From the perspective of academic and small research laboratories， the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells， resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression， however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines， we describe a method to stably integrate antibo... More
From the perspective of academic and small research laboratories， the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells， resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression， however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines， we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing. Initially， the antibody variable light (VL) chain is deleted by multiplexed Cas9 cleavage; subsequently， the variable heavy (VH) chain is replaced by a fluorescent reporter gene (mRuby) by Cas9-assisted homology-directed repair (HDR). This cell line is customized by replacing mRuby with a synthetic antibody (consisting of a VL， light constant region and VH) by once again using Cas9-assisted HDR. Due to hybridomas' inherent ability to surface display and secrete antibodies， they provide a suitable host for both the selection and the production process， substantially streamlining the process for stable cell line generation， and thus we refer to this platform as plug-and-(dis)play (PnP) hybridomas.
