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 Arabidopsis thaliana

• Published in: Cells (Basel, Switzerland) Vol. 10; no. 8; p. 2137
• Main Authors: Permyakova, Natalya V, Marenkova, Tatyana V, Belavin, Pavel A, Zagorskaya, Alla A, Sidorchuk, Yuriy V, Uvarova, Elena A, Kuznetsov, Vitaliy V, Rozov, Sergey M, Deineko, Elena V
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.08.2021; MDPI
• Subjects: Arabidopsis; Arabidopsis - metabolism; Arabidopsis Proteins - metabolism; Arabidopsis thaliana; Cell culture; Cell Culture Techniques; Cell cycle; Cell lines; Cloning; deltaferon; Deoxyribonucleic acid; DNA; DNA methylation; Endonuclease; Epigenetics; gene editing; Gene expression; Genetic engineering; Genomes; Histone H3; histone H3.3 gene; Histones; Histones - metabolism; Homology; Insertion; Interferon; knock-in; Plasmids; Proteins; histone H3.3 gene; Arabidopsis; cell culture; gene editing; deltaferon; knock-in
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells10082137

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 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 ( ) 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 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.