4. DISCUSSION
We have shown that CRISPR-mediated deletion of distal ovalbumin promoter
in DF1 cells (DF1 +/OVA Pro ∆) induces the expression
of Ovalbumin mRNA (Figures 1, 2). In addition, in cells with this
kind of promoter, we inserted a promoterless reporter in theOvalbumin gene (DF1 +/OVA Pro ∆-Tg (promoterless
dsRed)) and registered the expression of the reporter protein (DsRed2
fluorescence) (Figure 3). In this study, we showed that a chicken
non-oviduct cell line with deletion of distal promoter sequences can
serve as a chicken production model for steroid-independent expression
of a transgene driven by endogenous ovalbumin promoter (Figure 4).
Transgenesis has become an important technique for generating
biopharmaceutical products. The application of effective promoters is
essential for achieving high expression levels and well-structured
recombinant proteins. Although constitutive strong promoters have been
extensively used to drive the expression of transgenes, they increase
the metabolic burden of host cells, resulting in cell debilitation and
cell population reduction in culture. Utilization of constitutive strong
promoters might also lead to toxicity for the host cell due to the
activation of unfolded protein response and aggregation of misfolded
proteins in the host cells (Z. Liu, Tyo, Martinez, Petranovic, &
Nielsen, 2012). In this regard, researchers have tried to discover the
proper promoters for the continuous production of recombinant proteins
at a convenient rate, exclusive to a preferred cell kind, and with
appropriate posttranslational modifications and proper protein folding.
Tissue-specific ovalbumin promoter has been one of the novel candidates
for the large-scale production of pharmaceutical proteins. The synthesis
of several therapeutic proteins under the control of regulatory
sequences from the chicken Ovalbumin gene has been reported (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 et al., 2018; T. S. Park et al.,
2015; Zhu et al., 2005). Although the regulatory elements in theOvalbumin gene are well characterized out of their genomic
context (Dougherty et al., 2009; Dougherty & Sanders, 2005; Haecker et
al., 1995; Kato et al., 1992; Kaye et al., 1984, 1986; Monroe &
Sanders, 2000; H. M. Park et al., 2000; Sanders & McKnight, 1988;
Schimke et al., 1975; Schweers et al., 1990; Sensenbaugh & Sanders,
1999; Wang et al., 1989), it is not clear what regulatory sequences of
the ovalbumin promoter are sufficient and efficient enough for inducing
oviduct-specific expression of exogenous genes in the bioreactor
chickens. In plasmid constructs, various lengths of chicken ovalbumin
promoter fragments and, 5’ and 3’ flanking regions have been fused to
the exogenous genes in order to induce gene expression. Some reports
suggest that the inclusion of two major regulatory elements residing in
the chicken ovalbumin promoter, a steroid-dependent regulatory element
(SDRE, −900 to −732) and a negative regulatory element (NRE, −308 to
−88) is sufficient to induce oviduct-specific expression of a
therapeutic protein (S. C. Kwon et al., 2010; S G Lillico et al., 2007).
These two regulatory elements are critical for appropriate regulation ofOvalbumin gene expression (Gaub, Dierich, Astinotti, Touitou, &
Chambon, 1987; Nordstrom, Dean, & Sanders, 1993; Sanders & McKnight,
1988; Schweers et al., 1990; Schweers & Sanders, 1991). The SDRE is
required for responsiveness to steroid hormones (i.e., estrogen,
progesterone, androgen, and glucocorticoids) (Schimke et al., 1975) and
the NRE, acts as a bifunctional element, cooperating with SDRE to
activate Ovalbumin gene expression in the presence of steroids in
the oviduct tissue, and repressing the Ovalbumin gene
transcription in the absence of steroids in the oviduct and non-oviduct
cells (Gaub et al., 1987; Haecker et al., 1995; Sanders & McKnight,
1988; Sensenbaugh & Sanders, 1999).
In an attempt to improve the expression level of the transgene ex
situ (out of the native genomic context), additional regulatory
sequences comprising the ovalbumin exon 1, intron 1, and the beginning
of exon 2 were included in the promoter construct (S G Lillico et al.,
2007). Zhu et al. utilized either 7.5 kb and 15 kb of the 5’ flanking
region, and 15.5 kb of the 3’ flanking region from the Ovalbumingene to direct transgene expression ex situ . Although these
regions contained all oviduct-specific regulatory elements, the ectopic
expression of the transgene was detected in non-oviduct tissue of the
chimeric chicken, and also germline transmission did not occur under the
conditions of this study (Zhu et al., 2005). In the other studies, it
was assumed that inclusion of the estrogen-responsive enhancer element
(ERE), normally located approximately 3.3 kb upstream from the
transcription start site (Figure 1A) (Kato et al., 1992) in the
ovalbumin promoter-driven construct would increase the expression level
of transgene (M. S. Kwon et al., 2018; S G Lillico et al., 2007). On the
contrary, the results of the study failed to prove any increase in the
level of recombinant protein produced in the transgenic chickens (S G
Lillico et al., 2007). Herron et al. reintroduced an additional
regulatory sequence between ERE and SDRE in their construct to enhance
the expression level of protein in the egg white (Herron et al., 2018).
The ovalbumin promoter (ranging from 1.35 kb to 3.0 kb) which have been
used in most of ex situ (in a non-native site of the genome, or
in a plasmid construct) studies so far, contains five main conserved
sites which have been identified in chicken and other avian species
(Woodfint, Hamlin, & Lee, 2018). However, the progressive
identification of other farther regulatory elements associated with
oviduct specificity (Kodama et al., 2012) and the complexity of gene
expression regulation, have inevitably led to the use of ovalbumin
promoter in situ (in its original genomic position).
Oishi et al. were the first and remain the only group to report the
successful pruduction of pharmaceutical proteins driven by endogenous
ovalbumin promoter in the egg white of transgenic chickens (Oishi et
al., 2018). Low number of reports is due to the challenges in the
generation of transgenic chickens. Although transgenic chicken
bioreactors are valuable tools for the production of human recompinant
proteins containing appropriate posttranslational modifications,
generating of founder transgenic chicken is relatively difficult,
inefficient and time consuming. Thus, the use of alternative cell
production systems, for example chicken non-oviduct cell lines, would
seem desirable to overcome these obstacles.
Previous studies on the precise
characterization of the regulatory properties of the Ovalbumingene demonstrated that deletion of the SDRE and the NRE, as well as the
linker between them, increases
chloramphenicol
acetyltransferase (CAT) activity on a plasmid (Haecker et al., 1995;
Sanders & McKnight, 1988; Sensenbaugh & Sanders, 1999). These studies
indicated that a cooperation between multiple distal regulatory and
promoter-proximal regions confers oviduct-specific Ovalbuminexpression. Deletion of regulatory elements upstream of −80 abolished
the tissue-specific expression of Ovalbumin in primary oviduct
cell cultures, while basal expression increased to levels seen with
estrogen-induced genes containing a SDRE (Haecker et al., 1995; H. M.
Park et al., 2000; Sanders & McKnight, 1988). A few reports showed that
the expression of the reporter CAT gene was induced by the ovalbumin
proximal promoter (−87 to +9) in primary oviduct cell and non-oviduct
cell cultures such as LMH/2A (Table 3) (Dean, Jones, & Sanders, 1996;
Haecker et al., 1995; Monroe & Sanders, 2000; Muramatsu et al., 1998;
H. M. Park et al., 2000; Schweers et al., 1990; Sensenbaugh & Sanders,
1999).
Although previous transfection experiments with
truncated ovalbumin promoter-CAT
reporter (OvCAT) constructs have tried to mimic the activity of the
endogenous ovalbumin promoter in the oviduct and non-oviduct cells,
there is not any report on the in situ deletion of the regulatory
sequences of ovalbumin promoter and thier effects on the levels ofOvalbumin gene expression. In this study, we show that thein situ deletion of distal ovalbumin promoter results in the
upregulation of Ovalbumin transcript in chicken DF1 cell line.
Our RT-qPCR analysis upon deletion of the distal ovalbumin promoter
including two major regulatory elements, the SDRE and the NRE
(DF1+/OVA Pro ∆ cells), indicated an increased level
of expression of ovalbumin, ~104 fold
higher than the Ovalbumin transcript levels in WT DF1 (Figures
2). Deletion of a 962-bp region (−1044 to −82 bp) containing the distal
promoter elements completely abolished tissue-restricted and
hormone-dependent expression of the Ovalbumin gene. It has been
reported that chicken ovalbumin upstream promoter (COUP) site (−85 to
−73) represses basal Ovalbumin expression in the absent of
steroids and is required for induction by steroids (Figure 1A) (H. M.
Park et al., 2000). Although previous reports have shown that the
deletion of the COUP site in OvCAT constructs increases transcriptional
activity in the absence of the NRE and confirm its repression role on
the basal gene expression, our data clearly show that, without the NRE,
transcriptional activity is increased even when COUP site is present.
This finding suggests that the opposing effect of COUP site on the
transcriptional activity depends on the native genomic context and
perhaps to other regulatory elements in wild-type composition.
In our DF1+/OVA Pro ∆ cells, althogh the core promoter
elements (TATA box and the initiator element (INR)containing sufficient
information for the initiation of transcription) have been remained
intact, we cannot rule out the regulatory role of alternative promoters
in the genome (Ayoubi & Van De Ven, 1996). Kodama et al. have found
several TATA-like and other promoter motifs located at a position around
−1800 bp (Kodama et al., 2012). Muramatsu et al. demonstrated that the
sequence from −3200 to −2800 act as a tissue-specific silencer-like
which represses the Ovalbumin gene expression in non-oviduct
tissue (Figure 1A) (Muramatsu et al., 1998). However, the presence of
this sequence did not inhibit the transcriptional activity under our
experimental conditions. Our results support this notion that the
transcriptional regulation is not determined only by promoter regions,
but involves multiple native features in the local genomic context
including enhancers, insulators, DNA binding regulatory proteins such as
transcription factors and repressors, nucleosome positioning, histone
modifications, non-coding RNA, the three-dimensional organization of
genes, and epigenetic mechanisms (Andersson & Sandelin, 2019; Gibcus &
Dekker, 2012).
In conclusion, our study demonstrates the potential for producing
recombinant proteins in chicken cell lines as an appropriate alternative
to mammalian cell culture systems. This accomplishment of
hormonally-independent expression of the transgene driven by the
endogenous regulatory mechanism(s) overcomes the limitation of cloned
promoters, where the promoter regulatory sequences have to be taken out
of their cis context and spatial organization into a plasmid. Use
of CRISPR technology enables precise deletion or mutagenesis of
regulatory sequences in the native genomic context, showing great
promise to better understand, regulate, and exploit the native
biological elements of gene regulation.