Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for p...Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.展开更多
The ability of human embryonic stem cells(hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valu...The ability of human embryonic stem cells(hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are power-ful tools for tracking cell types of interest in differentiated cell populations.Here,we describe the construction of modular lentivectors containing different tissue-specific promoters(Tα1 ofα-tubulin;aP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla greenfluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to be traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.展开更多
基金National Institute of Health (S11 NS43499)RCMI (G12RR/AI03061, USA. )
文摘Lentiviral vectors have drawn considerable attention recently and show great promise to become important delivery vehicles for future gene transfer manipulation. In the present study we have optimized a protocol for preparation of human immunodeficiency virus type-1 (HIV-1)-based defective lentiviral vectors (DLV) and characterized these vectors in terms of their transduction of different cells. Transient co-transfection of 293T packaging cells with DNA plasmids encoding lentiviral vector constituents resulted in production of high-titer DLV (0.5-1.2 × 107IU/mL), which can be further concentrated over 100-fold through a single step ultracentrifugation. These vectors were capable of transducing a variety of cells from both primate and non-primate sources and high transduction efficiency was achieved using concentrated vectors. Assessment of potential generation of RCV revealed no detection of infection by infectious particles in DLV-transduced CEM, SupT-1 and MT-2 cells. Long-term culture of transduced cells showed a stable expression of transgenes without apparent alteration in cellular morphology and growth kinetics. Vector mobilization to untransduced cells mediated by wild-type HIV-1 infection was confirmed in this test. Challenge of transduced human T-lymphocytes with wild-type HIV-1 showed these cells are totally resistant to the viral infection. Considering the effective gene transfer and stable gene expression, safety and anti-HIV activity, these DLV vectors warrant further exploration for their potential use as a gene transfer vehicle in the development of gene therapy protocols.
文摘The ability of human embryonic stem cells(hESCs)to undergo indefinite self-renewal in vitro and to produce lineages derived from all three embryonic germ layers both in vitro and in vivo makes such cells extremely valuable in both clinical and research settings.However,the generation of specialized cell lineages from a mixture of differentiated hESCs remains technically difficult.Tissue specific promoter-driven reporter genes are power-ful tools for tracking cell types of interest in differentiated cell populations.Here,we describe the construction of modular lentivectors containing different tissue-specific promoters(Tα1 ofα-tubulin;aP2 of adipocyte Protein 2;and AFP of alpha fetoprotein)driving expression of humanized Renilla greenfluorescent protein(hrGFP).To this end,we used MultiSite gateway technology and employed the novel vectors to successfully monitor hESC differentiation.We present a versatile method permitting target cells to be traced.Our system will facilitate research in developmental biology,transplantation,and in vivo stem cell tracking.
