We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein （EGFP） was expressed by using third-generation self-inactive HIV-based lentiviral vectors....We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein （EGFP） was expressed by using third-generation self-inactive HIV-based lentiviral vectors. In our experiments, lentiviruses were injected into 204 fertilized eggs, from which 30 （15%） chickens were hatched. The exogenous gene was detected in the genomes of 16 out of 30 （53%） chickens. The green fluorescence signal was observed directly in various body parts, and was particularly significant in the testes. The transgenes were also found in the offspring of these chickens. The results indicate that HIV-based lentiviral vectors can be used to generate transgenic birds economically and effectively [ Current Zoology 55 （5）： 383 - 387,2009].展开更多
This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and id...This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.展开更多
Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission effici...Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.展开更多
Objective:To construct a recombinant lentiviral vector that co-express ZsGreen and BDNF and establish an Adipose derived stem cell (ADSC) line with stable BDNF expression down- regulation.Methods: BDNF were designed a...Objective:To construct a recombinant lentiviral vector that co-express ZsGreen and BDNF and establish an Adipose derived stem cell (ADSC) line with stable BDNF expression down- regulation.Methods: BDNF were designed and inserted into the lentiviral vector p HBLV-CMVIE-Zs Green-Puro. After identification by DNA sequencing, the lentiviral vectors carrying BDNF were packaged in 293 cells. The transfection efficiency was observed under fluorescence microscope;the lentiviral particles were collected to infect mouse ADSCs, and BDNF level were assessed using real-time PCR and Western blotting.Results: DNA sequencing demonstrated successful construction of the BDNF lentivirus vectors. Real- time PCR and Western blotting showed a high level BDNF mRNA and protein.Conclusion: We have successfully established an ADSC model with stable BDNF expression which establish the basis for the potential application of ADSCs-BDNF in the treatment of AD.展开更多
This was a single-arm,multicenter,open-label phase I trial.Lentiviral vectors(LV)carrying the ABCD1 gene(LV-ABCD1)was directly injected into the brain of patients with childhood cerebral adrenoleukodystrophy(CCALD),an...This was a single-arm,multicenter,open-label phase I trial.Lentiviral vectors(LV)carrying the ABCD1 gene(LV-ABCD1)was directly injected into the brain of patients with childhood cerebral adrenoleukodystrophy(CCALD),and multi-site injection was performed.The injection dose increased from 200 to 1600 lL(vector titer:1×10^(9) transduction units per mL(TU/mL)),and the average dose per kilogram body weight ranges from 8 to 63.6 lL/kg.The primary endpoint was safety,dose-exploration and immunogenicity and the secondary endpoint was initial evaluation of efficacy and the expression of ABCD1 protein.A total of 7 patients participated in this phase I study and were followed for 1 year.No injectionrelated serious adverse event or death occurred.Common adverse events associated with the injection were irritability(71%,5/7)and fever(37.2-38.5℃,57%,4/7).Adverse events were mild and selflimited,or resolved within 3 d of symptomatic treatment.The maximal tolerable dose is 1600 lL.In 5 cases(83.3%,5/6),no lentivirus associated antibodies were detected.The overall survival at 1-year was 100%.The ABCD1 protein expression was detected in neutrophils,monocytes and lymphocytes.This study suggests that the intracerebral injection of LV-ABCD1 for CCALD is safe and can achieve successful LV transduction in vivo;even the maximal dose did not increase the risk of adverse events.Furthermore,the direct LV-ABCD1 injection displayed low immunogenicity.In addition,the effectiveness of intracerebral LV-ABCD1 injection has been preliminarily demonstrated while further investigation is needed.This study has been registered in the Chinese Clinical Trial Registry(https://www.chictr.org.cn/,registration number:ChiCTR1900026649).展开更多
In this editorial we comment on the article by Wei et al,published in the recent issue of the World Journal of Clinical Oncology.The authors investigated the role of Transmembrane 9 superfamily member 1(TM9SF1)protein...In this editorial we comment on the article by Wei et al,published in the recent issue of the World Journal of Clinical Oncology.The authors investigated the role of Transmembrane 9 superfamily member 1(TM9SF1)protein in bladder cancer(BC)carcinogenesis.Lentiviral vectors were used to achieve silencing or overexpression of TM9SF1 gene in three BC cell lines.These cell lines were then subject to cell counting kit 8,wound-healing assay,transwell assay,and flow cytometry.Proliferation,migration,and invasion of BC cells were increased in cell lines subjected to TM9SF1 overexpression.TM9SF1 silencing inhibited proliferation,migration and invasion of BC cells.The authors conclude that TM9SF1 may be an oncogene in bladder cancer pathogenesis.展开更多
目的构建过表达内质网应激标志蛋白葡萄糖调节蛋白78(Glucose-regulated protein 78,GRP78)的人乳腺癌细胞系HS578T、MDA-MB-231,为探讨GRP78在乳腺癌发生发展中的作用机制提供基础。方法采用分子克隆技术构建表达GRP78的慢病毒载体pCDH...目的构建过表达内质网应激标志蛋白葡萄糖调节蛋白78(Glucose-regulated protein 78,GRP78)的人乳腺癌细胞系HS578T、MDA-MB-231,为探讨GRP78在乳腺癌发生发展中的作用机制提供基础。方法采用分子克隆技术构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒,采用PCR法扩增质粒,后使用EcoRⅠ、NotⅠ限制性内切酶切割质粒,对酶切产物进行测序鉴定,成功构建pCDH-CMV-GRP78-HA-EF1-Puro质粒。取对数生长期293FT细胞系(克隆分离株),在培养皿中加入pCDH-CMV-GRP78-HA-EF1-Puro混合液,培养48 h收集含GRP78慢病毒颗粒的上清液,保存备用。另取对数生长期人乳腺癌细胞系HS578T及MDA-MB-231,置于含GRP78慢病毒颗粒上清液的培养基中培养,培养48 h时在培养基中加入1∶500稀释嘌呤霉素,筛选GRP78过表达的HS578T、MDA-MB-231细胞系。加入嘌呤霉素培养48 h采用Western Blotting法检测HS578T、MDA-MB-231细胞GRP78。采用激光共聚焦显微镜对HS578T、MDA-MB-231细胞中GRP78蛋白进行定位。结果构建表达GRP78的慢病毒载体pCDH-CMV-GRP78-HA-EF1-Puro质粒。GRP78过表达的HS578T、MDA-MB-231细胞系在78 kD左右可见明显GPR78蛋白电泳条带。HS578T细胞系中存在GRP78蛋白表达,主要分布在细胞质中。结论成功构建GRP78过表达的MDA-MB-231、HS578T细胞系,GRP78主要表达于细胞质中。展开更多
基金supported by National High Technology Research and Development Program of China(863 Key Program,No.2007AA100504)Anhui Natural Science Foundation(No.050410201)
文摘We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein （EGFP） was expressed by using third-generation self-inactive HIV-based lentiviral vectors. In our experiments, lentiviruses were injected into 204 fertilized eggs, from which 30 （15%） chickens were hatched. The exogenous gene was detected in the genomes of 16 out of 30 （53%） chickens. The green fluorescence signal was observed directly in various body parts, and was particularly significant in the testes. The transgenes were also found in the offspring of these chickens. The results indicate that HIV-based lentiviral vectors can be used to generate transgenic birds economically and effectively [ Current Zoology 55 （5）： 383 - 387,2009].
文摘This study examined the differentiation character and pluripotency of mesenchymal stem cells (MSCs) under different conditions. Adult MSCs were initially isolated from the bone marrow of rats, cultured in vitro and identified by flow cytometry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was detected by RT-PCR analysis. Then MSCs were transfected with lenti- viral vectors pGC-FU-Sox9-EGFP. Enhanced green fluorescence protein (EGFP) expression and trans- fection efficiency were determined by fluorescence microscopy. The results demonstrated that EGFP caused no effect on the multilineage potential of adult MSCs. Sox9 gene expression of high level was maintained stable in the transfected MSCs and induced MSCs to differentiate into chondrocytes. Ag- gracan was positive in chondrogenic lineages and the expression of aggracan and type Ⅱ collagenwas significantly increased during MSCs chondrogenic differentiation. It was concluded that Sox9 gene-modified adult MSCs may be promising candidate cells for further studies on tissue engineering. EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.
基金the National Transgenic Breeding Project of China(2016ZX08009003006)National Natural Science Foundation of China(31672411)Discipline Innovative Engineering Plan(B12008)。
文摘Recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. However, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. In this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells(PGCs) to generate transgenic chickens. The lentiviral vectors were pseudotyped with a modified Sindbis virus envelope protein(termed M168) and conjugated with an antibody specific to PGC membrane proteins. We demonstrated that these optimized M168-pseudotyped lentiviral vectors conjugated with SSEA4 antibodies successfully targeted transduction of PGCs in vitro and in vivo. Compared with the control, 50.0%–66.7% of chicken embryos expressed green fluorescent protein(GFP) in gonads transduced by the M168-pseudotyped lentivirus. This improved the targeted transduction efficiency by 30.0%–46.7%. Efficient chimerism of exogenous genes was also observed. This targeting technology could improve the efficiency of germ line transmission and provide greater opportunities for transgenic poultry studies.
基金The National Natural Science Foundation of China.Project No:(81660224,81100246)Hainan Provincial Key Research and Development Project.Project No:(ZDYF2017120,ZDYF2016110)+2 种基金National College’s Students’Innovation and Entrepreneurship Training Program Project.Project No:(201711810010,HYCX2016010,HYCX2018032)Hainan Provincial Health and Family Planning Industry Research Project.Project No:(18A200115)Haikou People’s Hospital Project.Project No:(2018-SNP-01-003).
文摘Objective:To construct a recombinant lentiviral vector that co-express ZsGreen and BDNF and establish an Adipose derived stem cell (ADSC) line with stable BDNF expression down- regulation.Methods: BDNF were designed and inserted into the lentiviral vector p HBLV-CMVIE-Zs Green-Puro. After identification by DNA sequencing, the lentiviral vectors carrying BDNF were packaged in 293 cells. The transfection efficiency was observed under fluorescence microscope;the lentiviral particles were collected to infect mouse ADSCs, and BDNF level were assessed using real-time PCR and Western blotting.Results: DNA sequencing demonstrated successful construction of the BDNF lentivirus vectors. Real- time PCR and Western blotting showed a high level BDNF mRNA and protein.Conclusion: We have successfully established an ADSC model with stable BDNF expression which establish the basis for the potential application of ADSCs-BDNF in the treatment of AD.
基金supported by the Capital’s Funds for Health Improvement and Research(2022-1-5081)the National Key Research and Development Program of China(2023YFC2706304)+1 种基金Shenzhen Geno-Immune Medical InstituteBeijing Meikang Biotechnology Co.,LTD.
文摘This was a single-arm,multicenter,open-label phase I trial.Lentiviral vectors(LV)carrying the ABCD1 gene(LV-ABCD1)was directly injected into the brain of patients with childhood cerebral adrenoleukodystrophy(CCALD),and multi-site injection was performed.The injection dose increased from 200 to 1600 lL(vector titer:1×10^(9) transduction units per mL(TU/mL)),and the average dose per kilogram body weight ranges from 8 to 63.6 lL/kg.The primary endpoint was safety,dose-exploration and immunogenicity and the secondary endpoint was initial evaluation of efficacy and the expression of ABCD1 protein.A total of 7 patients participated in this phase I study and were followed for 1 year.No injectionrelated serious adverse event or death occurred.Common adverse events associated with the injection were irritability(71%,5/7)and fever(37.2-38.5℃,57%,4/7).Adverse events were mild and selflimited,or resolved within 3 d of symptomatic treatment.The maximal tolerable dose is 1600 lL.In 5 cases(83.3%,5/6),no lentivirus associated antibodies were detected.The overall survival at 1-year was 100%.The ABCD1 protein expression was detected in neutrophils,monocytes and lymphocytes.This study suggests that the intracerebral injection of LV-ABCD1 for CCALD is safe and can achieve successful LV transduction in vivo;even the maximal dose did not increase the risk of adverse events.Furthermore,the direct LV-ABCD1 injection displayed low immunogenicity.In addition,the effectiveness of intracerebral LV-ABCD1 injection has been preliminarily demonstrated while further investigation is needed.This study has been registered in the Chinese Clinical Trial Registry(https://www.chictr.org.cn/,registration number:ChiCTR1900026649).
文摘In this editorial we comment on the article by Wei et al,published in the recent issue of the World Journal of Clinical Oncology.The authors investigated the role of Transmembrane 9 superfamily member 1(TM9SF1)protein in bladder cancer(BC)carcinogenesis.Lentiviral vectors were used to achieve silencing or overexpression of TM9SF1 gene in three BC cell lines.These cell lines were then subject to cell counting kit 8,wound-healing assay,transwell assay,and flow cytometry.Proliferation,migration,and invasion of BC cells were increased in cell lines subjected to TM9SF1 overexpression.TM9SF1 silencing inhibited proliferation,migration and invasion of BC cells.The authors conclude that TM9SF1 may be an oncogene in bladder cancer pathogenesis.
