INTRODUCTION
In recent decades, the global demand for therapeutic proteins has
considerably increased. Although there are various platforms for the
production of recombinant proteins, the avian production system, due to
its significant advantages over other systems, has been taken into more
consideration (Demain & Vaishnav, 2009; Houdebine, 2018; Ivarie, 2003,
2006; Maksimenko, Deykin, Khodarovich, & Georgiev, 2013; Raju, Briggs,
Borge, & Jones, 2000). Recombinant human proteins produced in chicken
cells in comparison to the produced proteins in plant, bacterial, or
non-human mammalian cells, contain posttranslational modifications
(including glycosylation) that are much more similar to those in
proteins produced by human cells (Zhu et al., 2005). The proper
posttranslational modifications are essential for successful therapeutic
efficacy, prevention of unintended immune response, the long half-life
of proteins, and biological activity in vivo (Elliott et al.,
2004; Kodama et al., 2008; Rapp, Harvey, Speksnijder, Hu, & Ivarie,
2003).
Oligosaccharides on IgG in human and chicken are similar and comprise
mainly N-acetylneuraminic acid (NANA), whereas other species contain
N-glycolylneuraminic acid (NGNA) and NANA (e.g. rabbit), or only NGNA
(e.g. sheep, goats, and cow) (Simon G. Lillico, McGrew, Sherman, &
Sang, 2005; Raju et al., 2000; T. Shantha Raju, 2003). Therefore, avian
expression systems represent desirable platforms for the production of
recombinant human proteins.
In the early attempts to produce foreign proteins in avian systems,
viral vectors containing a constitutive promoter (such as CVM promoter)
were used to drive the expression. The use of these constitutive strong
promoters had several disadvantages including variations in the level of
protein expression, improper folding of the protein product, the
possibility of promoter silencing, and toxicity due to their expression
in a wide range of tissues. Thus, there has been an increasing trend
toward the use of regulated promoters. Among these, the native
hormone-dependent promoters have demonstrated to be efficient in
transgene expression. For example, the ovalbumin promoter with its
regulatory sequences has been used in the cultured primary oviduct cells
or in the transgenic chickens for the production of exogenous proteins
(Byun et al., 2011; Cao et al., 2015; Herron et al., 2018; Kodama et
al., 2012; M. S. Kwon et al., 2018; S. C. Kwon et al., 2010; S G Lillico
et al., 2007; T. Liu et al., 2015; Oishi, Yoshii, Miyahara, & Tagami,
2018; T. S. Park et al., 2015; Zhu et al., 2005). Despite the successful
achievements and progress in this field, primary oviduct cell culture is
difficult to perform and needs experimental requirements including
efficient transfection of the primary cells, cell-cell and
cell-substratum interactions, and dependency to steroid hormones
(Yoshimura & Oka, 1990). Furthermore, the generation of transgenic
chicken is elaborate, time-consuming, and requires highly skilled
personnel for embryo-manipulation.
Hence, using a hormone-independent promoter to drive the transgene
expression in a chicken cell line might be an easy and quick alternative
method of production platform for human proteins. This system would
allow us to use the significant advantages of production in chicken
cells.
The regulatory sequences of the ovalbumin promoter, which has been
extensively used in the chicken production systems are
well-characterized (Dougherty, Park, & Sanders, 2009; Dougherty &
Sanders, 2005; Haecker, Muramatsu, Sensenbaugh, & Sanders, 1995; Kato
et al., 1992; Kaye, Bellard, Dretzen, Bellard, & Chambon, 1984; Kaye et
al., 1986; Monroe & Sanders, 2000; H. M. Park, Haecker, Hagen, &
Sanders, 2000; Sanders & McKnight, 1988; Schimke, McKnight, Shapiro,
Sullivan, & Palacios, 1975; Schweers, Frank, Weigel, & Sanders, 1990;
Sensenbaugh & Sanders, 1999; Wang et al., 1989). Although, there are
many reports on the cis-acting regulatory sequences responsible for
ovalbumin gene expression, other factors including trans-acting
regulatory elements, nucleosomal rearrangements, histone modifications,
the chromosomal structure of the gene locus, and the three-dimensional
(3D) nuclear organization may play critical roles for its proper
expression in oviduct cells (Bellard, Dretzen, Bellard, Oudet, &
Chambon, 1982). Previous plasmid-based studies have shown that the
deletion of the steroid-dependent regulatory element (SDRE; −900 to
−732) and negative regulatory element (NRE; −308 to −88), as well as the
linker between them in the ovalbumin promoter, increases the reporter
gene activity driven by that (Haecker et al., 1995; Sanders & McKnight,
1988; Sensenbaugh & Sanders, 1999). These reports demonstrated that the
presence of ovalbumin proximal promoter (−87 to +9) is sufficient for an
estroid-independent expression. This led us to hypothesize that thein situ deletions of SDRE and NRE elements in the genome of a
chicken non-oviduct cell line may also lead to the steroid-independent
ovalbumin gene upregulation, while the trans-acting regulatory elements
are still able to exert their effects.
In this study, we have deleted the ovalbumin distal promoter including
SDRE and NRE elements in the genome of the DF1 fibroblast cell line via
CRISPR/Cas9 system, and have analyzed the increased expression of theOvalbumin gene and the induced activity of an inserted
transgene.