Setsuro Tech Inc. delivers genome-edited culture cells produced by the VIKING method, which are instrumental tools to analyze gene and protein functions. Our genome-edited cell production covers major cell lines such as Hela, HepG2, HEK293F, etc. Moreover, Setsuro Tech Inc. offers a package service that includes the production of genome-edited cells and gene and/or protein analysis using produced genome-edited cells. Please contact us if you want to save your resource and time!
A donor vector containing a drug resistance marker is inserted at the target site to disrupt the target gene.
A donor vector containing a drug resistance marker is inserted into one allele of the target sequence, and another allele is knocked out via a frameshift caused by the indel.
Vectors are mainly knocked-in at a safe harbor site (AAVS1).
By knocking-in a gene to a safe harbor site, the target protein can be stably expressed.
Point mutants are generated in cultured cells through two steps as indicated below:
1. Knock-out of an endogenous gene
2. Knock-in of a point mutation sequence to a safe harbor site.
* Due to knock-in to a safe harbor site, the point mutation-induced gene is no longer expressed endogenously. Tips: This method can be used for “humanization.”
The VIKING method is an improved version of knock-in technology using non-homologous end joining (NHEJ), that dramatically reduces unintended mutations (off-target effects) by optimizing the ratio of vector introduction.
This method uses three types of vectors: target genome truncation vectors, donor vectors, and donor truncation vectors, which work in concert to enable insertion of almost the entire vector sequence without damaging the genome.
Encoded drug resistance markers facilitate the selection of cells and allow for the insertion of large vectors of more than 10 kbp. The drawback to this method is its ineffectiveness at deleting a few bases; additionally, a vector sequence containing the drug resistance marker is inserted into the genomic sequence.
- Hashimoto, M. and Takemoto, T*. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci. Rep. 5, 11315; doi: 10.1038/srep11315 (2015).
- Hashimoto M, Yamashita Y, Takemoto T*.Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 418: 1-9 (2016).
- Tanihara F, Takemoto T*, Kitagawa E, Rao S, Do L, Onishi A, Yamashita Y, Kosugi C, Suzuki H, Sembon S, Suzuki S, Nakai M, Hashimoto M, Yasue A, Matsuhisa M, Noji N, Fujimura T, Fuchimoto Di, Otoi T*. Somatic cell reprogramming-free generation of genetically modified pigs. Science Advances. 2 (9) e1600803 (2016).
- Sawatsubashi S*, Joko Y, Fukumoto S, Matsumoto T, Sugano SS*. Development of versatile non-homologous end joining-based knock-in module for genome editing. Sci Rep. 2018 Jan 12;8(1):593.