Production of biopharmaceuticals in plant expression systems is becoming a promising alternative to existing platforms based on mammalian and bacterial cells. Moreover, some recombinant proteins, such as immunotoxins or regulatory proteins, are toxic to mammalian cells. Protein production in mammalian cells is expensive due to the high cost of the culture media components. Mammalian cells are necessary for the production of complex proteins because bacteria cannot form disulfide bonds efficiently and neither bacteria nor yeast can add human-like post-translational modifications (PTMs) in the secretory pathway. More than 50% of all pharmaceutical recombinant proteins are synthesized in mammalian cells. Today, recombinant proteins are mainly produced in traditional prokaryotic and eukaryotic systems such as Escherichia coli, several yeast species, and mammalian cell cultures. The ever-growing demand for recombinant proteins stimulates the development of various expression systems for the production of these proteins that meet the existing stringent standards. They are mainly used as pharmaceuticals for diagnostics, for the vaccination of humans or animals, and as drugs or monoclonal antibodies. Recombinant proteins are foreign proteins produced in various expression systems. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high. 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. 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. 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. For the targeted insertion, we selected the region of the histone H3.3 gene ( HTR5) with a high constitutive level of expression. 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. 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.
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