Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BACKGROUND: Maize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop. RESULTS: In this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression­an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments. CONCLUSIONS: This paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.

Gene stacking as a strategy to confer characteristics of agronomic importance in plants by genetic engineering / Empilhamento gênico como estratégia para conferir características de importância agronômica em plantas via engenharia genética

ABSTRACT: Gene stacking refers to the introduction of two or more transgenes of agronomic interest in the same plant. The main methods for genetically engineering plants with gene stacking involve (i) the simultaneous introduction, by the co-transformation process, and (ii) the sequential introduction of genes using the re-transformation processes or the sexual crossing between separate transgenic events. In general, the choice of the best method varies according to the species of interest and the availability of genetic constructions and preexisting transgenic events. We also present here the use of minichromosome technology as a potential future gene stacking technology. The purpose of this review was to discuss aspects related to the methodology for gene stacking and trait stacking (a gene stacking strategy to combine characteristics of agronomical importance) by genetic engineering. In addition, we presented a list of crops and genes approved commercially that have been used in stacking strategies for combined characteristics and a discussion about the regulatory standards. An increased number of approved and released gene stacking events reached the market in the last decade. Initially, the most common combined characteristics were herbicide tolerance and insect resistance in soybean and maize. Recently, commercially available varieties were released combining these traits with drought tolerance in these commodities. New traits combinations are reaching the farmer's fields, including higher quality, disease resistant and nutritional value improved. In other words, gene stacking is growing as a strategy to contribute to food safety and sustainability.

RESUMO: O empilhamento gênico se refere a introdução de dois ou mais transgenes de interesse agronômico na mesma planta. Os principais métodos de produção de plantas geneticamente modificadas com empilhamento gênico envolvem (i) a introdução simultânea, pelo processo de co-transformação, e (ii) a introdução sequencial de genes, pelos processos de re-transformação ou por cruzamento entre eventos transgênicos. Em geral, a escolha do melhor método varia de acordo com a espécie de interesse e a disponibilidade de construções genéticas e eventos transgênicos preexistentes. Também é apresentado aqui o uso da tecnologia de minicromossomos como tecnologia potencial de empilhamento gênico. O objetivo desta revisão é discutir aspectos relacionados à metodologia para o empilhamento de genes a combinação de características (obtida via empilhamento de genes de interesse agronômico) via engenharia genética. Além de discutir, é apresentado uma lista de culturas e genes aprovados comercialmente que tem sido usado em estratégias de empilhamento e uma discussão sobre normas regulatórias. Um número maior de eventos com empilhamento de genes foi aprovado e liberado no mercado na última década. Inicialmente, a combinação das características de tolerância a herbicidas e resistência a insetos era a mais popular, principalmente em soja e milho. Recentemente, estas características combinadas com tolerância a seca nessas culturas foram liberadas comercialmente. Novas características combinadas estão entrando na lavoura, incluindo aumento da qualidade, resistência a doenças e aumento do valor nutricional. Em outras palavras, o empilhamento gênico está crescendo como tecnologia para contribuir para a segurança alimentar e sustentabilidade.

Construction of human PKD2 lentivirus to rectify polycystin-2 expression and Wnt/β-catenin signaling pathways in the Pkd2-null cell lines / 中华泌尿外科杂志

Objective To establish PKD2 gene recombinant lentivirus and to investigate its restorative effects on polycystin-2 and Wnt/β-catenin signaling pathways in Pkd2-null cell lines.Methods From August 2016 to February 2017,PKD2 gene was cleaved from the pcDNA3.1-TM-PKD2 plasmid and was inserted into the pLV-sfGFP 2A Puro by restriction enzymes Xba Ⅰ and Xho Ⅰ.The recombinant pLV-sfGFP-PKD2 plasmid was sequenced to verify a correct construction.Then we obtained recombinant lentiviruses by co-transfecting 293T cells with recombinant plasmid and packaging plasmids.B3/D3 (Pkd2 +/-) and B2/E8 (Pkd2-/-) cell lines were used to evaluate the effectiveness of lentivirus,they were divided into experimental group,control group,blank group and treated with pLV-sfGFP-PKD2 virus,pLV-sfGFP virus or culture media,respectively.The expression of PC2 was detected by Western blotting and immunofluorescence staining.Finally the cell proliferation was evaluated by detecting of proliferating cell nuclear antigen.The changes of Wnt/β-catenin signaling pathway were evaluated by real-time quantitative PCR.Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-sfGFP-PKD2 was constructed successfully.After infected with pLV-sfGFP-PKD2 virus,the expression of PC2 in the experimental group B2 and E8 cells(0.668 ±0.013,0.763 ±0.021) was restored to the normal level,compared with control group B3 and D3 cells,respectively (0.687 ± 0.015,P =0.164;0.776 ± 0.008,P =0.409).The proliferative activity in experimental group B2 cells(0.573 ±0.010) was significantly lower than that in control group B2 cells (0.848 ±0.031,P ＜0.01),and was returned to the level of blank group B3 cells (0.585 ±0.017,P =0.369).Reexpression of PKD2 in experimental group B2 cells also reduced the expression of Wnt7a,β-catenin,back to blank group B3 cells' level(0.037 ±0.005 vs.0.037 ±0.004,P=0.969;0.554 ±0.008 vs.0.571 ±0.013,P =0.64).Conclusions The recombinant pLV-sfGFP-PKD2 lentivirus has been constructed successfully.The lentivirus could rectify the absence of PC2 in PKD2-null cell lines,by which the hyperactivated Wnt/β-catenin signaling pathway and the abnormal cell proliferation caused by PC2 deficiency could be also restored to normal levels.

Expression and identification of VEGF165 in bone marrow mesenchymal stem cells of rhesus / 天津医药

Objective To detect the transferred vascular endothelial growth factor (VEGF)165 gene expression in rhesus autologous bone marrow mesenchymal stem cells (MSCs), and to explore the functional viability of transgenic MSCs. Methods MSCs from rhesus bone were isolated by Ficoll, which were used to detect the phenotype. After the culturing, the expression vector pcDNA-eGFP-VEGF165 was transfected into bone marrow MSCs. Fluorescence microscope and flow cytometry were used to detect the enhanced green fluorescent protein (eGFP) expression. At the same time, the phenotype in transfected MSCs was also indentified. The VEGF165 expression level was detected by RT-PCR. Results The highly purified MSCs were collected successfully. The transfected MSCs and daughter cells showed expressions of eGFP and VEGF165, which also remained the characteristics of MSCs. Conclusion The VEGF165 gene that is transfected into MSCs can maintain characteristics of MSCs, and stably express foreign genes.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

Detecting adventitious transgenic events in a maize center of diversity

Background: The genetic diversity of maize in Peru includes several landraces (within race clusters) and modern open pollinated and hybrid cultivars that are grown by farmers across various regions, thereby making this country a secondary center of diversity for this crop. A main topic of controversy in recent years refers to the unintended presence of transgenic events in locally grown cultivars at main centers of crop diversity. Peru does not yet have biosafety regulations to control or permit the growing of genetically modified crops. Hence, the aim of this research was to undertake a survey in the valley of Barranca, where there were recent claims of authorized transgenic maize grown in farmers fields as well as in samples taken from feed storage and grain or seed trade centers. Results: A total of 162 maize samples (134 from fields, 15 from local markets, eight from the collecting centers of poultry companies, from the local trading center and four samples from seed markets) were included for a qualitative detection by the polymerase chain reaction (PCR) of Cauliflower Mosaic Virus (CaMV) 35S promoter (P35S) and nopaline synthase terminator (Tnos) sequences, as well as for six transgenic events, namely BT11, NK603, T25, 176, TC1507 and MON810. The 134 maize samples from farmers fields were negative for Cry1Ab delta-endotoxin insecticidal protein and enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) using lateral flow strips. The PCR analysis did not detect any of the six transgenic events in samples from farmers fields, local markets, seed trading shops and the local collecting center. There were four transgenic events (T25, NK603, MON810 and TC1507) in grain samples from the barns of poultry companies. Conclusions: This research could not detect, at the 95 percent probability level, transgenes in farmers' fields in the valley of Barranca. The four transgenic events in grain samples from barns of poultry companies...

Expression of nitric oxide synthase in the Aβ transgenic drosophila / 中国医师杂志

ObjectiveTo investigate the expression and effects of nitric oxide synthase in the Abeta transgenic drosophila.MethodsmRNA was obtained from 20 Abeta transgenic drosophila and 20 wild type drosophila,and cDNA was synthesized by reverse transcription.The expression of nitric oxide synthase mRNA was detected by RT-PCR.ResultsThe expression of nitric oxide synthase mRNA (0.020 ± 0.006)in the Abeta transgenic drosophila was significantly lower than that in the wild type drosophila ( 1.000 ±0.149),there had significant difference between Abeta transgenic drosophila and wild type drosophila ( P ＜ 0.05)..ConclusionsNitric oxide generated by nitric oxide synthase was an important messenger molecule,and it was closely related to Alzheimer's disease.

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector / Transferência de genes heterólogos para células corneanas epiteliais primárias utilizando vetor lentivírus

PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP) reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80 percent confluency) prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS). We evaluated the transduction efficiency (TE) over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells). RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1 percent) and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7 percent. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

OBJETIVO: Avaliar a transferência de genes heterólogos expressando a proteí­na "Green Fluorescent Protein" (GFP) para células corneanas epiteliais primárias ex vivo utilizando vetor lentivírus. MÉTODOS: Tecido corneoescleral de coelhos foi usado para obtenção de suspensão de células corneanas epitelias. As células foram semeadas na densidade de 5×10³ células/cm² e expandidas por 5 dias até uma confluência de 70-80 por cento antes de serem transduzidas. A transferência genética foi monitorada por microscopia fluorescente e por um separador de células ativadas por fluorescência. Foram avaliadas a eficiência de transdução ao longo do tempo e o efeito dose-resposta de diferentes quantidades de partículas virais. Um grupo de células foi analisado pelo separador de células ativadas por fluorescência para avaliar a transdução de células com fenótipo de cé­lulas tronco do epitélio corneano (baseado na exclusão do corante "Hoechst dye"). RESULTADOS: Os vetores lentivírus foram efetivos na transdução de cé­lulas corneanas epiteliais primárias de coelhos ex vivo. Fotodocumentação das células vivas demonstrou células epiteliais de morfologia normal e expressando o gene fluorescente (GFP). A eficiência de transdução ao longo do tempo foi maior no quinto dia após a transdução (14,1 por cento) e demonstrou uma tendência à estabilidade a partir do oitavo dia após a transdução. O número de células transduzidas foi dose-dependente e atingiu 7 por cento com as maiores concentrações de partículas virais. Quando analisadas pelo separador de células ativadas por fluorescência para detecção de células transduzidas e também de células que excluíram o corante "Hoechst dye", foi detectado que células com fenótipo de células tronco do epitélio corneano ("side-population") também foram transduzidas. CONCLUSÕES: Os vetores lentivirais podem transferir genes heterolólogos para células corneanas epiteliais primárias expandidas ex vivo de forma eficiente. Os genes foram expressos de forma estável ao longo do tempo e puderam ser transferidos tanto para células epiteliais maduras como para presumíveis células tronco epiteliais. A eficiência de transdução foi obtida de forma dose-dependente.

Effect of Ad. mTGF-β1-gene transfection on ischemia-reperfusion injury to the cardiac allografts of rat in vitro / 中华胸心血管外科杂志

Objective Isohemia-reperfusion injury oecurred during heart transplantation may result in failure of grafts and the death of receivers perioperatively. Over expression of TGF-β1 in the myocardium therapeutically was shown to be help-ful in limiting the reperfusion injury to the grafts. The study was designed to investigate the role of Ad. mTGF-β1 gene transfec-tion during ischemia-reperfusion injury in vitro after heart transplantation in rats and the possible mechanisms. Methods The model of heterotopic cardiac transplantation was established by Heron's technique with cuff vessel anastomosis. Animals were divided into 3 groups: in group A ( n =6, control group), the donor hearts were perfusod with 6 ml of Stanford University cardio-plegic solution via coronary arteries at 4℃ for about 40 minutes; in group B ( n =6, vector alone group), the donor hearts were perfused with 6 ml of Stanford University cardioplegic solution containing 5 × 10~9 plaque-forming units( pfu)/gram of the vec-tor, and in group C (study group), the donor hearts were perfused with the solution containing 5 × 10~9 pfu/gram vector with mTGF-β1. The donor hearts were observed with an electro-microscope. The expression of mTGF-β1 in the grafts was identified with immunohistochemical staining. Gene products expressed in tissues were quantified by one step RT-PCR. Activities of SOD ,MDA ,MPO in the grafts were measured. Results At 8 hours after transplantation, mTGF-β1 and its expression were de-tected by means of RT-PCR and immunohistochemical staining in the rats of group C. Expression scores of foreign gene were significantly higher in groups A and B. The apoptotic index of the myocardial cells in group C was lower than those in groups A and B. The activity of SOD was higher in group C than those in groups A and B, though the activities of MDA and MPO were decreased. Conclusion The study demonstrated that gent transfer in vitro via coronary artery was effective. Ad. mTGF-β1 gene transfection in vitro ameliorates the myocardial ischemia-reperfusion injury in rats, for which heart transplantation was per formed, increases the activity of SOD, and decreases the activities of MDA, MPO.

Influence of the lentiviral vectors-mediated mouse genetic engineering Treg after allogeneic bone marrow transplantation on graft-versus-hostdiseaseinmice / 中华器官移植杂志

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneie bone marrow transplantation (allo-BMT)on graft-versus-host disease (GVHD) in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) converted CD4~+ CD25~- T cells from Balb/c mice into engineered Tregs in vitro.An allo-BMT model of Balb/c→C57BL/6 mice was established.Mice were randomly assigned into four groups:(1) The recipients in engineering Treg group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenoeytes plus 5×10~6 genetic engineering Treg;(2)The recipients in transplantation control group were iniected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytes;(3) The recipients in radiation group were injected with 0.2 ml RPMI 1640;(4)The recipients in empty vector control group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytas plus 5×10~6 empty vector transduced CD4~+ CD25~- T cells.Survival time,clinical GVHD Score or histopathological analysis(skin,liver and small intestine) were observed after allo-BMT.Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured Results The mean survival times in radiation group, transplantation control group,erIgineering Treg group and empty vector control group were (8.8±0.6),(36.7±2.5),(51.6±4.0) and (34.1±2.3)days respectively.The survival time in engineering Treg group was signiticantly prolonged as compared with other groups as judged by the log-rank test(P<0.05).Histopathological ahalysis in several target organs (skin,liver and small intestine)confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased compared to transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD during allo-BMT in mice

Long-term and stable expression of transgene mediated by piggyBac transposon in gynecological malignant tumor cells / 中华妇产科杂志

Objective To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer.Methods Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK).By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B.The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency.The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR).The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay.The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line.The insertion sites of foreign gene tranferred by PB transposon in genome were analyzed by inverse PCR.Results (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully.(2) Using three different transfective reagents, PB trausposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1 B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7 ± 9.2) %]was higher than that of lipofectamine 2000 [(54.1 ± 11.4) %]and jetPEI [(46.5 ± 7.4) %, all P < 0.05] ; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfeetion efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7 ± 9.2) %, (74.4 ± 8.9) % and (83.2 ± 9.7) % respectively, which all were higher than that on HEC-1B [(39.5 ± 8.7) %, P < 0.05] .(3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines.(4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 μg/ml respectively.Inhibitory effect of GCV (10 μg/ml) on SKOV3 transfected with pPB/TK was (86 ± 9) %, which was superior to that transfected with pORF-HSVtk alone [(52 ± 12)%, P < 0.05] .(5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites, mRFP1 expression still could be detected in three months after transfected.Conclusions PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression.It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.

Influence of the lentiviral vectors-mediated mouse genetic engineering Treg on GVHD and GVL effect after allogeneic bone marrow transplantation in mice / 中华器官移植杂志

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneic bone marrow transplantation (alloBMT) on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) transformed CD4 + CD25- T cells from Balb/c mice into engineered Tregs in vitro. An allo-BMT model of Balb/c→C57BL/6 mice was established. The recipients were given lethal X-ray total body irradiation before transplantation.Mice were randomly assigned into five groups and each group contained 10 recipients: (1) The recipients in radiation group were injected with 0.2 ml RPMI 1640; (2) The recipients in leukemia control group were injected with 5 × 106 donor bone marrow cells and 500 mouse T-cell leukemia/lymphoma cells (EL4 cells); (3) The recipients in transplantation control group were injected with 5 × 106 donor bone marrow cells and 5 × 106 splenocytes plus 500 EL4 cells; (4) The recipients in engineering Treg group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 genetic engineering Treg; (5) The recipients in empty vector control group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 empty vector-transduced CD4+ CD25- T cells. Survival time, clinical GVHD score or histopathological analysis (skin, liver and small intestine) were observed after allo-BMT. Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured. Results The mean survival time in radiation group, leukemia control group, transplantation control group,engineering Treg group and empty vector control group was ( 10. 3 ± 1.5), (20. 7 ± 1.9), (26. 0 ±4.3), (49. 0 ± 17. 7) and (24. 4 ± 4. 1 ) days respectively. The survival time in engineering Treg group was significantly prolonged as compared with other groups as judged by the log-rank test (P＜0. 05).Histopathological analysis in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD or leukemia were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased as compared with transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD without affecting GVL activity during allo-BMT in mice.

Construction of eucaryotic expression plasmid carrying the recombinant rabbit TGF β1 gene and self-induction of rabbit articular synoviocytes into chondrocytes in vitro / 中华创伤杂志

Objective To investigate the experimental methods of transferring the synoviocytes with the reconstructed pcDNA3.1-TGF-β1 gene by the liposomes and study the feasibility of self-induction of synoviocytes to the chondrocytes in vitro so as to provide a scientific and experimental basis for the further gene enhanced tissue engineering research in articular cartilage repair.Methods Synoviocytes were cultivate in vitro and purified to construct the eucaryotic expression plasmid carrying the recombinant rabbit TGF-β1 gene.By means of Lipofectamine 2000,the synoviocytes were transfected with pcDNA3.1-TGF-β1 (experimental group) and with pcDNA3.1 ( + ) blank plasmid (control group).The synoviocytes free from transfection was set as blank group.After 48 hours of transfection,the cells were screened by G418.Representative sections from among the positive clones were used for RT-PCR assay of instant expression of TGF-β1.The other sections were used for immunohistochemical analysis with antibodies to TGF β1.Screening of cells by G418 was continued for 12 days of cell counting and drawing the growth curve.The antigens of TGF-β1 and collagen Ⅱ were examined every week three weeks after transfection.All images were processed by using analysis instrument (Image-Pro Plus V6.0).Statistical analysis was conducted with SPSS 13.0 software package.The difference between groups was tested by using variance ( ANOVA) analysis.Results The transfection efficiency in experimental group was 18% ,with temporary decreased living activity of the transfected cells shown by growth curve.The cell population decreased to 3.6 × 104/ml four days after transfection.After 72 hours of transfection,the positive fragment of TGF-β1 was detected by RT-PCR assay,and immunohistochemical staining of antibiotics to TGF-β1 showed positive particles only in the experimental group.After three weeks of tranfection,the immunohistochemical analysis with antibodies to TGF-β1 and type Ⅱ collagen showed that there were positive particles in the transfected cells in the experimental group,with no positive particle in the control and blank groups.According to the results of Image-Pro Plus V6.0,PU value of anti-TGF-β1 was (19.04±1.26) seven days after transfection,while that of control and blank groups were (4.07 ± 0.65)and (3.23 ±0.56) respectively,with statistical difference (P<0.05).PU value of anti-type Ⅱ collagen in experimental group,control group and blank group was (13.74 ± 1.27),(4.62 ±0.56) and (3.93 ± 0.38) 14 days after transfection,with statistical difference ( P < 0.05).Conclusions Transfection of the rabbit articular synoviocytes with the reconstructed pcDNA3.1-TGF-β1 gene can be successfully accomplished by using Lipofectamine 2000 method.The transfected synoviocytes can express TGF-β1 and excrete type Ⅱ collagen,have chondrogenic potential when transfected by pcDNA3.1-TGF-β1 gene and can be used as candidate cells for repair of articular cartilage.

Inhibitory effect of IGF-1 gene on motoneurons apoptosis in anterior horn after acute spinal cord injury in adult rats / 吉林大学学报(医学版)

Objective To identify the effect of insulin-like growth factor-1(IGF-1) gene transfer on motoneurons apoptosis in anterior horn after acute spinal cord injury in adult rats.Methods Thirty-six male Wistar rats were randomly divided into 3 groups: IGF-1 group,model group and control group.The SCI models were made by cutting half spinal cord of rats on the T_8 segment by ophthalmogy scissors.Lipofectamine and recombinant plasmid pcDNA-hIGF-1 complexes were injected into the injury spinal cord by microinjector in IGF-1 group while pcDNA3.1,LipfectAmine and saline were administered in model group.The expression of IGF1 was detected by immunohistochemistry method,the apoptosis of the motoneurons in anterior horn was detected by TUNEL,and moting function of hindlimbs was evaluated by BBB locomotor scale.Results Compared with model group and control group,the expression of IGF-1 was increased significantly(P

Detection of Segregation of Foreign Genes in Transgenic Wheat Lines by Multiplex Polymerase Chain Reaction / 中国生物工程杂志

The method of multiplex PCR was set up to identify two or three transgenes in one reaction such as uidA and bar; uidA and IDx5 or uidA, bar and 1Dx5 genes. Three sets of primer pairs which was specific to each of these three genes respectively were designed and synthesized. Recombinant plasmids pAHC25 and p1Dx5 harboring uidA + bar and 1Dx5 gene separately were used as template DNA in the process of optimizing an multiplex PCR reaction. The optimal annealing temperature for uidA and bar MPCR is range from 57. 1℃-62. 3℃ , for uidA and 1Dx5 is range from 60℃ to 60. 6℃ , and for uidA、bar and 1Dx5 range from 57. 0℃-58. 4℃. The amount of template for MPCR is twice as much as that for simplex PCR, while the concentration of primers is the same with simplex one. Less than 50bp MPCR products can be separated clearly by 10% non-denaturalized polyacrylamid gel electrophoresis. Fourteen transgenic wheat lines were tested by multiplex and simplex PCR respectively, which shows the same results and hence presents that MPCR is the reliable, rapid and high-effective approach to detect foreign genes from transgenic plant.

The use of bioluminescent image in monitoring islet graft in vivo after islet transplantation / 中华器官移植杂志

Objective To demonstrate the superiority and feasibility of using bioluminescent image to monitor the islet graft after islet transplantation.Methods Diabetic models were established by intraperitoneal injection of streptomycin into mature male C57BL/6 mice.Islets were harvested from the pancreas of C57BL/6 and Bclb/c mice by digestion and purification,and transfected with Lueiferase gene.The mouse diabetic models were divided into iso-transplantaion group (n=20) and allo-transplantation group (n=7).The islets of C57BL/6 were transplanted into iso-transplantaion mice with different doses (10,50,110 and 200,n=5 in every dose),and Bclb/c mouse islets were transplanted into allo-transplantation group.The islets were transplanted into the subcutaneous fat tis- sue near left scapula.The receptor mice were scanned with CCD camera to get bioluminescent images at different scheduled time points,and the changes in random blood glucose of allo-transplantation group were observed.Results On day 6 after transplantation,the scanning image showed that the pi- xel intensity from the region of interest (ROI) was increased with the increased number of islet grafts and they had a positive correlation.The random blood glucose was reduced to the normal level in the first 2 days,and then increased again to the diabetic level on 11 days averagely,while pixel intensity from the ROI reached the peak on day 6-7,and then reduced rapidly after islet transplantation in allo- transplantation group.The beginning of pixel intensity reduction occurred on day (6.14?0.90), while that of the random blood glucose raise occurred on day (10.00?0.82) after transplantation,and the former alteration occurred obviously earlier than the latter (P

In vivo Recombinant Adenovirus-mediated p53 Gene Therapy in a Syngeneic Rat Model for Colorectal Cancer

The p53 gene has a significant role in controlling genomic stability of cancer. The purpose of this study was to evaluate the tumor response of allograft colorectal tumor treated with Ad5CMV-p53 in a syngeneic rat model. Two weeks after the inoculation of WB-2054-M5 tumor cells in the flank of rats, rats were randomly assigned by tumor size to one of three groups (n=18 in each): phosphate buffered saline (PBS), Ad5CMV, and Ad5CMV-p53. Recombinant adenovirus or PBS was administered through intratumoral injection at three divided doses every other day for 4 weeks. Apoptosis of the tumors was evaluated using TUNEL assay. After 2 and 4 weeks of treatment, the volume (cm3) of tumors in PBS, Ad5CMV, and Ad5CMV-p53 was as follows: 2 week: 1.66 +/-0.43, 1.40 +/-0.47, 0.75 +/-0.26 (p<0.001), 4 week: 4.41 +/-0.88, 3.93 +/-1.86, 2.33 +/-0.51 (p<0.001). Tumor growth showed no statistically significant difference between the PBS and Ad5CMV groups (6-week vol. p=0.32). The TUNEL assay results revealed more apparent apoptotic cells in Ad5CMV-p53-treated tumors than in other groups. Growth of allograft colorectal cancer in the syngeneic rat model was significantly suppressed by intratumoral Ad5CMV-p53 gene therapy. These results demonstrate that gene replacement therapy with p53 may provide a novel modality of treatment in conjunction with other present treatments for metastatic colorectal cancer.

Effects of human DAF and CD59 co-expression in vascular endothelia in transgenic mice on heart work during human plasma peffasion / 中华器官移植杂志

Objective To construct transgenic mice tissue-specifically co-expressing human DAF/CD59 in the vascular endothelia and to investigate the ability to protect against human comple- ment-mediated attack.Methods Transgenic mice were generated by co-microinjection of hDAF/CD59 expression constructs driven by the human intercellular adhesion molecule-2(ICAM-2)promoter.PCR and Southern blot of genomic DNA were used to assess the presence of hDAF/CD59 in the genome of the founders,and the expression at protein level was measured by flow cytometry.Immunohistochemis- try was used to detect the distribution of hDAF/CD59.An ex vivo perfusion model was used to com- pare hearts from these hDAF/CD59 transgenic mice with hDAF hearts.Results After microinjection of genes,11 of 135 mice born were shown to be double-transgenic,and human DAF/CD59 were ex- pressed on the surface of leucocytes in 6 of the 11 DAF/CD59-integrated mice.Expression levels of 6 founders ranged from 80% to 95% of that in human leucocytes.Human DAF/CD59 were strongly expressed in the vascular endothelia of heart,kidney,with little or no positive staining observed in non- endothelial cells.Compared to hDAF hearts that maintained approximately 20% maximum work during perfusion with 20% human plasma,these endothelial-specific hDAF/CD59 hearts were further protected with work maintained at 40% of the maximum level during 60 min.Conclusion The intro- duced hDAF/CD59 genes have been integrated and specifically expressed in the vascular endothelia. The endothelial co-expression of hDAF/CD59 can provide greater protection against human complement-mediated attack than the expression of hDAF alone.

An experimental study on the role of CTLA4Ig gene transfected DCs in the induction of immune tolerance / 中华器官移植杂志

Objective To investigate the possibility of DCs transfected with CTLA4Ig cDNA by retrovirus vector to induce antigen specific hyporesponsiveness.Methods The modified DCs(CTLA4Ig-DCs) were prepared by transferring the DCs from cultured rat BM cells with the constructed retro-virus CTLA4Ig vector.The CTLA4Ig gene expression was detected on the prepared DCs by RT-PCR and Dot-ELISA methods.The influence of the modified DCs on mixed lymphocyte reaction(MLR) intensity was determined by T cell proliferation.Results The CTLA4Ig gene could be transferred successfully to DCs by retrovirus vector,which was confirmed by RT-PCR and Dot-ELISA methods.As compared with control group,DCsRev could significantly and antigen-specifically inhibit MLR in vitro in a dose-and time-dependent manner.The number of DCRev from 10~(3) to 10~(4) could reach the maximal inhibition by(69.12 %).On the other hand,the inhibition capacity of DCsRev was increased from(48 h) to 12 h prior to adding stimulating cells and the maximal inhibition was(98.3 %) at(12 h).Analysis of T cell proliferation revealed that donor-specific inhibition could be induced by DCsRev in an ex-vivo model.But this kind of inhibition was not lifetime.Conclusions The CTLA4Ig gene could be transferred successfully to DCs by retrovirus vector.This kind of DCs lost capacity of stimulating MLR,and could inhibit T cell proliferation,which might be responsible for the antigen-specific suppression induced by DCsRev.

Effect of intraspinal grafting of adenovirus-mediated brain derived neurotrophic factor ex vivo transgene myoblasts cells and methylprednisolone on expression of caspase-3 after spinal cord injury / 中华创伤杂志

Objective To investigate the effect of intraspinal grafting of brain derived neurotrophic factor (BDNF) ex vivo transgene myoblasts cells and methylprednisolone on caspase-3 expression after spinal cord injury (SCI). Methods A total of 120 experimental rats were divided into Group A (spinal cord contusion injury group), Group B (grafting of BDNF ex vivo transgene myoblasts cells group), Group C (methylprednisolone intravenous injection group) and Group D (grafting of BDNF ex vivo transgene myoblasts cells and methylprednisolone intravenous injection group). At days 1, 3, 7, 14 and 28 respectively after SCI, the expression of caspase-3 was measured immunohistochemically for quantitative analysis via a computer image analysis system. The motion functional recovery of the rats was observed by praxiologic and electrophysiologic examination. Results Positive expression cells of caspase-3 were found in all groups, with number from the highest to the lowest in order of Group A, Group B, Group C and Group D (P