Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery.

ABSTRACT: Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor contai

SUBMITTER: Yamazaki K 

PROVIDER: S-EPMC9758134 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature