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A targeting frequency of 6.25% was observed yielding a total of five targeted ES cell clones.
The 94% targeting frequency in A. aculeatus was high enough for practical use in gene replacement.
However, the targeting frequency for approximately 1-kb homologous sequences was lower than in N. crassa (Ninomiya et al. [2004]).
Data from yeast show a 10-fold decrease in the targeting frequency whereas mammalian cells have an elevated correction frequency.
Arginine auxotrophs were found in one of 32 transformants of pyrG13 (targeting frequency of 3%) and in 29 of 31 transformants of MR12 (94%).
This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.
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Optimal targeting frequencies are often obtained with constructs, including more than 10 kb of homology.
Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases.
The AaargB gene was replaced with a selection marker using the AaargB deletion cassette 1 in pyrG13 and MR12 to elucidate the gene targeting frequencies.
Finally, we used a chromosomal reporter system in CHO-K1 cells to compare the gene targeting frequencies induced by natural and engineered meganucleases.
Pre-infusion maps provide expected targeting frequencies for gene loci and for specific elements within each locus.
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