Introduction
Biopharmaceuticals have revolutionized the treatment of a wide range of
diseases, and are increasingly being used in medicines
(Kesik-Brodacka, 2018). Chinese hamster
ovary (CHO) cells have been the most common hosts for the production of
therapeutic proteins (Lai, Yang, & Ng,
2013). Traditional cell line development in CHO cells is based on
random integration of recombinant constructs into the genome, resulting
in clones with variable expression (Lai et
al., 2013). Heterologous expression within cell pools requires the
selection of multiple clones and then following a tedious screening
procedure (Li, Vijayasankaran, Shen, Kiss,
& Amanullah, 2010). Moreover, due to lack of control over the genome
integration site, protein productivity of selected clones may decrease
over time, leading to clonal heterogeneity
(Coates et al., 2005). To overcome such
possible shortcomings, site-specific integration (SSI) has been proposed
for use in cell line development (N. K.
Hamaker & K. H. Lee, 2018). SSI integrates gene-of-interest (GOI) into
a predetermined genomic hotspot to enable stable and robust transgene
expression. This makes it possible to generate isogenic clones with
consistent productivity and stability, consequently reducing clone
screening and selection time (N. K.
Hamaker & K. H. Lee, 2018).
Recombinase-mediated cassette exchange (RMCE) is a procedure in reverse
genetics allowing precise integration of a genomic target cassette
(Turan, Zehe, Kuehle, Qiao, & Bode,
2013). Specifically, it allows double-reciprocal crossover events
between two pairs of heterospecific recombinase target sites using a
site-specific recombinase including Tyr-recombinases (e.g. Cre or Flp)
and Ser-recombinases (e.g. ΦC31 or Bxb1)
(Turan et al., 2013).Thus, RMCE exchanges
only a preexisting gene cassette with an analogous one carrying GOI
without changing cellular characteristics
(Turan et al., 2013). It is the most
advanced platform to establish new clones with similar productivity and
characteristics compared to existing clones
(Turan et al., 2013). Cre/Lox-based RMCE
functions exceptionally well, exhibiting cassette exchange events with
high fidelity (Gidoni, Srivastava, &
Carmi, 2008; Schnütgen, Stewart, von
Melchner, & Anastassiadis, 2006). Cre recombinase allows for
repetitive cumulative gene targeting to predefined genomic loci in
mammalian cells using different target sites such as Lox5171 and LoxP
(Gidoni et al., 2008;
Schnütgen et al., 2006).
In this study, we aimed to evaluate whether the Cre/Lox-based RMCE
constitutes an effective strategy to acquire clones from existing ones
established by CRISPR/Cas9-mediated SSI at a genomic hotspot allowing
stable expression. Here, we report that Cre/Lox-based RMCE generates new
clones by exchanging only a existing cassette with a targeting cassette
carrying GOI. Our results will open up a new paradigm of commercial
protein production by repeatedly establishing cell lines with
predictable productivity and properties.