Journal: Human Gene Therapy Methods

Article Title: Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

doi: 10.1089/hgtb.2014.135

Figure Lengend Snippet: In vivo and ex vivo detection of luciferase after intradermal injections of LV or rAAV2 serotype 1, 2, 5, 6, 8, or 9 into xenografted human skin. (a) In vivo imaging of luciferase activity after intradermal vector administration. Shown are representative images from days 7, 14, 21, and 28 of xenotransplanted mice in groups injected intradermally with LV/PGK-FLuc, LV/CMV-FLuc, rAAV2/1-CMV-FLuc, rAAV2/2-CMV-FLuc, rAAV2/5-CMV-FLuc, rAAV2/6-CMV-FLuc, rAAV2/8-CMV-FLuc, rAAV2/9-CMV-FLuc, or negative controls. (b) Bioluminescence from injected xenografted human skin was recorded on days 3, 7, 14, 21, and 28 and the results are based on the signal intensity computed from a standardized region of interest. The experiment was performed twice, and data for each group were pooled from the two experiments (LV, rAAV2/1, rAAV2/2, rAAV2/6, rAAV2/8, and negative controls, n = 4; rAAV2/5 and rAAV2/9, n = 3). (c) Ex vivo detection of luciferase activity in human skin xenografts exposed to LV or rAAV2 serotype 1, 2, 5, 6, 8, or 9. Shown are representative images of the human skin xenografts after excision of these immediately after the mice were killed on day 28. (d) Depiction of average radiance after normalization to 102 for LV/CMV-FLuc in the human skin xenografts ex vivo on day 28 (n = 3). (e) Bioluminescence intensity depicted as the average luciferase activity in lysates of the excised human skin grafts normalized to total protein concentration (n = 3). LV, lentiviral vector; rAAV, recombinant adeno-associated viral vectors; p/sec/cm2/sr, photons per second per centimeter squared per steradian. Data are presented as means + SD.

Article Snippet: Luciferase activity was analyzed with luciferase assay reagent II (Promega) in 96-well arrays and detected with a multisample plate-reading luminometer (Berthold).

Techniques: In Vivo, Ex Vivo, Luciferase, In Vivo Imaging, Activity Assay, Plasmid Preparation, Injection, Protein Concentration, Recombinant

Journal: Human Gene Therapy Methods

Article Title: Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

doi: 10.1089/hgtb.2014.135

Figure Lengend Snippet: rAAV2/6 transduction of neighboring tissues but not of the skin graft after intradermal vector injection. (a) In vivo imaging of luciferase expression after intradermal vector administration. Representative images show xenotransplanted mice (day 28) in groups injected intradermally with LV/CMV-FLuc (left) or rAAV2/6-CMV-FLuc (middle), as well as a saline-injected negative control mouse (right). (b) Ex vivo detection of luciferase activity in human skin xenografts exposed to LV/CMV-FLuc or rAAV2/6-CMV-FLuc and in a graft from a negative control mouse injected with saline. Shown are representative images of the human skin xenografts excised immediately after the mice were killed on day 28. LV, lentiviral vector; rAAV, recombinant adeno-associated viral vectors; p/sec/cm2/sr, photons per second per centimeter squared per steradian. Data are presented as means + SD.

Article Snippet: Luciferase activity was analyzed with luciferase assay reagent II (Promega) in 96-well arrays and detected with a multisample plate-reading luminometer (Berthold).

Techniques: Transduction, Plasmid Preparation, Injection, In Vivo Imaging, Luciferase, Expressing, Saline, Negative Control, Ex Vivo, Activity Assay, Recombinant

Journal: Human Gene Therapy Methods

Article Title: Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

doi: 10.1089/hgtb.2014.135

Figure Lengend Snippet: High-dose rAAV2/6 transduction increases luciferase expression in the neighboring tissues but not in the skin graft after intradermal vector injection. (a) In vivo imaging of luciferase expression after intradermal vector administration. Representative images show xenotransplanted mice (day 28) in groups injected intradermally with LV/CMV-FLuc (left, mice 1–4; same vector dose as in previous experiments), a saline-injected negative control mouse (left, mouse 5), or high-dose (HD) rAAV2/6-CMV-FLuc (right, mice 1–5; 5-fold increased vector dose compared with previous experiments). Overexposed images are shown on the right-hand side. (b) In vivo imaging of luciferase expression in the mice shown in (a) after graft removal. After the mice were killed, the human skin xenografts were excised and new imaging was performed on the dead mice without grafts. Overexposed images are shown on the right-hand side. (c) Ex vivo detection of luciferase activity in the central part of the human skin xenografts (4-mm punch biopsy) exposed to LV/CMV-FLuc (left, grafts 1–4), saline-injected negative control (left, graft 5), or HD rAAV2/6-CMV-FLuc (right, grafts 1–5). Depiction of the average radiance in central graft tissue ex vivo from the punch biopsies are shown on the right-hand side. Data are presented as means + SD. ***p < 0.01 (p = 0.0026). LV, lentiviral vector; rAAV, recombinant adeno-associated viral vectors; HD, high dose; p/sec/cm2/sr, photons per second per centimeter squared per steradian.

Article Snippet: Luciferase activity was analyzed with luciferase assay reagent II (Promega) in 96-well arrays and detected with a multisample plate-reading luminometer (Berthold).

Techniques: Transduction, Luciferase, Expressing, Plasmid Preparation, Injection, In Vivo Imaging, Saline, Negative Control, Imaging, Ex Vivo, Activity Assay, Recombinant

Journal: Molecular Therapy. Nucleic Acids

Article Title: Enhanced Tailored MicroRNA Sponge Activity of RNA Pol II-Transcribed TuD Hairpins Relative to Ectopically Expressed ciRS7-Derived circRNAs

doi: 10.1016/j.omtn.2018.09.009

Figure Lengend Snippet: Superior miR-7 Suppression by miR7-Specific TuDs Compared to ciRS-7 (A) Schematic representation of ciRS-7 and TuD-7 expressed from the CMV RNA pol II promoter. SA and SD refer to splice acceptor site and splice donor site, respectively. (B) Schematic representation of the psiCHECK reporter plasmid designated miR-7 target. The miR-7 target reporter vector encodes a perfect miR-7 target site in the 3′ UTR of an RLuc reporter gene, whereas the vector designated no miRNA target encodes an RLuc reporter gene without any miRNA target sites in the 3′ UTR. Furthermore, the psiCHECK reporter plasmid encodes a FLuc reporter gene used as a control for potential variations in transfection efficiencies. Dual-Glo luciferase assays evaluating the miR-7 suppression potentials of TuD-7 and ciRS-7 in HeLa (C) and HEK293 (D) cells. The transfections were made using equal molar amounts of plasmids. Data are depicted as mean ± SEM; ***p < 0.001.

Article Snippet: Two days after transfection, luciferase expression levels were measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to manufacturer’s protocol on a multisample platereading luminometer (Berthold, Bad Wildbad, Germany).

Techniques: Plasmid Preparation, Transfection, Luciferase

Journal: Molecular Therapy. Nucleic Acids

Article Title: Enhanced Tailored MicroRNA Sponge Activity of RNA Pol II-Transcribed TuD Hairpins Relative to Ectopically Expressed ciRS7-Derived circRNAs

doi: 10.1016/j.omtn.2018.09.009

Figure Lengend Snippet: Modest Enhancement of miR-7 Suppression by ciRS-7 Carrying a miR7-Specific TuD Dual-Glo luciferase assays evaluating miR-7 suppression by ciRS-7 carrying TuD-7 in either (A) MCS5 (ciRS7-MCS5-TuD) or MCS3 (ciRS7-MCS3-TuD), (B) an eGFP-WPRE-TuD-7 expression cassette (ciRS7-eGFP-WPRE-TuD), or (C) WPRE in either MCS5 (ciRS7-MCS5-WPRE) or MCS3 (ciRS7-MCS3-WPRE). Data are depicted as mean ± SEM; *p < 0.05; ***p < 0.001; ns, not significant.

Article Snippet: Two days after transfection, luciferase expression levels were measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to manufacturer’s protocol on a multisample platereading luminometer (Berthold, Bad Wildbad, Germany).

Techniques: Luciferase, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Enhanced Tailored MicroRNA Sponge Activity of RNA Pol II-Transcribed TuD Hairpins Relative to Ectopically Expressed ciRS7-Derived circRNAs

doi: 10.1016/j.omtn.2018.09.009

Figure Lengend Snippet: Higher RNA Expression Levels of TuD-7 Than ciRS-7 (A) Schematic representation of ciRS-7 encoding primer and probe binding sites for eGFP-specific TaqMan qPCR (eGFP-qPCR) or probe binding sites for northern blot (eGFP-Northern). TaqMan qPCR (B) and northern blot (C) evaluating ciRS-7 and TuD-7 expression levels in HeLa cells. (D) Quantifications of band intensities from the northern blot shown. The same RNA samples were used for the eGFP-specific TaqMan qPCR and northern blot as well as the ciRS-7 specific TaqMan qPCR shown in Figure S2 C. (E) Dual-Glo luciferase assay comparing miR-7 suppression mediated by ciRS-7, ciRS7-TuD7, and TuD-7 at varying plasmid dosages. Except for the Dual-Glo luciferase assay shown in (E), the transfections were made using equal molar amounts of plasmids. (B and E) Data are depicted as mean ± SEM; ***p < 0.001.

Article Snippet: Two days after transfection, luciferase expression levels were measured using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to manufacturer’s protocol on a multisample platereading luminometer (Berthold, Bad Wildbad, Germany).

Techniques: RNA Expression, Binding Assay, Northern Blot, Expressing, Luciferase, Plasmid Preparation, Transfection