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Xian-Yang Zhang

Publications and source records attributed to Xian-Yang Zhang.

7 recordsLinked to original sources
PubMed2005

Improved tagging strategy for protein identification in mammalian cells.

BACKGROUND: The tagging strategy enables full-length endogenous proteins in mammalian cells to be expressed as green fluorescent fusion proteins from their authentic promoters. RESULTS: We describe improved genetic tools to facilitate protein tagging in mammalian cells based on a mobile genetic element that harbors an artificial exon encoding a protein tag. Insertion of the artificial exon within introns of cellular genes results in expression of hybrid proteins consisting of the tag sequence fused in-frame to sequences of a cellular protein. We have used lentiviral vectors to stably introduce enhanced green fluorescent protein (EGFP) tags into expressed genes in target cells. The data obtained indicate that this strategy leads to bona fide tripartite fusion proteins and that the EGFP tag did not affect the subcellular localization of such proteins. CONCLUSION: The tools presented here have the potential for protein discovery, and subsequent investigation of their subcellular distribution and role(s) under defined physiological conditions, as well as for protein purification and protein-protein interaction studies.

Animals↗
PubMed2005

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases.

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.

Animals↗
PubMed2005

Altering the tropism of lentiviral vectors through pseudotyping.

The host range of retroviral vectors including lentiviral vectors can be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses. Such particles possess the tropism of the virus from which the GP was derived. For example, to exploit the natural neural tropism of rabies virus, vectors designed to target the central nervous system have been pseudotyped using rabies virus-derived GPs. Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Pseudotypes involving VSV-G have become effectively the standard for evaluating the efficiency of other pseudotypes. This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity. Particular attention is paid to publications of successfully targeting a specific organ or cell types.

Animals↗
PubMed2004

Stable transgene expression in tumors and metastases after transduction with lentiviral vectors based on human immunodeficiency virus type 1.

The relatively low efficiency of target cell transduction and variations in the stability of transgene expression by retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are major impediments to the use of such vectors in cancer gene therapy approaches. The present study was designed to investigate the stability and efficiency of transgene expression in human lung and breast cancer cell lines transduced with vectors based on human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo in nude mouse models of metastasis. H460 lung carcinoma cells and MDA-MB-231 breast carcinoma cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-Gal), respectively. Transduced H460 cells were administered to nude mice by either intravenous or subcutaneous injection and MDA-MB-231 cells were implanted orthotopically into the mammary fat pad of such mice to induce primary tumor and metastatic lung tumor formation. High-level EGFP expression was maintained in transduced H460 cells in metastatic lung nodules for up to 6 weeks and transgene expression in vitro persisted for at least 23 days after retrieval of EGFP-positive H460 cells from the lungs of tumor-bearing mice and subsequent cultivation in vitro. Likewise, beta-Gal expression levels in metastatic MDA-MB-231 cells in lungs remained high for up to 11 weeks. Southern blot analyses carried out with DNA from lung nodules showed that proviral DNAs in H460 cells were maintained stably over many cell generations and during subsequent reimplantation in vivo. However, molecular analyses revealed variations in transgene copy numbers and expression levels among individual lung clones. These results demonstrate the usefulness of HIV-1-based lentiviral vectors for sustained and stable transgene expression in human lung and breast cancer cell lines in vitro and in vivo.

Animals↗
PubMed2004

Transduction of bone-marrow-derived mesenchymal stem cells by using lentivirus vectors pseudotyped with modified RD114 envelope glycoproteins.

Bone-marrow-derived mesenchymal stem cells (MSCs) have attracted considerable attention as tools for the systemic delivery of therapeutic proteins in vivo, and the ability to efficiently transfer genes of interest into such cells would create a number of therapeutic opportunities. We have designed and tested a series of human immunodeficiency virus type 1 (HIV-1)-based vectors and vectors based on the oncogenic murine stem cell virus to deliver and express transgenes in human MSCs. These vectors were pseudotyped with either the vesicular stomatitis virus G (VSV-G) glycoprotein (GP) or the feline endogenous virus RD114 envelope GP. Transduction efficiencies and transgene expression levels in MSCs were analyzed by quantitative flow cytometry and quantitative real-time PCR. While transduction efficiencies with virus particles pseudotyped with the VSV-G GP were found to be high, RD114 pseudotypes revealed transduction efficiencies that were 1 to 2 orders of magnitude below those observed with VSV-G pseudotypes. However, chimeric RD114 GPs, with the transmembrane and extracellular domains fused to the cytoplasmic domain derived from the amphotropic Moloney murine leukemia virus 4070A GP, revealed about 15-fold higher titers relative to the unmodified RD114 GP. The transduction efficiencies in human MSCs of HIV-1-based vectors pseudotyped with the chimeric RD114 GP were similar to those obtained with HIV-1 vectors pseudotyped with the VSV-G GP. Our results also indicate that RD114 pseudotypes were less toxic than VSV-G pseudotypes in human MSC progenitor assays. Taken together, these results suggest that lentivirus pseudotypes bearing alternative Env GPs provide efficient tools for ex vivo modification of human MSCs.

Animals↗
PubMed2002

Lentiviral vectors for sustained transgene expression in human bone marrow-derived stromal cells.

Bone marrow-derived mesenchymal stromal cells (MSCs) have attracted attention as potential platforms for the systemic delivery of therapeutic proteins in vivo following gene transfer using oncogenic retroviruses. However, the major limitations of this strategy include low levels of gene transfer and a general lack of long-term transgene expression. We have investigated the expression of several transgenes in MSCs following HIV-1 lentiviral vector-mediated gene transfer. Vectors containing a variety of strong promoters driving enhanced green fluorescence protein (EGFP) and coral (Discosoma sp.)-derived red fluorescent protein (DsRed) reporter genes pseudotyped with the vesicular stomatitis virus-G (VSV-G) glycoprotein were able to transduce cultured MSCs with high efficiency. Transduction efficiencies and transgene expression levels in MSCs were found to be higher with lentiviral vectors than with a vector based on the murine stem cell virus pseudotyped with VSV-G. Transgene expression was maintained in culture for at least 5 months. HIV-1-based lentiviral vectors were able to transduce clonogenic mesenchymal progenitor cells, which were capable of maintaining transgene expression by their MSC progeny, over several cell divisions and during differentiation into adipocytes, indicating that terminal adipocyte cell differentiation was unaffected by lentivirus-mediated reporter gene transfer. Collectively these results suggest that lentivirus-mediated gene transfer strategies provide an efficient tool for ex vivo modification of MSCs that does not interfere with differentiation.

Bone Marrow Cells↗