Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of <i>Arabidopsis thaliana</i>
Natalya V. Permyakova,
Tatyana V. Marenkova,
Pavel A. Belavin,
Alla A. Zagorskaya,
Yuriy V. Sidorchuk,
Elena A. Uvarova,
Vitaliy V. Kuznetsov,
Sergey M. Rozov,
Elena V. Deineko
Affiliations
Natalya V. Permyakova
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Tatyana V. Marenkova
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Pavel A. Belavin
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Alla A. Zagorskaya
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Yuriy V. Sidorchuk
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Elena A. Uvarova
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Vitaliy V. Kuznetsov
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Sergey M. Rozov
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Elena V. Deineko
Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.