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Use of Mutated FLP Recognition Target (FRT) Sites for the Exchange of Expression Cassettes at Defined Chromosomal Loci
Abstract
Using the FLP/FRT system for site-specific recombination and the wild-type recognition site (FRT) in conjunction with certain mutant FRT sites, it becomes possible to provoke, with high yield, a double-reciprocal crossover event in cultured mammalian cells. It is demonstrated that this technology enables a targeting of expression cassettes to appropriate chromosomal reference sites in the recipient cell to improve the concepts of reverse genetics. The design of mutant FRT sites promoting such a process will be delineated. Our results show that the five spacer mutations tested are functional as the wild type but differ in the extent of their cross-recombination, which has to be minimized for their simultaneous usage.
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“The pigs were engineered to carry 69 genomic edits, using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-mediated nonhomologous end joining and homology-directed repair30,31, and recombinase-mediated cassette exchange32 (Fig. 2a, Extended Data Fig. 1a and Supplementary Table 2).”
From Design and testing of a humanized porcine donor for xenotransplantation (2023) · cites this paper as [32]
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Design and testing of a humanized porcine donor for xenotransplantation
Anand, R. P., Layer, J. V., Heja, D., Hirose, T., Lassiter, G., Firl, D. J., Paragas, V. B., Akkad, A., Chhangawala, S., Colvin, R. B., et al. (2023) · Nature · doi:10.1038/s41586-023-06594-4 · Cited by 302
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Functional Access to Neuron Subclasses in Rodent and Primate Forebrain
Mehta, P., Kreeger, L., Wylie, D. C., Pattadkal, J. J., Lusignan, T., Davis, M. J., Turi, G. F., Li, W.-K., Whitmire, M. P., Chen, Y., et al. (2019) · Cell Reports · doi:10.1016/j.celrep.2019.02.011 · Cited by 67
Metadata
- Authors
- Schlake, T., & Bode, J.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1994-01-01
- Volume
- 33
- Issue
- 43
- Pages
- 12746-12751
- ISSN
- 0006-2960
- 1520-4995
- Cited by
- 272
- References
- 0
- Language
- en
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