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- Laurie, K. L., et al.
(författare)
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Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector
- 2007
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Ingår i: Gene Therapy. - : Springer Science and Business Media LLC. - 0969-7128 .- 1476-5462. ; 14:23, s. 1623-1631
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Tidskriftsartikel (refereegranskat)abstract
- The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk- E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 30 enhancer sequence from Ig genes. The Igk- E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19(+) cells, but not by CD3(+), CD11b(+), CD11c(+) or Gr-1(+) cells. CD19(+) cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19(+) cells, while eGFP was expressed by both spleen focus forming virus and cytomegalovirus constitutive promoters in CD19(+) and CD3(+) lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.
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| 2. |
- Tiede, Karen, et al.
(författare)
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Considerations for environmental fate and ecotoxicity testing to support environmental risk assessments for engineered nanoparticles.
- 2009
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Ingår i: Journal of chromatography. A. - : Elsevier BV. - 0021-9673. ; 1216:3, s. 503-9
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Tidskriftsartikel (refereegranskat)abstract
- There is an increasing concern over the safety of engineered nanoparticles (ENPs) to humans and the environment and it is likely that the environmental risks of these particles will have to be tested under regulatory schemes such as REACH. Due to their unique properties and the fact that their detection and characterisation in complex matrices is challenging, existing analytical methods and test approaches for assessing environmental risk may not be appropriate for ENPs. In this article we discuss the challenges associated with the testing of ENPs to generate data on persistence, mobility, bioavailability and ecotoxicity in the environment. It is essential that careful consideration is given to the selection of the test material, the test system (including test vessels and study media) and the test exposure conditions. During a study it is critical that not only the concentration of the ENP is determined but also its characteristics (e.g. size, shape, degree of aggregation and dissolution). A range of analytical techniques is available including microscopy-based approaches (e.g transmission and scanning electron microscopy), dynamic light scattering, and size separation approaches (e.g. field flow fractionation and hydrodynamic chromatography) coupled to detection methods such as inductively coupled plasma MS. All of these have their disadvantages: some are unable to distinguish between ENPs and natural interferences; some techniques require sample preparation approaches that can introduce artefacts; and others are complex and time-consuming. A combination of techniques is therefore needed. Our knowledge in this area is still limited, and co-ordinated research is required to gain a better understanding of the factors and processes affecting ENP fate and effects in the environment as well as to develop more usable, robust and sensitive methods for characterisation and detection of ENPs in environmental systems.
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