engleski

Creation of citrine-tagged Fancd2 expressing mice using programmable nucleases

FANCD2 is a gene of the Fanconi anemia (FA) pathway, involved in DNA damage response and repair. The core complex is composed of eight FANC proteins and two additional Fanconi anemia-associated proteins (FAAPs). They are particularly responsive to interstrand cross- links, which occur due to accumulation of toxic substances or during treatment with some cancer therapy drugs. The core complex activates FANCD2 and FANCI by monoubiquitination, and DNA repair machinery is recruited to repair the damage. To facilitate the analysis of FANCD2 function, we used the TALEN or CRISPR gene editing technology to create a mouse model expressing a citrine-tagged version of the FANCD2 protein. Citrine gene was inserted behind the start codon of the Fancd2 gene using either TALENs and CRISPRs in two different approaches. The donor vector was assembled using ligation-independent cloning, and contained homology arms for the Fancd2 region surrounding the ATG site. For TALENs, the right TALEN includes the ATG site of the Fancd2 gene. For CRISPRs, we used two sgRNA combined with the nickase version of Cas9, with one of the sgRNAs including the ATG. TALENs or CRISPRs combined with the insetion-construct were injected into zygotes. Mouse embryonic fibroblasts were developed and the activity of citrine-tagged Fancd2 was examined.

gene editing nucleases; Fancd2; citrine; mouse model

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