Journal: Disease Markers

Article Title: KIF23 Promotes Gastric Cancer by Stimulating Cell Proliferation

doi: 10.1155/2019/9751923

Figure Lengend Snippet: Knockdown of KIF23 significantly inhibited the proliferation in gastric cancer cells. (a) Photographs of the colony formation assay showed MGC-803 (a) and SGC-7901 cells (b) that were transfected with the control or KIF23 shRNA. Cultivation lasted 2 weeks. (b) The MTT assay revealed the difference between the control and KIF23-depleted gastric cancer cells. (c) The western blot assay showed that the expression level of Ki67 was markedly decreased in MGC-803 and SGC-7901 cells. (d) The western blot assay revealed that the PCNA expression was significantly downregulated in MGC-803 and SGC-7901 cells. Results are presented as mean ± SD ( ∗ P < 0.05).

Article Snippet: Ready-to-package AAV shRNA clones of KIF23 were bought from Addgene plc.

Techniques: Knockdown, Colony Assay, Transfection, Control, shRNA, MTT Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

doi: 10.1101/488585

Figure Lengend Snippet: AAV yields after sucrose treatment in a large scale production. AAVpro 293T cells were triple transfected with a transfer plasmid, a plasmid encoding rep and serotype specific cap genes and a plasmid encoding the adenoviral helper sequences. The following day media was replaced with fresh DMEM with or without 0.1M sucrose. Cells and media were harvested 96h post transfection and viral particles purified via iodixanol gradient ultracentrifugation followed by concentration and buffer exchange. For each serotype between 2 (AAV2) and 4 (rAAV2-retro and AAV5) different preps were prepared and the range and average number of AAV genome copies (GC) calculated and plotted, A. B, the average fold change of sucrose to untreated viral prep was calculated and plotted.

Article Snippet: For the large-scale HYPERFlask M sucrose studies the following plasmids were packaged in the rAAV2-retro (Addgene 81070, gift of Dr. Alla Karpova) serotype; pGP-AAV-syn-jGCaMP7f-WPRE (Addgene 104488, gift of Dr. Douglas Kim) AAV-pgk-Cre (Addgene 24593, gift of Dr. Patrick Aebischer) pAAV-hsyn-Jaws-KGC-GFP-ER2 (Addgene 65014, gift of Dr. Ed Boyden) and pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40 (Addgene 100853, gift of Dr. Douglas Kim).

Techniques: Transfection, Plasmid Preparation, Purification, Concentration Assay, Buffer Exchange

Journal: bioRxiv

Article Title: Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

doi: 10.1101/488585

Figure Lengend Snippet: AAVpro 293T cells were triple transfected with a plasmid encoding jGCaMP7f (Addgene number 104488), a plasmid encoding rep and rAAV2-retro cap genes (Addgene number 81070) and a plasmid encoding the adenoviral helper sequences. The following day media was replaced with fresh DMEM with or without 0.1M of the indicated sugar. Cells and media were harvested 96h post transfection and viral particles purified via iodixanol gradient ultracentrifugation followed by concentration and buffer exchange. A, following buffer exchange, the level of sucrose or sorbitol was measured in the final preparations as compared to a standards of known concentration. B, Neuro2a cells were transduced with approximately 1.5 × 10 6 vector particles per cell from the untreated and sucrose treated viral preparations. GCaMP7f expression was assessed 96h post transduction. C, purity of the final untreated and sucrose treated preparations was assessed via SDS-PAGE followed by silver staining.

Article Snippet: For the large-scale HYPERFlask M sucrose studies the following plasmids were packaged in the rAAV2-retro (Addgene 81070, gift of Dr. Alla Karpova) serotype; pGP-AAV-syn-jGCaMP7f-WPRE (Addgene 104488, gift of Dr. Douglas Kim) AAV-pgk-Cre (Addgene 24593, gift of Dr. Patrick Aebischer) pAAV-hsyn-Jaws-KGC-GFP-ER2 (Addgene 65014, gift of Dr. Ed Boyden) and pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40 (Addgene 100853, gift of Dr. Douglas Kim).

Techniques: Transfection, Plasmid Preparation, Purification, Concentration Assay, Buffer Exchange, Transduction, Expressing, SDS Page, Silver Staining

Journal: bioRxiv

Article Title: Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

doi: 10.1101/488585

Figure Lengend Snippet: A, AAVpro 293T were left untreated (top panels) or treated with 0.1M sucrose (bottom panels) for 72 hours. The level of endocytosis of untreated and sucrose-treated cells was assessed via internalization of the pH sensitive pHrodo green dextran dye. B, AAVpro 293T cells were transduced with CAG-GFP rAAV2-retro in the absence (top panels) or presence (bottom panels) of 0.1M sucrose. 72h later, cells GFP expression was assessed by direct fluorescence.

Article Snippet: For the large-scale HYPERFlask M sucrose studies the following plasmids were packaged in the rAAV2-retro (Addgene 81070, gift of Dr. Alla Karpova) serotype; pGP-AAV-syn-jGCaMP7f-WPRE (Addgene 104488, gift of Dr. Douglas Kim) AAV-pgk-Cre (Addgene 24593, gift of Dr. Patrick Aebischer) pAAV-hsyn-Jaws-KGC-GFP-ER2 (Addgene 65014, gift of Dr. Ed Boyden) and pAAV.Syn.Flex.NES-jRGECO1a.WPRE.SV40 (Addgene 100853, gift of Dr. Douglas Kim).

Techniques: Transduction, Expressing, Fluorescence