High efficiency polyoma DNA transfection of chloroquine treated cells

Luthman, Holger; Magnusson, Göran

doi:10.1093/nar/11.5.1295

Cited by 693 publications

(312 citation statements)

References 28 publications

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“…We found that TAT-pK-mediated gene transfer is affected by either of these agents, suggesting that transduction relies on the endocytic pathway. These results are similar to past reports showing that gene transfer via receptor-mediated endocytosis (Cotten et al, 1990) or mediated by DEAE-dextran (Luthman and Magnusson, 1983) is markedly enhanced with endosomotrophic agents, such as chloroquine. However, our data contradict other studies on TAT-peptide-mediated protein transduction (Mann and Frankel, 1991;Derossi et al, 1996;Vivès et al, 1997;Elliott and O'Hare, 1997) and TAT-phage-mediated gene transfer (Eguchi et al, 2001) that do not depend on endosomotrophic reagents.…”

Section: Discussionsupporting

confidence: 92%

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

et al. 2004

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Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.

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Section: Discussionsupporting

confidence: 92%

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

et al. 2004

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“…Activity Assays All expression constructs were transfected into COS-7 cells using a DEAE-dextran procedure as modified by Luthman and Magnusson (1983). After 2-3 days, conditioned media from these cultures were applied to 5-day-old triplkate cultures of chick myotubes for 14-20 hr.…”

Section: Methodsmentioning

confidence: 99%

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution

Rüegg

Tsim

Horton

et al. 1992

Neuron

239

198

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SummaryWe isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. 80th proteins are the result of alternative splicing of the product of the agrin gene, but, unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrinrelated protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.

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“…[14][15][16] FACS analysis for EGFP expression in these cells indicated that exposure to chloroquine increased the transient expression from 12 to 50% (Figure 2b). However, after selection with G418 for stably expressing hMSCs, only one colony was visualized on one plate.…”

Section: Electroporation Of Mscs a Peister Et Almentioning

confidence: 99%

Stable transfection of MSCs by electroporation

et al. 2004

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Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 ms in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neo r ). After electroporation of 10 6 cells with 10 mg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 mg/ml G418.The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.

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High efficiency polyoma DNA transfection of chloroquine treated cells

Cited by 693 publications

References 28 publications

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

The agrin gene codes for a family of basal lamina proteins that differ in function and distribution

Stable transfection of MSCs by electroporation

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