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1.
Hepatol Int ; 14(1): 96-104, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31832976

RESUMO

BACKGROUND: The diagnostic and prognostic values of glypican3 (GPC3) and glutamine synthetase (GS) proteins in hepatocellular carcinoma (HCC) have been reported, but their specificity and sensitivity remain low. Here, we applied RNAscope to improve HCC early pathological and differential diagnosis by estimating GPC3 and GS mRNAs. METHODS: We performed RNAscope and immunohistochemistry (IHC) to detect GPC3 and GS biomarkers on the tissue sections of 194 cases, including high- and low-grade liver dysplastic nodules; highly, moderately, and poorly differentiated HCCs; intrahepatic cholangiocarcinomas (ICCs); metastatic HCC; and carcinomas from other organs. RESULTS: The results showed that all the cases that were negative for GPC3 by RNAscope were also negative for this protein by IHC. The use of RNAscope assay improved the GPC3 and GS specificity and sensitivity by 20-30%. Hence, HCC shows early recognition and upgrades the metastatic HCC differentiation by 23% compared with IHC (p = 0.0001, 0.0064). Meanwhile, all liver cirrhosis, cholangiocytes and non-HCC samples were negative for GPC3 and GS except lymphocytes in lymphomas, and 2 (8.3%) out of the 24 ICC samples but not in the cancer cells. CONCLUSION: RNAscope for GPC3 and GS panel was highly specific and sensitive for the pathological identification of dysplastic nodules, early stages of HCCs, and would differentiate them from HCCs and metastatic tumors compared with IHC.

2.
Artigo em Inglês | MEDLINE | ID: mdl-31794839

RESUMO

PURPOSE: To compare long-term survival outcomes and sequelae between children and adult nasopharyngeal carcinoma (NPC) in the era of intensity-modulated radiotherapy (IMRT). METHODS AND MATERIALS: Data on 285 NPC patients aged ≤ 18 years old at diagnosis and treated with IMRT between January 2004 and November 2016 were retrospectively reviewed. Propensity score matching method was adopted to screen matched adult NPC patients at a ratio of 1:3. Survival outcomes and treatment-related toxicities between children and adult groups were compared. RESULTS: In total, 159 children and 477 adult NPC patients were included in this study. The 5-year overall survival (OS), distant metastasis-free survival (DMFS), locoregional relapse-free survival (LRRFS), and disease-free survival (DFS) between children and adult were 89.2% vs 83.6% (P = 0.144), 88.7% vs 83.5% (P = 0.124), 96.4% vs 89.1% (P = 0.013), and 86.5% vs 77.3% (P = 0.021), respectively. Subgroup analyses revealed that the young age was an independent prognostic factor of OS, DMFS and LRRFS in advanced N stage (N2-3) group, and DFS in advanced T stage (T3-4) group, N2-3 and stage III-IVA groups. The most common sequela was ototoxicity (68.9%) in children patients and xerostomia (70.8%) in adult patients. Adult survivors had a significantly higher incidence of grade 3-4 late toxicities in xerostomia (17.6% vs. 8.9%, P = 0.004), skin dystrophy (9.3% vs. 3.7%, P = 0.022), neck fibrosis (8.3% vs. 4.4%, P < 0.001) and radiation encephalopathy (0.8% vs. 0, P = 0.006). Children survivors were more likely to develop grade 3-4 growth retardation and endocrine insufficiency (3.0% vs. 0.3%, P = 0.014). CONCLUSIONS: Children NPC patients achieved significantly better survival outcomes but less late toxicities than adult patients. However, we should pay great attention to growth problems of children survivors.

3.
Crit Rev Oncol Hematol ; 135: 30-38, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30819444

RESUMO

Epstein-Barr virus (EBV) is believed to be a pathogen causing a number of human cancers, but the pathogenic mechanisms remain unclear. An increasing number of studies have indicated that EBV-encoded microRNAs (EBV miRNAs) are expressed in a latency type- and tumor type-dependent manner, playing important roles in the development and progression of EBV-associated tumors. By targeting one or more genes of the virus and the host, EBV miRNAs are responsible for the deregulation of a variety of viral and host cell biological processes, including viral replication, latency maintenance, immune evasion, cell apoptosis and metabolism, and tumor proliferation and metastasis. In addition, some EBV miRNAs can be used as excellent diagnostic, prognostic and treatment efficacy predictive biomarkers for EBV-associated tumors. More importantly, EBV miRNA-targeting therapeutics have emerged and have been developing rapidly, which may open a new era in the treatment of EBV-associated tumors in the near future.


Assuntos
Infecções por Vírus Epstein-Barr/genética , MicroRNAs/genética , Neoplasias/patologia , Neoplasias/virologia , Infecções Tumorais por Vírus/genética , Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4/genética , Humanos , RNA Viral/genética , Evasão Tumoral/genética , Infecções Tumorais por Vírus/complicações , Latência Viral/genética , Replicação Viral/genética
4.
Virology ; 529: 144-151, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30710798

RESUMO

Circular RNAs (circRNAs) are a novel class of non-coding RNA molecules in eukaryotic organisms that have potentially important roles in gene regulation. Nevertheless, whether viruses can encode circRNA is still uncertain. To examine whether large genome DNA viruses can generate circRNA during the infection of human cells, we performed RNA sequencing of ribosomal RNA-depleted total RNA from Epstein-Barr virus (EBV)-infected cell lines, including SNU-719, AGS-EBV, C666-1 and Akata. We identified an EBV-encoded circRNA, ebv_circ_RPMS1, that consists of the head-to-tail splicing of exons 2-4 from the RPMS1 gene. Furthermore, we demonstrated that ebv_circ_RPMS1 was localized in both cytoplasm and nuclei. Given that circRNAs shape gene expression by titrating microRNAs, regulating transcription and/or interfering with splicing, we identified a novel viral regulator of host and/or viral gene expression.


Assuntos
Herpesvirus Humano 4/genética , RNA não Traduzido/genética , RNA Viral/genética , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Experimentais/virologia
5.
Chin J Cancer ; 34(11): 531-7, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26370236

RESUMO

INTRODUCTION: Brain metastasis is common in relapsed neuroblastoma patients, but the characteristics of brain metastasis remain largely unknown. This study aimed to investigate the status of brain metastasis with neuroblastoma in South China. METHODS: In this retrospective case-based study, 106 patients with stage 4 neuroblastoma from the Department of Pediatric Oncology in Sun Yat-sen University Cancer Center between January 2004 and May 2013 were included. The incidence, risk factors, and survival status of these patients were reviewed and analyzed. RESULTS: Of the 106 patients, 11 (10.4%) developed brain metastasis, accounting for 20.0% of 55 patients with relapse or progression. The age at initial diagnosis of the 11 patients ranged from 2 to 10 years (median 4 years), which was younger than that of the patients without brain metastasis (median 5 years, range 1-10 years, P=0.073). The male to female ratio of the 11 patients was 8:3, which was not significantly different from that of the patients without brain metastasis (P=0.86). Patients with brain metastasis had higher lactate dehydrogenase levels than those without brain metastasis, but the differences were not significant (P=0.076). Eight patients died, and 3 patients survived. The median interval from the initial diagnosis to the development of brain metastasis was 18 months (range 6-32 months). The median survival was 4 months (range 1 day to 29 months) after the diagnosis of brain metastasis. The median interval from the manifestation of brain metastasis to death was 3 months (range 1 day to 11 months). CONCLUSIONS: High-risk factors for brain metastasis in cases of neuroblastoma include bone marrow involvement and a younger age at initial diagnosis. Nevertheless, multiple treatment modalities can improve disease-free survival.


Assuntos
Progressão da Doença , Mortalidade , Metástase Neoplásica , Neuroblastoma , Fatores de Risco , Fatores Etários , Protocolos de Quimioterapia Combinada Antineoplásica , Encéfalo , Neoplasias Encefálicas , Criança , China , Intervalo Livre de Doença , Feminino , Humanos , Incidência , L-Lactato Desidrogenase , Masculino , Recidiva Local de Neoplasia , Estudos Retrospectivos
6.
World J Gastroenterol ; 21(18): 5582-90, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-25987783

RESUMO

AIM: To compare the prognostic ability of inflammation scores for patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE). METHODS: Data of 224 consecutive patients who underwent TACE for unresectable HBV-related HCC from September 2009 to November 2011 were retrieved from a prospective database. The association of inflammation scores with clinicopathologic variables and overall survival (OS) were analyzed, and receiver operating characteristic curves were generated, and the area under the curve (AUC) was calculated to evaluate the discriminatory ability of each inflammation score and staging system, including tumor-node-metastasis, Barcelona Clinic Liver Cancer, and Cancer of the Liver Italian Program (CLIP) scores. RESULTS: The median follow-up period was 390 d, the one-, two-, and three-year OS were 38.4%, 18.3%, and 11.1%, respectively, and the median OS was 390 d. The Glasgow Prognostic Score (GPS), modifed GPS, neutrophil-lymphocyte ratio, and Prognostic Index were associated with OS. The GPS consistently had a higher AUC value at 6 mo (0.702), 12 mo (0.676), and 24 mo (0.687) in comparison with other inflammation scores. CLIP consistently had a higher AUC value at 6 mo (0.656), 12 mo (0.711), and 24 mo (0.721) in comparison with tumor-node-metastasis and Barcelona Clinic Liver Cancer staging systems. Multivariate analysis revealed that alanine aminotransferase, GPS, and CLIP were independent prognostic factors for OS. The combination of GPS and CLIP (AUC = 0.777) was superior to CLIP or GPS alone in prognostic ability for OS. CONCLUSION: The prognostic ability of GPS is superior to other inflammation scores for HCC patients undergoing TACE. Combining GPS and CLIP improved the prognostic power for OS.


Assuntos
Carcinoma Hepatocelular/terapia , Quimioembolização Terapêutica , Técnicas de Apoio para a Decisão , Hepatite B/diagnóstico , Neoplasias Hepáticas/terapia , Neutrófilos , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores/sangue , Proteína C-Reativa/análise , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Distribuição de Qui-Quadrado , Bases de Dados Factuais , Feminino , Hepatite B/complicações , Hepatite B/mortalidade , Humanos , Mediadores da Inflamação/sangue , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Contagem de Plaquetas , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Curva ROC , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Albumina Sérica/análise , Albumina Sérica Humana , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
7.
Neurosci Lett ; 498(3): 227-31, 2011 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-21605627

RESUMO

The neuroprotective effects of Jatrorrhizine from Coptidis Rhizoma against hydrogen peroxide (H(2)O(2))-induced rat pheochromocytoma line PC12 injury and its potential mechanisms were evaluated in the present study. When cells were exposed to H(2)O(2) (200 µM) for 12h, there was a significant reduction in cell survival and activity of antioxidant enzyme (SOD and HO-1) and LDH release. In addition, increased ROS production, declined MMP and increased production of malondialdehyde (MDA) were observed. Preincubation of cells with Jatrorrhizine (0.01-10.0 µM) 24h prior to H(2)O(2) exposure markedly elevated cell viability and activities of antioxidant enzyme (SOD and HO-1), prevented LDH release and lipid peroxidation (MDA) production, attenuated the decrease of MMP and scavenged ROS formation. Jatrorrhizine also attenuated caspase-3 activation of the downstream cascade following ROS. Our results suggest that Jatrorrhizine holds potential for neuroprotective effects against H(2)O(2)-induced injury.


Assuntos
Berberina/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Fármacos Neuroprotetores/farmacologia , Animais , Apoptose/efeitos dos fármacos , Berberina/farmacologia , Caspase 3/metabolismo , Relação Dose-Resposta a Droga , Heme Oxigenase-1/metabolismo , L-Lactato Desidrogenase/metabolismo , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Ratos , Espécies Reativas de Oxigênio , Superóxido Dismutase/metabolismo
8.
Gynecol Oncol ; 121(1): 174-80, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21163514

RESUMO

OBJECTIVE: The study was to investigate the role of EFEMP1 in angiogenesis and growth of cervical carcinoma in vivo. METHODS: Effects of EFEMP1 on proliferation of Hela cells and HUVECs, invasion of Hela cells and migration of HUVECs, and adhesion of Hela cells to HUVECs were evaluated by MTT, Transwell chamber assay and adhesion assay, respectively. EFEMP1 overexpression in Hela cells was achieved by stable EFEMP1 gene transfection into Hela cells by Lipofectamin™ 2000 and the effectiveness of transfection was verified with western-blotting. The effect of EFEMP1 transfection upon the VEGF expression of Hela cells was detected with ELISA. The nude mouse models bearing cervical cancer were established with Hela cells transfected with EFEMP1 gene to observe the role of EFEMP1 in angiogenesis and growth of cervical cancer in vivo. VEGF expression and microvascular density of cervical cancer tissues were detected with immunohistochemistry and CD34 labeling respectively to elucidate the pathway by which EFEMP1 influences the growth of cervical cancer. RESULTS: Proliferation and invasion of Hela cells were promoted by the EFEMP1 protein. The EFEMP1 gene transfection into Hela cells was successful and EFEMP1 gene obtained stable high expression in Hela cells. Compared to the control, the tumors with EFEMP1 overexpression showed a faster growth rate and had a higher level of VEGF expression and microvascular density. EFEMP1 gene transfection elevated the VEGF protein level in Hela cells and EFEMP1 protein facilitated the adhesion of Hela cells to HUVECs. However, no direct effect of EFEMP1 was observed on proliferation, migration and tube formation of HUVECs. CONCLUSIONS: EFEMP1 promoted the angiogenesis and accelerated the growth of cervical carcinoma in vivo through a VEGF up-regulation pathway.


Assuntos
Proteínas da Matriz Extracelular/biossíntese , Neoplasias do Colo do Útero/metabolismo , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Feminino , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteínas Recombinantes/farmacologia , Transfecção , Transplante Heterólogo , Neoplasias do Colo do Útero/irrigação sanguínea , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
9.
Nan Fang Yi Ke Da Xue Xue Bao ; 28(8): 1331-4, 2008 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-18753052

RESUMO

OBJECTIVE: To evaluate the adsorption and desorption of epirubicin (EADM) by carbon-coated iron nanocrystals (CCIN). METHODS: EADM standard curve was generated. After thorough mixture of CCIN and EADM with sonication, the mixture solution was centrifuged at high speed to obtain dissociated EADM for evaluating the adsorption capacity of CCIN. A dialyzer was used to evaluate the desorption of drug-loaded CCIN particles in different media (PBS, normal saline, or distilled water), at different temperatures, and with different quantities of loaded drug. RESULTS: The adsorption of EADM by CCIN presented linear adsorption before saturation and saturation adsorption, with an adsorption saturation point of about 160 microg/mg. The desorption of EADM from CCIN particles was affected by such factors as the extraction media, temperature, and quantity of the loaded drug. Compared to distilled water, PBS and normal saline improved the release rate of EADM from the drug-loaded CCIN particles. Higher temperature also contributed to higher release rate of EADM. Higher release rate of EADM occurred after the CCIN particles adsorbed greater amount of EADM. CONCLUSION: CCIN shows an EADM adsorption pattern of Langmuir isotherm adsorption. Such factors as higher temperature, PBS solution, higher speed of medium replacement, and more drug adsorbed all contribute to a higher release rate of EADM.


Assuntos
Carbono/química , Epirubicina/química , Ferro/química , Nanopartículas/química , Adsorção , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Epirubicina/farmacocinética
10.
Acta Biochim Biophys Sin (Shanghai) ; 38(7): 507-13, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16820867

RESUMO

To study the minimal length required for the secretion of recombinant proteins and silk proteins in posterior silk gland, the signal peptide (SP) of the fibroin heavy chain (FibH) of silkworm Bombyx mori was systematically shortened from the C-terminal. Its effect on the secretion of protein was observed using enhanced green fluorescent protein (EGFP) as a reporter. Secretion of EGFP fusion proteins was examined under fluorescence microscope. FibH SPs with lengths of 20, 18, 16 and 12 a.a. can direct the secretion of the reporter, yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP was shortened to 12 a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP. When 16 a.a. of the FibH SP were used, the secretion of fusion protein was normal and the cleavage site was between the Gly-Ser linker and Met, the starting amino acid of EGFP. These findings are applicable for the expression of foreign proteins in silkworm silk gland. The cleavage site of the SP is discussed and compared with the predictive results of the SignalP 3.0 online prediction program.


Assuntos
Fibroínas/química , Fibroínas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Animais , Baculoviridae/genética , Bombyx/fisiologia , Fibroínas/genética , Fibroínas/isolamento & purificação , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de Proteína
11.
Biochem Biophys Res Commun ; 341(4): 1203-10, 2006 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-16466694

RESUMO

In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.


Assuntos
Bombyx/genética , Fibroínas/genética , Sinais Direcionadores de Proteínas/genética , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Fibroínas/metabolismo , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Acta Biochim Biophys Sin (Shanghai) ; 37(12): 819-25, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16331326

RESUMO

The gene encoding fibroin light chain protein (FibL) is specifically expressed in the posterior silk gland of silkworm and repressed in other tissues. The binding sites of several transcription factors involved in the silk gland transcription specificity of fibl promoter have been recognized, including SGFB, PSGF and BMFA. Here we report the leak expression of the enhanced green fluorescent protein (EGFP) reporter gene in tissues other than the posterior silk gland in vivo when under the control of a shortened fibl promoter with deletion of the 5' terminal 41 bp sequence, which is located at -650 nt to -610 nt upstream of the fibl transcription starting site. Assay of silk gland specificity of the promoters was performed by observation of green fluorescence in tissues of silkworm larvae following inter-haemocoelic injection of recombinant Autographa californica multiple nuclear polyhedrosis virus carrying the EGFP reporter gene controlled by different lengths of fibl promoters. Our results indicated that availability of the binding sites of several known factors, including SGFB, PSGF and BMFA, is not sufficient for intact silk gland transcription specificity of fibl promoter, and there are possible inhibitor binding sites in the 41 bp sequence (-650 nt to -610 nt) upstream of the transcription starting site which may be required to repress the activity of fibl promoter in other tissues.


Assuntos
Bombyx/genética , Fibroínas/genética , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Animais , Sítios de Ligação , Linhagem Celular , Genes Reporter , Larva/citologia , Larva/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-12053199

RESUMO

A gene unit, which encoded fibroin-like peptide, was synthesized and constructed. The unit was multimerized to about 2 400 bp using BamHI and BglII at each end of the unit, then was fused with gfp reporter gene. The fusion gene, flanked by the 5'and 3'sequence of the fibroin heavy chain gene of silkworm Bombyx mori, was transferred into the eggs of silkworm by electroporation. After the silkworms developed and spinned silk, 73 out of about 5 400 cocoons were brighter than normal ones under UV light. The protein extracted from the brighter cocoon could react with the GFP polyclonal antibodies. Genomic DNA from these silkworms and their progenies were analyzed. The integration of gfp gene into genomic DNA of silkworm and the occurrence of expected homologous recombination event had been proved by Southern hybridization. It was shown that gfp-fibroin like fusion gene had integrated into the genomic DNA of silkworm by homologous recombination and the phenotype of "brighter cocoon" could be used to select transgenic silkworms.

14.
Artigo em Inglês | MEDLINE | ID: mdl-12136190

RESUMO

The plasmid pGL2Rz including ribozyme gene was linearized and introduced into early eggs of silkworm (G(0)) by gene gun. The luciferase activity in blood of the G(1) generation was detected, then the resistant silkworm was selected by NPV infection from G(2) generation. The transgenic silkworm resistant against NPV 10 times more than control ones was got at the G(4) generation. PCR and Southern blotting proved that the ribozyme gene was integrated into the genome of silkworm multicopily. The expression of ribozyme was also detected by RT-PCR in pupa. The results showed that the transgenic silkworm strain of anti-NPV ribozyme has been got.

15.
Artigo em Inglês | MEDLINE | ID: mdl-12136199

RESUMO

The replacement of fibroin heavy chain gene in silkworm by site directed homologous recombination was studied The DNA fragment consisting of an IE promoter driving green fluorescent protein (GFP) gene as reporter flanked by pieces of the 5' and 3' sequences of the fibroin heavy chain gene of silkworm at two sides was transferred into silkworm eggs by electroporation Green fluorescent flecks were seen on three silkworms fifth instar among five thousand silkworms under UV light PCR analysis proved that the GFP gene was integrated into the genome of silkworm Southern hybridization of genomic DNA of one transgenic silkworm showed that the fibroin heavy chain gene was successfully knocked out and replaced by the reporter gene The transgenic silkworms could grow up to the fifth instar as normal but they could not spin silk while control ones do.

16.
Artigo em Inglês | MEDLINE | ID: mdl-12142923

RESUMO

Neomycin resistance gene (neo(R)) flanked by 5' and 3' regions of fibroin H-chain gene of silkworm (Bombyx mori.L.) was transferred into eggs of silkworm by gene gun in the early period of fertilization. The larvae were fed with an artificial diet containing neomycin in early 24 hours post transfettion, and some of them survived. The neo(R) encoding sequence in G(2) generation derived from the survivals was detected by Southern blotting. The results indicated that neo(R) could be used as a selective marker for studies on transgenic silkworm.

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