Experiment: Testing AAV5 for activation of tdTomato in HEK293T cells
PI: Guang-Ping Gao, PhD
Description: AAV shuttle plasmids expressing SpCas9 and guide RNAs targeting the Ai9 transgene were tested in HEK293T cells by transient transfection. Both delivery and gene editing were detected by fluorescence.
Delivery Assays:
- Three plasmids 1) AAV vector expressing dual sgRNAs and eGFP, 2) AAV vector expressing SpCas9 and 3) Ai9 transient reporter assay plasmid were transiently transfected into HEK293T cells. Images were taken for GFP expression after 72 hours
- Two plasmids 1) an unspecified Cre plasmid and 2) Ai9 transient reporter assay plasmid were transiently transfected into HEK293T cells. Images were taken for GFP expression after 72 hours
- Two plasmids 1) AAV vector expressing dual sgRNAs and eGFP and 2) Ai9 transient reporter assay plasmid were transiently transfected into HEK293T cells. Images were taken for GFP expression after 72 hours
Editing Assay:
- Two plasmids 1) AAV vector expressing dual sgRNAs and eGFP and 2) Ai9 transient reporter assay plasmid were transiently transfected into HEK293T cells. Images were taken for tdTomato expression after 72 hours
- Three plasmids 1) AAV vector expressing dual sgRNAs and eGFP, 2) AAV vector expressing SpCas9 and 3) Ai9 transient reporter assay plasmid were transiently transfected into HEK293T cells. Images were taken for tdTomato expression after 72 hours
- Two plasmids 1) an unspecified Cre plasmid and 2) Ai9 transient reporter assay plasmid were transiently transfected into HEK293T cells. Images were taken for tdTomato expression after 72 hours
Parent Project: Develop Combinatorial Non-Viral and Viral CRISPR Delivery for Lung Diseases
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Other experiments in this project: 2
Download: Submitted files
Results |
| Editing Efficiency | Delivery Efficiency | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Condition | Sex | Editor | Model | Delivery | Target Locus | Guide | Vector | Dosage | Signal | Signal | Image |
| Condition 1 | Female | HEK-293T with Ai9 transient reporter assay | Lipofectamine 3000 | Ai9/Ai14 reporter transgene | sgAi9R; sgAi9L | pH509 AAVsc-u6-sgAI9L-U6-AI9R-U1A-EGFP (1) | 2 micrograms of plasmid into 80,000 cells/well | ||||
| Condition 2 | Female | SpCas9 | HEK-293T with Ai9 transient reporter assay | Lipofectamine 3000 | Ai9/Ai14 reporter transgene | sgAi9R; sgAi9L | pAAV.pU1a-SpCas9; pH509 AAVsc-u6-sgAI9L-U6-AI9R-U1A-EGFP (1) | 2 micrograms of plasmid into 80,000 cells/well | |||
| Condition 3 | Female | Cre recombinase | HEK-293T with Ai9 transient reporter assay | Lipofectamine 3000 | 2 micrograms of plasmid into 80,000 cells/well | ||||||
| Develop AAV dual sgRNA vector for Ai9 LSL-tdTomato reporter |
 |
Methods: 12well plate, 80,000cells per well. Lipo 3000. total 2ug plasmid per well (SpCas9 : sgRNA : Ai9 plasmid=1:1:1, equal mole). 72hours later take image. |
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