Structured Review

Vector Biolabs adenovirus expressing gfp
Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of <t>GFP</t> fluorescence of AV ‐ CMV ‐ GFP ( AV ‐ GFP ) transduced cultured‐intact islets ( CULT ) and nontransduced control (Cont) as well as LV ‐ CMV ‐ GFP ( LV ‐ GFP ) transduced pseudoislets ( PSEU ) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μ m. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV ‐ GFP ( AV ‐C) and pseudoislets treated with LV ‐ GFP / LV ‐ShScramble ( LV ‐P) prepared from four donors. (D–G) Comparison of LV ‐shScramble (Scr) and LV ‐sh GCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM . n = 5 donors. (E) Representative insulin secretion in response to 16.7 <t>mmol/L</t> glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1 ). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. * P
Adenovirus Expressing Gfp, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets"

Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets

Journal: Physiological Reports

doi: 10.14814/phy2.13907

Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of GFP fluorescence of AV ‐ CMV ‐ GFP ( AV ‐ GFP ) transduced cultured‐intact islets ( CULT ) and nontransduced control (Cont) as well as LV ‐ CMV ‐ GFP ( LV ‐ GFP ) transduced pseudoislets ( PSEU ) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μ m. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV ‐ GFP ( AV ‐C) and pseudoislets treated with LV ‐ GFP / LV ‐ShScramble ( LV ‐P) prepared from four donors. (D–G) Comparison of LV ‐shScramble (Scr) and LV ‐sh GCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM . n = 5 donors. (E) Representative insulin secretion in response to 16.7 mmol/L glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1 ). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. * P
Figure Legend Snippet: Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of GFP fluorescence of AV ‐ CMV ‐ GFP ( AV ‐ GFP ) transduced cultured‐intact islets ( CULT ) and nontransduced control (Cont) as well as LV ‐ CMV ‐ GFP ( LV ‐ GFP ) transduced pseudoislets ( PSEU ) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μ m. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV ‐ GFP ( AV ‐C) and pseudoislets treated with LV ‐ GFP / LV ‐ShScramble ( LV ‐P) prepared from four donors. (D–G) Comparison of LV ‐shScramble (Scr) and LV ‐sh GCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM . n = 5 donors. (E) Representative insulin secretion in response to 16.7 mmol/L glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1 ). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. * P

Techniques Used: Transduction, Microscopy, Fluorescence, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

Related Articles

Transduction:

Article Title: p53 regulates nuclear GSK-3 levels through miR-34-mediated Axin2 suppression in colorectal cancer cells
Article Snippet: .. Adenovirus expressing GFP (ad-GFP, #1060) and GFP-p53 (ad-GFP-p53, #1260) were purchased from Vector Biolabs. p53−/− HCT116 was transduced with adenoviral vectors by directly applying the diluted viruses to the culture medium at 100 multiplicity of infections as described previously. .. The transduction efficiency was determined by direct visualization using fluorescent microscopy of GFP-expressing cells.

Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets
Article Snippet: .. For cultured‐intact islet transduction, intact islets after overnight culture were resuspended in 0.1 mmol/L EGTA in serum free CRML and incubated with adenovirus expressing GFP under CMV promoter (Ad‐CMV‐GFP from Vector Biolabs, Malvern, PA) at 10,000 pfu/IEQ for 1 h at room temperature with mixing every 15 min before transferring to 10% HI‐FBS CRML for culture. ..

shRNA:

Article Title: Combined mutation of Vhl and Trp53 causes renal cysts and tumours in mice
Article Snippet: .. Cells were infected with adenoviruses expressing GFP (Vector Biolabs, 1060) or Cre-GFP (Vector Biolabs, 1700), retroviruses (LMP) expressing non-silencing hairpin or miR30-shRNA against Trp53 (Dickins et al, ), lentiviruses (LKO.1) expressing non-silencing hairpin (Addgene, 10879) or shRNA against Vhl (Open Biosystems, TRC0000009735) (Thoma et al, ). .. For lentiviral-mediated knockdown of Trp53 , we generated a vector (pLenti X1 Puro DEST, Addgene 17297) containing the U6 promoter (derived from pENTR/pSM2 (U6), Addgene 17387) driving expression of a previously described (Dickins et al, ) miR30 format shRNA against Trp53 (1224) or expressing an empty (ns) miR30 backbone.

Transferring:

Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets
Article Snippet: .. For cultured‐intact islet transduction, intact islets after overnight culture were resuspended in 0.1 mmol/L EGTA in serum free CRML and incubated with adenovirus expressing GFP under CMV promoter (Ad‐CMV‐GFP from Vector Biolabs, Malvern, PA) at 10,000 pfu/IEQ for 1 h at room temperature with mixing every 15 min before transferring to 10% HI‐FBS CRML for culture. ..

Infection:

Article Title: Combined mutation of Vhl and Trp53 causes renal cysts and tumours in mice
Article Snippet: .. Cells were infected with adenoviruses expressing GFP (Vector Biolabs, 1060) or Cre-GFP (Vector Biolabs, 1700), retroviruses (LMP) expressing non-silencing hairpin or miR30-shRNA against Trp53 (Dickins et al, ), lentiviruses (LKO.1) expressing non-silencing hairpin (Addgene, 10879) or shRNA against Vhl (Open Biosystems, TRC0000009735) (Thoma et al, ). .. For lentiviral-mediated knockdown of Trp53 , we generated a vector (pLenti X1 Puro DEST, Addgene 17297) containing the U6 promoter (derived from pENTR/pSM2 (U6), Addgene 17387) driving expression of a previously described (Dickins et al, ) miR30 format shRNA against Trp53 (1224) or expressing an empty (ns) miR30 backbone.

Article Title: Evidence for a cytoplasmic microprocessor of pri-miRNAs
Article Snippet: .. Floxed cells were infected with Adenovirus expressing GFP or GFP/Cre (vector biolabs #1060 and #1700, respectively) at an MOI of 500 and subsequently treated six days post-Adenovirus infection as described. .. For serum starvation, cells were washed and incubated with serum free media for 24 h prior to infection.

Incubation:

Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets
Article Snippet: .. For cultured‐intact islet transduction, intact islets after overnight culture were resuspended in 0.1 mmol/L EGTA in serum free CRML and incubated with adenovirus expressing GFP under CMV promoter (Ad‐CMV‐GFP from Vector Biolabs, Malvern, PA) at 10,000 pfu/IEQ for 1 h at room temperature with mixing every 15 min before transferring to 10% HI‐FBS CRML for culture. ..

Cell Culture:

Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets
Article Snippet: .. For cultured‐intact islet transduction, intact islets after overnight culture were resuspended in 0.1 mmol/L EGTA in serum free CRML and incubated with adenovirus expressing GFP under CMV promoter (Ad‐CMV‐GFP from Vector Biolabs, Malvern, PA) at 10,000 pfu/IEQ for 1 h at room temperature with mixing every 15 min before transferring to 10% HI‐FBS CRML for culture. ..

Expressing:

Article Title: p53 regulates nuclear GSK-3 levels through miR-34-mediated Axin2 suppression in colorectal cancer cells
Article Snippet: .. Adenovirus expressing GFP (ad-GFP, #1060) and GFP-p53 (ad-GFP-p53, #1260) were purchased from Vector Biolabs. p53−/− HCT116 was transduced with adenoviral vectors by directly applying the diluted viruses to the culture medium at 100 multiplicity of infections as described previously. .. The transduction efficiency was determined by direct visualization using fluorescent microscopy of GFP-expressing cells.

Article Title: Combined mutation of Vhl and Trp53 causes renal cysts and tumours in mice
Article Snippet: .. Cells were infected with adenoviruses expressing GFP (Vector Biolabs, 1060) or Cre-GFP (Vector Biolabs, 1700), retroviruses (LMP) expressing non-silencing hairpin or miR30-shRNA against Trp53 (Dickins et al, ), lentiviruses (LKO.1) expressing non-silencing hairpin (Addgene, 10879) or shRNA against Vhl (Open Biosystems, TRC0000009735) (Thoma et al, ). .. For lentiviral-mediated knockdown of Trp53 , we generated a vector (pLenti X1 Puro DEST, Addgene 17297) containing the U6 promoter (derived from pENTR/pSM2 (U6), Addgene 17387) driving expression of a previously described (Dickins et al, ) miR30 format shRNA against Trp53 (1224) or expressing an empty (ns) miR30 backbone.

Article Title: RNase III nucleases from diverse kingdoms serve as antiviral effectors
Article Snippet: .. Adenoviruses expressing GFP or Cre were purchased from Vector Biolabs (Cat# 1768 and 1779). .. Cell culture and reagents Drosophila cells (DL1) were grown, maintained and treated with dsRNA as previously described , .

Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets
Article Snippet: .. For cultured‐intact islet transduction, intact islets after overnight culture were resuspended in 0.1 mmol/L EGTA in serum free CRML and incubated with adenovirus expressing GFP under CMV promoter (Ad‐CMV‐GFP from Vector Biolabs, Malvern, PA) at 10,000 pfu/IEQ for 1 h at room temperature with mixing every 15 min before transferring to 10% HI‐FBS CRML for culture. ..

Article Title: Evidence for a cytoplasmic microprocessor of pri-miRNAs
Article Snippet: .. Floxed cells were infected with Adenovirus expressing GFP or GFP/Cre (vector biolabs #1060 and #1700, respectively) at an MOI of 500 and subsequently treated six days post-Adenovirus infection as described. .. For serum starvation, cells were washed and incubated with serum free media for 24 h prior to infection.

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    Vector Biolabs gfp expressing adenovirus ad rgd gfp icre
    Targeted cells remained RNA foci-negative after removal of the puromycin selectable marker. (A) Following infection with Ad <t>(RGD)-GFP-iCre,</t> floxed puromycin resistant gene cassettes were removed in both colonies 2 and colony 6. The size of the PCR products
    Gfp Expressing Adenovirus Ad Rgd Gfp Icre, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Biolabs replication deficient adenoviruses expressing gfp
    Extra‐endothelial DLL4 signalling inhibits HemECs angiogenesis. (A) HemECs were co‐cultured with normal human dermal fibroblasts and treated with conditioned media from <t>CHO</t> cells, untransduced (negative) or transduced with <t>GFP</t> (Ad.GFP) or sDLL4 adenovirus (ad.DLL4) and VEGF‐A 165 a or VEGF‐A 165 b; the cells were then stained for VE‐cadherin. (B–D) Quantifications of (A): VEGF‐A 165 a, not VEGF‐A 165 b, induced endothelial tube extension, branching and coverage in the assay ( n = 3; p
    Replication Deficient Adenoviruses Expressing Gfp, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Vector Biolabs adenovirus expressing cre recombinase
    GATA4 expression is inversely correlated with RANKL mRNA. (A, B, C) Bone marrow from WT and OCN-cKO mice was differentiated for two weeks with β-glycerophosphate and ascorbic acid and then mRNA was obtained. (D, E, F) Calvarial osteoblasts were isolated and the silenced with lentivirus to Gata4 (shG4) or control (shC) lentivirus. The cells were differentiated for two weeks with β -glycerophosphate and ascorbic acid, in vitro . (G, H, I) GATA4 fl/fl bone marrow treated with Adenovirus GFP (adv-GFP) or Adenovirus Cre <t>recombinase</t> (Adv-CRE). qPCR was performed with primers to Gata4 (A, D, G), RANKL (B, E, H) and osteoprotegerin (OPG: C, F, I). qPCR data was normalized to β-actin (Actb) mRNA. Student’s t-test: * P
    Adenovirus Expressing Cre Recombinase, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Vector Biolabs adenovirus expressing gfp
    Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of <t>GFP</t> fluorescence of AV ‐ CMV ‐ GFP ( AV ‐ GFP ) transduced cultured‐intact islets ( CULT ) and nontransduced control (Cont) as well as LV ‐ CMV ‐ GFP ( LV ‐ GFP ) transduced pseudoislets ( PSEU ) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μ m. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV ‐ GFP ( AV ‐C) and pseudoislets treated with LV ‐ GFP / LV ‐ShScramble ( LV ‐P) prepared from four donors. (D–G) Comparison of LV ‐shScramble (Scr) and LV ‐sh GCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM . n = 5 donors. (E) Representative insulin secretion in response to 16.7 <t>mmol/L</t> glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1 ). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. * P
    Adenovirus Expressing Gfp, supplied by Vector Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/adenovirus expressing gfp/product/Vector Biolabs
    Average 88 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    adenovirus expressing gfp - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

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    Targeted cells remained RNA foci-negative after removal of the puromycin selectable marker. (A) Following infection with Ad (RGD)-GFP-iCre, floxed puromycin resistant gene cassettes were removed in both colonies 2 and colony 6. The size of the PCR products

    Journal: Stem cells (Dayton, Ohio)

    Article Title: Genome Modification Leads to Phenotype Reversal in Human Myotonic Dystrophy type 1 iPS-cell Derived Neural Stem Cells

    doi: 10.1002/stem.1970

    Figure Lengend Snippet: Targeted cells remained RNA foci-negative after removal of the puromycin selectable marker. (A) Following infection with Ad (RGD)-GFP-iCre, floxed puromycin resistant gene cassettes were removed in both colonies 2 and colony 6. The size of the PCR products

    Article Snippet: Cells were treated at 70% confluence with Cre and GFP expressing adenovirus (Ad (RGD)-GFP-iCre, Vector Biolabs, Philadelphia, PA) at a multiplicity of infection (MOI) of 10.

    Techniques: Marker, Infection, Polymerase Chain Reaction

    Extra‐endothelial DLL4 signalling inhibits HemECs angiogenesis. (A) HemECs were co‐cultured with normal human dermal fibroblasts and treated with conditioned media from CHO cells, untransduced (negative) or transduced with GFP (Ad.GFP) or sDLL4 adenovirus (ad.DLL4) and VEGF‐A 165 a or VEGF‐A 165 b; the cells were then stained for VE‐cadherin. (B–D) Quantifications of (A): VEGF‐A 165 a, not VEGF‐A 165 b, induced endothelial tube extension, branching and coverage in the assay ( n = 3; p

    Journal: The Journal of Pathology

    Article Title: Altered ratios of pro‐ and anti‐angiogenic VEGF‐A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma

    doi: 10.1002/path.4715

    Figure Lengend Snippet: Extra‐endothelial DLL4 signalling inhibits HemECs angiogenesis. (A) HemECs were co‐cultured with normal human dermal fibroblasts and treated with conditioned media from CHO cells, untransduced (negative) or transduced with GFP (Ad.GFP) or sDLL4 adenovirus (ad.DLL4) and VEGF‐A 165 a or VEGF‐A 165 b; the cells were then stained for VE‐cadherin. (B–D) Quantifications of (A): VEGF‐A 165 a, not VEGF‐A 165 b, induced endothelial tube extension, branching and coverage in the assay ( n = 3; p

    Article Snippet: Adenovirus Chinese hamster ovary (CHO) cells were infected with recombinant, replication‐deficient adenoviruses expressing GFP or sDLL4 under a CMV promoter (Vector Biolabs) at 100 MOI.

    Techniques: Cell Culture, Transduction, Staining

    GATA4 expression is inversely correlated with RANKL mRNA. (A, B, C) Bone marrow from WT and OCN-cKO mice was differentiated for two weeks with β-glycerophosphate and ascorbic acid and then mRNA was obtained. (D, E, F) Calvarial osteoblasts were isolated and the silenced with lentivirus to Gata4 (shG4) or control (shC) lentivirus. The cells were differentiated for two weeks with β -glycerophosphate and ascorbic acid, in vitro . (G, H, I) GATA4 fl/fl bone marrow treated with Adenovirus GFP (adv-GFP) or Adenovirus Cre recombinase (Adv-CRE). qPCR was performed with primers to Gata4 (A, D, G), RANKL (B, E, H) and osteoprotegerin (OPG: C, F, I). qPCR data was normalized to β-actin (Actb) mRNA. Student’s t-test: * P

    Journal: Bone

    Article Title: GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation

    doi: 10.1016/j.bone.2018.07.014

    Figure Lengend Snippet: GATA4 expression is inversely correlated with RANKL mRNA. (A, B, C) Bone marrow from WT and OCN-cKO mice was differentiated for two weeks with β-glycerophosphate and ascorbic acid and then mRNA was obtained. (D, E, F) Calvarial osteoblasts were isolated and the silenced with lentivirus to Gata4 (shG4) or control (shC) lentivirus. The cells were differentiated for two weeks with β -glycerophosphate and ascorbic acid, in vitro . (G, H, I) GATA4 fl/fl bone marrow treated with Adenovirus GFP (adv-GFP) or Adenovirus Cre recombinase (Adv-CRE). qPCR was performed with primers to Gata4 (A, D, G), RANKL (B, E, H) and osteoprotegerin (OPG: C, F, I). qPCR data was normalized to β-actin (Actb) mRNA. Student’s t-test: * P

    Article Snippet: Adenovirus expressing Cre-recombinase (catalog # 1700) (or GFP (as a control, catalog #1060) was purchased from Vector Biolabs (Malvern, PA) and used at an MOI of 10.

    Techniques: Expressing, Mouse Assay, Isolation, In Vitro, Real-time Polymerase Chain Reaction

    Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of GFP fluorescence of AV ‐ CMV ‐ GFP ( AV ‐ GFP ) transduced cultured‐intact islets ( CULT ) and nontransduced control (Cont) as well as LV ‐ CMV ‐ GFP ( LV ‐ GFP ) transduced pseudoislets ( PSEU ) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μ m. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV ‐ GFP ( AV ‐C) and pseudoislets treated with LV ‐ GFP / LV ‐ShScramble ( LV ‐P) prepared from four donors. (D–G) Comparison of LV ‐shScramble (Scr) and LV ‐sh GCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM . n = 5 donors. (E) Representative insulin secretion in response to 16.7 mmol/L glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1 ). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. * P

    Journal: Physiological Reports

    Article Title: Delivery of sh RNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets. Delivery of shRNA via lentivirus in human pseudoislets provides a model to test dynamic regulation of insulin secretion and gene function in human islets

    doi: 10.14814/phy2.13907

    Figure Lengend Snippet: Lentivirus‐mediated transduction of pseudoislets. (A) Representative microscopy images of GFP fluorescence of AV ‐ CMV ‐ GFP ( AV ‐ GFP ) transduced cultured‐intact islets ( CULT ) and nontransduced control (Cont) as well as LV ‐ CMV ‐ GFP ( LV ‐ GFP ) transduced pseudoislets ( PSEU ) and nontransduced control (Cont) on Day 2, 4, 7, and 9 after transduction. Scale bar = 200 μ m. (B) Perifusion profiles of islets from (A). Glucose ramp (Glu) is indicated by the bar at the top. (C) Ratio of stimulation index for the first phase over the second phase of insulin secretion determined as in methods were compared between cultured‐intact islets treated with AV ‐ GFP ( AV ‐C) and pseudoislets treated with LV ‐ GFP / LV ‐ShScramble ( LV ‐P) prepared from four donors. (D–G) Comparison of LV ‐shScramble (Scr) and LV ‐sh GCK (ShGck) transduced pseudoislets. (d) qPCR determined GCK expression levels in Scr and ShGck pseudoislets and expressed using PPIB as internal control. Mean ± SEM . n = 5 donors. (E) Representative insulin secretion in response to 16.7 mmol/L glucose and 30 mmol/L KCl at indicated time in Scr and ShGck pseudoislets. Mean ± SEM of duplicates samples in donor 7 (Table 1 ). (F) Area under the curve (AUC) of insulin secretion during the first glucose ramp determined taking insulin secretion at base line as 1. (G) AUC of insulin secretion during KCl ramp determined taking insulin secretion at base line as 1. Mean ± SEM. n = 3 donors. * P

    Article Snippet: For cultured‐intact islet transduction, intact islets after overnight culture were resuspended in 0.1 mmol/L EGTA in serum free CRML and incubated with adenovirus expressing GFP under CMV promoter (Ad‐CMV‐GFP from Vector Biolabs, Malvern, PA) at 10,000 pfu/IEQ for 1 h at room temperature with mixing every 15 min before transferring to 10% HI‐FBS CRML for culture.

    Techniques: Transduction, Microscopy, Fluorescence, Cell Culture, Real-time Polymerase Chain Reaction, Expressing