcoma cell lines (KHOS and U-2OS) and a lentiviral-based short hairpin RNA (shRNA) human kinase library. CellTiter 96(R)AQueous One Solution Cell Proliferation Assay was used to measure cell growth and survival. We identified several kinases, including human polo-like kinase (PLK1), which inhibit cell growth and induce apoptosis in osteosarcoma cells when knocked down. cDNA rescue and synthetic siRNA assays confirm that the observed phenotypic changes result from the loss of PLK1 gene expression. Furthermore, a small molecule inhibitor to PLK1 inhibited osteosarcoma cell growth and induced apoptosis. Western blot analysis confirmed that PLK1 is highly expressed and activated in several osteosarcoma cell lines as well as in resected tumor samples. Immunohistochemistry analysis showed that patients with high PLK1 tumor expression levels correlated with significantly shorter survival than patients with lower levels of tumor PLK1 expression. These results demonstrate the capability and feasibility of a high-throughput screen with a large collection of lentiviral kinases and its effectiveness in identifying potential drug targets. The development of more potent inhibitors that target PLK1 may open doors to a new range of anti-cancer strategies in osteosarcoma.
Assuntos
Proteínas de Ciclo Celular/farmacologia , Osteossarcoma/enzimologia , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , RNA/análise , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indóis/farmacologia , Lentivirus/genética , Osteossarcoma/patologia , Fenóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Sarcoma/enzimologia , Regulação para Cima , Quinase 1 Polo-LikeRESUMO
Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Técnicas de Inativação de Genes/métodos , Marcação de Genes/métodos , Mutagênese Sítio-Dirigida , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteína Jagged-1 , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Notch3 , Receptores Notch/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Proteínas Serrate-Jagged , Dedos de ZincoRESUMO
We have identified a novel human gene by transcriptional microarray analysis, which is co-regulated in tumors and angiogenesis model systems with VEGF expression. Isolation of cDNA clones containing the full-length VCC-1 transcript from both human and mouse shows a 119 amino acid protein with a 22 amino acid cleavable signal sequence in both species. Comparison of the protein product of this gene with hidden Markov models of all known proteins shows weak but significant homology with two known chemokines, SCYA17 and SCYA16. Northern analysis of human tissues detects a 1 kb band in lung and skeletal muscle. Murine VCC-1 expression can also be detected in lung as well as thyroid, submaxillary gland, epididymis, and uterus tissues by slot blot analysis. By quantitative real time RT-PCR 71% of breast tumors showed 3- to 24-fold up-regulation of VCC-1. In situ hybridization of breast carcinomas showed strong expression of the gene in both normal and transformed mammary gland ductal epithelial cells. In vitro, human microvascular endothelial cells grown on fibronectin increase VCC-1 expression by almost 100-fold. In addition, in the mouse angioma endothelial cell line PY4.1 the gene was over-expressed by 28-fold 6 h after induction of tube formation while quiescent and proliferating cells showed no change. VCC-1 expression is also increased by VEGF and FGF treatment, about 6- and 5-fold, respectively. Finally, 100% of mice injected with NIH3T3 cells over-expressing VCC-1 develop rapidly progressing tumors within 21 days while no growth is seen in any control mice injected with NIH3T3 cells containing the vector alone. These results strongly suggest that VCC-1 plays a role in angiogenesis and possibly in the development of tumors in some tissue types.
Assuntos
Quimiocinas/genética , Quimiocinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Sequência de Aminoácidos , Animais , Proliferação de Células , Células Cultivadas , Quimiocinas/química , Quimiocinas CXC , Sequência Conservada , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Transplante de Neoplasias , Neoplasias/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.