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Most of the tg animal models of AD are based on the targeted overexpression of single or multiple mutant molecules associated with familial AD (FAD) (Table 1, Fig. 3).
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Wild type molecules at the enzyme-specific sequence are digested, while mutant molecules are not.
Among the multiple mutant phenotypes, pathogenicity-defective mutants were our primary focus in this study.
All recombinant mutant molecules were activated with thrombin.
Multiple mutant versions of Cas9, including Cas9D10A, Cas9H840A, and a Cas9D10A,H840A double mutant, were tested.
Photoreceptor mutants were crossed with bas1-2 sob7-1, and multiple mutant combinations were isolated in F2 populations.
For this assay, the wild‐type and mutant signals represent the presence of wild‐type and mutant molecules, respectively.
PCR primers which flank the restriction site are then used to amplify the mutant molecules only.
In multiple mutants it is likely, that some of the remaining molecules could substitute for the deleted Rpf better than others and the probability of such successful substitution decreases in the order of single-double-triple mutants.
The multiple mutants showed denaturation temperatures up to 27 deg.
There, the pollen grains from tir1 and afb1afb2afb3 multiple mutants age prematurely (Cecchetti et al. 2008).
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