RESUMO
Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.
Assuntos
Homozigoto , Células-Tronco Embrionárias Murinas/fisiologia , Mutagênese/genética , Animais , Células Cultivadas , Elementos de DNA Transponíveis , Diploide , Citometria de Fluxo , Vetores Genéticos , Gentamicinas/farmacologia , Haploidia , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Poli A , Puromicina/farmacologia , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Activating Gs a mutations have been identified in most instances of fibrous dysplasia (FD). This mutation leads to consistently elevated intracellular cyclic adenosine monophosphate (cAMP) levels, with various biological consequences. The development of secondary sarcoma in FD is a rare but well-established phenomenon. This finding raised the possibility that a common gene mutation exists in these tumors. MATERIALS AND METHODS: The expression of the Gs a mutation was examined in 16 cell lines and 173 musculoskeletal tumor tissues, including 13 cases of FD, via RT-PCR and sequence analysis. RESULTS: No expression of a Gs a mutation was detected in any cell line or clinical tissue sample, excluding FD tissues. Direct sequence analysis demonstrated results identical to those of RT-PCR. CONCLUSION: Activating Gs a mutation rarely occurs in musculoskeletal tumors other than FD. The occurrence of most sarcomas displays no correlation with Gs a mutations.
Assuntos
Neoplasias Ósseas/genética , Displasia Fibrosa Óssea/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Neoplasias Musculares/genética , Mutação , Osteossarcoma/genética , Sarcoma/genética , Linhagem Celular Tumoral , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: The sensitivity of human tumor tissues to infection with recombinant adenoviruses correlates with the expression of the coxsackievirus and adenovirus receptor (CAR). CAR has been shown to function as the primary receptor for adenoviruses and to play a critical role in adenovirus entry into host cells. It is important for clinical gene therapy to determine the expression level of CAR in tumor tissues. EXPERIMENTAL DESIGN: We analyzed the expression of CAR mRNA in 154 musculoskeletal tumor tissues from 154 patients and 10 normal mesenchymal tissues from 3 patients using reverse transcription-PCR and real-time quantitative PCR. An adenovirus infection assay was performed in two cell lines that were established from CAR-positive osteosarcoma tissue and CAR-negative malignant fibrous histiocytoma tissue. RESULTS: Ninety-nine of 154 tumors were detected as CAR positive by reverse transcription-PCR. We found that the expression levels of CAR mRNA varied markedly between different tumors as determined by real-time quantitative PCR. CAR mRNA was expressed at high levels in osteosarcoma, Ewing's sarcoma, neurofibroma, and schwannoma; at intermediate levels in exostosis, giant cell tumor, liposarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor, and hemangioma; and at low levels in alveolar soft part sarcoma and desmoid. Whereas the osteosarcoma cell line that expressed a high level of CAR mRNA, like its parent tumor, had a high efficiency of adenovirus infection, the malignant fibrous histiocytoma cell line with almost undetectable expression of CAR mRNA, like its parent tumor, had a low efficiency of infection. CONCLUSIONS: Our data showed the great variations in CAR mRNA expression among human musculoskeletal tumors and mesenchymal tissues and implicated the potential usefulness of adenoviral vectors in gene therapy for osteosarcoma, Ewing's sarcoma, neurofibroma, and schwannoma. Efficient transduction with adenovirus for gene therapy could be realized in appropriate, sensitive tumor types.
Assuntos
Tumores Neuroectodérmicos/metabolismo , Osteossarcoma/metabolismo , RNA Mensageiro/metabolismo , Receptores Virais/biossíntese , Sarcoma de Ewing/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Células HeLa , Humanos , Mesoderma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
We report on a 50-year-old man with dystrophic localized amyloidosis who noticed a soft tumor in his left thigh about 20 years ago, after which the tumor has gradually enlarged. The multicystic tumor showed hemorrhage, hematoma, necrosis, fibrosis, and tiny nodules and various polymorphous granulomas were observed. One was rich in eosinophilic amorphous materials and cholesterol crystals, and was poor in cell reaction. Another was formed by granuloma consisting of multinucleated giant cells, foamy cells, and macrophages. Transitional granulomas between the two were also observed. The materials showed eosinophilia and red staining and apple-green birefringence in polarized light by alkaline Congo-red stain, and they were also resistant to potassium permanganate pretreatment. They were also positive for amyloid P component and consistently negative for amyloid A, kappa- and lambda-light chains, beta2-microglobulin, and transthyretin. Therefore, it was suggested that this might be an amyloid derived from the hematoma, which has not been reported to date.
Assuntos
Amiloidose/patologia , Tecido Conjuntivo/patologia , Cistos/patologia , Hematoma/patologia , Amiloide/metabolismo , Amiloidose/metabolismo , Amiloidose/cirurgia , Corantes , Vermelho Congo , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/cirurgia , Cistos/metabolismo , Cistos/cirurgia , Diagnóstico Diferencial , Hematoma/metabolismo , Hematoma/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias de Tecidos Moles/diagnóstico , Coxa da Perna , Resultado do TratamentoRESUMO
Liposclerosing myxofibrous tumor (LSMFT) is a benign fibro-osseous lesion that is characterized by mixture of histologic elements including lipoma, fibroxanthoma, myxoma, ischemic ossification, and fibrous dysplasia (FD)-like features. These tissue components are seen in the original reports of FD; however, the relationship between LSMFT and FD is not clear. Point mutation of the alpha subunit of G protein (Gs alpha), which increases cyclic adenosine monophosphate formation, has been recognized as the cause of McCune-Albright syndrome as well as polyostotic and monostotic FD of bone. Gs alpha mutation at the Arg201 codon in 2 patients of LSMFT was demonstrated in the present study. Although direct sequencing analysis using the fresh-frozen materials could not detect the mutation, the polymerase chain reaction fragmentation length polymorphism (PCR-RFLP) disclosed the missense point mutation Gs alpha at the Arg201 codon in 2 cases involving LSMFT. This result strongly suggests that a subset of LSMFT is a variant form of FD.