Application of a kind of rhododendron in gene editing
A gene editing and rhododendrin technology, applied in genetic engineering, introduction of foreign genetic material using vectors, and other methods of inserting foreign genetic material, etc., can solve problems such as insufficient promotion, mouse application, and single practical range of cell types. , to achieve the effect of improving efficiency
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Embodiment 1
[0027] Example 1: Rhododendrin affects DNA double-strand break repair pathway
[0028] A single reporter system for detecting DSB repair was used to further verify the effect of rhododendron on its pathway selection. Cells were pretreated with different concentrations of rhododendron (final concentrations were 0.1 μM, 1 μM, 5 μM, and 10 μM) for 24 h before nucleofection, and then Mix 5 μg pCMV-I-SceI and 15ng pDsRed2-N1 plasmids corresponding to 1×10 per plate 6 The cells were transfected. At this time, the rhododendron treatment was maintained in the medium until 72 hours later, the ratio of GFP+ / DsRed+ was analyzed by flow cytometry to detect the repair efficiency of rhododendron on HR and NHEJ.
[0029] The result is as figure 1 As shown, it can be seen that rhododendrin can promote homologous recombination repair (HR) by using the detection double-strand break repair reporter system, but has no effect on non-homologous end-joining repair (NHEJ).
Embodiment 2
[0030] Example 2: Rhododendrin promotes gene editing efficiency of targeted integration in human cells
[0031] 1. Rhododendrin can effectively promote the efficiency of targeted integration mediated by CRISPR / Cas9
[0032] HEK293FT cells were plated in a 6-well cell culture plate, and the above cells were treated for 24 hours with gradient concentrations of active small molecule compounds (final concentrations of Rhododendron were 0.1 μM, 1 μM, 5 μM, and 10 μM, respectively). Both SCR7 and RS-1 were currently The reported small molecule compounds that can effectively improve the targeting integration efficiency, the final concentrations used in this study: the final concentrations of SCR7 were 0.1 μM and 1 μM, respectively, and the final concentration of RS-1 was 10 μM. The mixture of sgRNA targeting AAVS1 site, Cas9 and donor carrier with mCherry fluorescence was transferred into the cells by electroporation, and the proportion of mCherry-positive cells was analyzed by flow ...
Embodiment 3
[0037] Example 3: Rhododendrin promotes target integration efficiency at the level of mouse stem cells (ES)
[0038] 1. Detect the gene editing efficiency of active small molecule compounds to specific sites of targeted integration
[0039]Mouse ES cells were spread in 6-well cell culture plates, and after 24 hours of culture, gradient concentrations of active small molecule compounds were used (the final concentrations of Rhododendrin were 0.1 μM, 1 μM, 5 μM, and 10 μM; the final concentrations of SCR7 were 0.1 μM and 1 μM; The final concentrations of RS-1 are 1 μM and 10 μM respectively) to act on the above cells for 24 hours, and transfer the sgRNA targeting the Actb site, Cas9 and donor carrier mixture with Puromycin selection into the cells by electroporation, and use 1 mg / ml after 48 hours Puromycin screened the gene editing products treated with different concentrations of active small molecule compounds, and after 96 hours, the screened clones were stained with Coomass...
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