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Texto completo SciELO Brasil
Covas, Dimas Tadeu
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Id: lil-713680
Autor: Freitas, Marcela Cristina Correa de; Fontes, Aparecida Maria; Fernandes, Andrielle de Castilho; Picanço-Castro, Virginia; Russo, Elisa Maria de Sousa; Covas, Dimas Tadeu.
Título: Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII
Fonte: Rev. bras. hematol. hemoter;36(3):213-218, May-Jun/2014. tab, graf.
Idioma: en.
Resumo: OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K. METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones. RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293. CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins. .
Descritores: Fator VIII
Integração Viral
Vírus da Leucemia Murina
Hemofilia A
Responsável: BR408.1 - Biblioteca da Faculdade de Medicina - BFM

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Id: lil-606640
Autor: Alrefaei, Yousef Noori; Okatcha, Tunika Ida; Skinner, Danielle Elaine; Brindley, Paul James.
Título: Progress with schistosome transgenesis
Fonte: Mem. Inst. Oswaldo Cruz;106(7):785-793, Nov. 2011. ilus.
Idioma: en.
Projeto: NIAID.
Resumo: Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.
Descritores: Técnicas de Transferência de Genes
Genoma Helmíntico/genética
Schistosoma japonicum/genética
Schistosoma mansoni/genética
-Animais Geneticamente Modificados
Elementos de DNA Transponíveis
DNA de Helmintos/genética
DNA Viral/genética
DNA Viral/isolamento & purificação
Vetores Genéticos
Vírus da Leucemia Murina/genética
Vírus da Leucemia Murina/isolamento & purificação
Glicoproteínas de Membrana/genética
Glicoproteínas de Membrana/isolamento & purificação
Interferência de RNA
Schistosoma japonicum/virologia
Schistosoma mansoni/virologia
Proteínas do Envelope Viral/genética
Proteínas do Envelope Viral/isolamento & purificação
Limites: Animais
Tipo de Publ: Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Responsável: BR1.1 - BIREME

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Id: lil-432711
Autor: Silva, Flávia Helena da; Dalberto, Tiago Pires; Nardi, Nance Beyer.
Título: Beyond retrovirus infection: HIV meets gene therapy
Fonte: Genet. mol. biol;29(2):367-379, 2006. ilus.
Idioma: en.
Resumo: The human immunodeficiency virus (HIV) is classified as a retrovirus because of its RNA genome and the fact that it requires reverse transcriptase to convert it into DNA. This virus belongs to the lentivirinae subfamily and is able to infect quiescent cells but is better known for its association with acquired immunodeficiency syndrome (AIDS) and can be described as one of the most effective vectors for gene transfer. Biosafety concerns are present whenever viral vectors are employed but are particularly pertinent to the development of HIV-based vectors. Insertional mutagenesis and the production of new replication-competent viruses (RCV) have been pointed to as major problems, but experimental data have shown that safe protocols can be developed for their production and application. Virological, evolutionary, immunological and cell biology studies must be conducted jointly to allow the clinical use of HIV vectors. This review will focus on the general properties, production and applications of retrovectors in gene therapy, with particular emphasis on those based on HIV systems.
Descritores: Terapia Genética
Infecções por Retroviridae/etiologia
Vírus da Leucemia Murina
-Vetores Genéticos
Genoma Viral
Síndrome de Imunodeficiência Adquirida/terapia
Limites: Humanos
Tipo de Publ: Revisão
Responsável: BR26.1 - Biblioteca Central

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