Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: Cat-specific primers for quantitative RT-PCR (GDNF = human).

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Quantitative RT-PCR

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: GDNF-transduced cNPCs: morphology and reporter gene expression. Feline NPCs transduced using a bicistronic lenti-GDNF-GFP vector and cultured under proliferation conditions (UM) for 60 days (p9–p26). Cellular growth, morphology, and GFP expression were monitored over this time period. In this figure, paired phase contrast ((a), (c), (e), (g), (i), (k)) and fluorescence ((b), (d), (f), (h), (j), (l)) micrographs of the same field are presented for each of 6 sequential time points, as indicated. Transduced cNPCs exhibited consistent mophologies, continued growth, and sustained GFP expression throughout the period examined. Bars = 100 μ m.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Gene Expression, Plasmid Preparation, Cell Culture, Expressing, Fluorescence

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: Growth properties of transduced versus nontransduced cNPCs. The growth of lenti-GDNF-GFP vector transduced cNPCs was compared to nontransduced cNPCs under proliferation conditions (UM). One flask of each type of cells was harvested and counted daily for 3 consecutive days. From this data it can be seen that the transduced cNPCs continued to proliferate despite overexpression of GDNF and that growth was similar to that of nontransduced cells out to day 2, after which the nontransduced cells exhibited relatively greater growth at the day 3 time point.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Plasmid Preparation, Over Expression

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: Flow cytometric analysis of GFP expression after induction of differentiation. Nontransduced cNPCs and lenti-GDNF-GFP vector transduced cNPCs cultured under proliferation conditions (UM) were compared to transduced cNPCs cultured for 10 days in Ultraculture-based medium without EGF or bFGF and containing 10% FBS in order to induce differentiation (UM-FBS). Curve A: nontransduced cNPCs as negative controls; curve B: lenti-GDNF-GFP transduced cNPCs and curve C: lenti-GDNF-GFP transduced cNPCs in UM-FBS. Induction of differentiation did not attenuate expression of the GFP reporter gene.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Plasmid Preparation, Cell Culture

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: ELISA analysis of GDNF production by transduced cNPCs. (a) Lenti-GDNF-GFP vector transduced and nontransduced cells at passage 17 (cNPCp17) were seeded equally, under identical conditions, and allowed to grow for 15 days in UM, over which period the cells were passaged 3 times. At the time of each passage, culture media conditioned over the prior 48 hours was collected for ELISA assay. The conditioned media from transduced cNPCs was substantially enriched for GDNF compared to nontransduced cells. (b) Lenti-GDNF-GFP transduced and nontransduced cNPCp17 cells were trypsinized, lysed, and subjected to ELISA. GDNF was markedly elevated in lysates of transduced cells.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: Effect of cell differentiation on transgene GDNF expression by ELISA. Lenti-GDNF-GFP vector transduced cNPCp24 cells were seeded equally in either UM (proliferation conditions) or Ultraculture-based medium without additional growth factors but containing 10% FBS (differentiation conditions, UM-FBS). Cultures were fed 24 hours prior to collecting GDNF conditioned media for ELISA assay at which time the cells were counted. ELISA data is presented as GDNF (ng) per million cells per day in order to further evaluate whether differentiation of transduced cNPCs had an influence on transgene expression. These data are consistent with sustained GDNF overexpression, confirming the flow cytometric data that showed no evidence of diminished reporter gene expression in the UM-FBS treated population of transduced cNPCs.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Cell Differentiation, Expressing, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Over Expression, Gene Expression

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: Expression profiles of cNPCs before and after transduction. The relative impact of GDNF overexpression on transcript expression levels was evaluated using qPCR analyses for a profile of 32 genes, which included β -actin as a housekeeping gene. Lenti-GDNF-GFP vector transduced cNPCp20 cells were compared to nontransduced cNPCp20 cells (with nontransduced cells set to 1.00). GDNF transcript level was over 14,000-fold higher in transduced versus nontransduced cells (note that Y -axis has break to accommodate value). The value for GDNF was vastly greater than any other changes in transcript level across the profile examined.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Transduction, Over Expression, Plasmid Preparation

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: GDNF expression by cNPCs before and after transduction and differentiation. Immunocytochemistry (ICC) was performed on cNPCs using a rabbit anti-human GDNF antibody to evaluate expression of GDNF at the protein level, before and after transduction and before and after exposure to growth factor deprived/FBS-containing differentiation conditions (UM-FBS). (a) Nontransduced cNPCp20 cultured in UM (proliferation conditions) exhibit baseline cytoplasmic labeling for GDNF (red). (b) Lenti-GDNF-GFP vector transduced cNPCs cultured in UM show increased intensity of GDNF (red) labeling. (c) Transduced cNPCs cultured in UM-FBS (differentiation conditions) are larger in size and show persistent overexpression of GDNF (red), that is, heterogeneously distributed among the profiles. Nuclear labeling = DAPI (blue), scale bar = 50 μ m.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Transduction, Immunocytochemistry, Cell Culture, Labeling, Plasmid Preparation, Over Expression

Journal: Journal of Ophthalmology

Article Title: Efficient Transduction of Feline Neural Progenitor Cells for Delivery of Glial Cell Line-Derived Neurotrophic Factor Using a Feline Immunodeficiency Virus-Based Lentiviral Construct

doi: 10.1155/2011/378965

Figure Lengend Snippet: Expression of NPC and lineage markers before and after transduction and differentiation. The effects of passage number, induction of differentiation and GDNF transgene expression on the expression of 5 markers was evaluated using ICC. Nontransduced and lenti-GDNF-GFP vector transduced cNPCp20 were cultured in UM or UM-FBS, then immunolabeled with specific antibodies. The changes in expression patterns seen predominantly reflected exposure to differentiation conditions (alternating columns), with little that might be attributable to passage number or lenti-GDNF-GFP transduction. Scale bar = 50 μ m.

Article Snippet: ELISA was performed using a human GDNF DuoSet ELISA kit and protocol (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Transduction, Plasmid Preparation, Cell Culture, Immunolabeling

Journal: Neuroreport

Article Title: Glial cell line-derived neurotrophic factor-secreting human neural progenitors show long-term survival, maturation into astrocytes, and no tumor formation following transplantation into the spinal cord of immunocompromised rats

doi: 10.1097/WNR.0000000000000092

Figure Lengend Snippet: Survival, differentiation, and sustained GDNF expression following long-term transplants of hNPCs. (a, b) Immunocytochemistry following in vitro differentiation shows hNPC GDNF (a) and control hNPCs (b) express the astrocyte marker GFAP (red) but only hNPCs GDNF express GDNF (green). (c, d) High magnification image of cells expressing Ku80 (green) and Ki67 (red) at 1 month (c) and 7.5 months (d) after transplantation. (e, j) Low magnification image of human cytoplasmic marker (SC121, red) and the motor neuron marker (ChAT, green) showing appropriate targeting of the transplanted cells to the ventral horn at 1 month (e) and 7.5 months (j) after transplantation. (f, k) Low and (g, l) high magnification images of cells expressing human-specific nestin (red) and human-specific GFAP (green) at 1 month (f, g) and 7.5 months (k, l) after transplantation. Note the increased GFAP expression and changes in morphology at 7.5 months. (h, i and m, n) GDNF expression is observed ipsilateral (h, m) but not contralateral (i, n) to the transplant at 1 month (h, i) and 7.5 months (m, n) after transplantation. Note the appearance of large cells stained for GDNF, which is presumably host motor neurons taking up GDNF secreted by the transplanted cells. (o, p) Stereological quantification of cells expressing Ku80 revealed a significant decrease in the number of grafted cells (o) and in cell density (p) at 1 month compared with 7.5 months after transplantation. (q) No significant difference in cell proliferation was observed at 1 month compared with 7.5 months after transplantation. (r–t) Quantification of cells expressing nestin and GFAP showed no difference in nestin expression (r, s) but a significant increase in GFAP-expressing cells at 7.5 months compared with 1 month after transplantation (t). Scale bars: (a)–(d), (g), (l), 10 μm; (e) and (f), (j) and (k), 75 μm; (h), (i), (m), (n), 100 μm. ChAT, choline acetyltransferase; GDNF, glial cell line-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; hNPCs, human neural progenitor cells. * P <0.05.

Article Snippet: For in vitro differentiation, cells were dissociated, plated onto laminin-coated glass coverslips for 7 days, fixed with 4% paraformaldehyde (PFA), and then stained with antibodies against glial fibrillary acidic protein (GFAP) (Z0334; Dako, Carpinteria, California, USA; 1/500), GDNF (BAF212; R&D Systems, Minneapolis, Minnesota, USA; 1/250), and a DAPI nuclear counterstain (D1306; Life Technologies).

Techniques: Expressing, Immunocytochemistry, In Vitro, Control, Marker, Transplantation Assay, Staining, Derivative Assay