Gene Targeting for Precision Glyco-Engineering: Production of Biopharmaceuticals Devoid of Plant-Typical Glycosylation in Moss Bioreactors
One of the main challenges for the production of biopharmaceuticals in plant-based systems is the modulation of plant-specific glycosylation patterns towards a humanized form. Posttranslational modifications in plants are similar to those in humans, but several differences affect product quality and efficacy and can also cause immune responses in patients. In the moss Physcomitrella patens highly efficient gene targeting via homologous recombination enables glyco-engineering to obtain suitable platform lines for the production of recombinant proteins and biopharmaceuticals. Here we describe the methods which are effective for creating gene targeting constructs and transgenic moss lines as well as confirming successful homologous integration of the constructs and modification of target gene expression.
Key wordsPhyscomitrella patens Gene targeting Homologous recombination Knockout construct Protoplast transformation Glyco-engineering Biopharmaceutical production Plant-made pharmaceuticals Molecular farming
This work was supported by contract research “Glykobiologie/Glykomik” of the Baden-Wuerttemberg Stiftung, by the Excellence Initiative of the German Federal and State Governments (EXC294 to R.R.), and EU-co-funded by INTERREG IV Project A17 “ITP-TIP” (ERDF). We thank Anne Katrin Prowse for proofreading of the manuscript.
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