Validation Experiment: Independent validation for Gao Delivery Team: Testing ssAAV5 delivered intratracheally for editing activity in lung epithelia in Ai9 mice
PI: Jason D Heaney, PhD
Description: AAV5 encoding CRISPR/Cas editing machinery were delivered to the lungs of reporter mice by intratracheal instillation. After 4 weeks incubation, the mice were dissected and the lungs imaged for the presence of tdTomato fluorescence, indicating successful editing. Editing calculated by dividing the number of tdTomato+ red cells by the number of nuclei in each airway
Delivery Assays:
PCR detection of AAV-delivered Cas9 and GFP by agarose gel electrophoresis
Editing Assay:
- Fluorescent imaging for tdTomato+ cells
- Editing efficiency is calculated by identifying airways in lungs sections by shape and DAPI-stained nuclei density. tdTomato-positive cells and total nuclei are counted in each airway. Airways are divided into small (<50000 square microns) and large (>50000 square microns)
Parent Project: BCM-Rice Resource for the Analysis of Somatic Gene Editing in Mice - Gao Validation (Gao, Guang-Ping )
Download: Submitted files
Original Experiment/s that are being validated:
Organ System Overview
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