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\begin{document}

\title{Screening of CHO-K1 endogenous promoters for expressing recombinant
proteins in mammalian cell cultures}



\author[1]{Ileana Tossolini}%
\author[2]{Agustina Gugliotta}%
\author[3]{Fernando López-Díaz}%
\author[2]{Ricardo Kratje}%
\author[2]{Claudio Prieto}%
\affil[1]{FBCB - UNL}%
\affil[2]{Universidad Nacional del Litoral}%
\affil[3]{Salk Institute for Biological Studies}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
For the production of recombinant protein therapeutics in mammalian
cells, a high rate of gene expression is desired and hence strong
viral-derived promoters are commonly used. However, they usually induce
cellular stress and can be susceptible to epigenetic silencing.
Endogenous promoters, which coordinates their activity with cellular and
bioprocess dynamics while at the same time they maintain high expression
levels, may help to avoid such drawbacks. In this work, endogenous
promoters were identified based on high expression levels in RNA-seq
data of CHO-K1 cells cultured in high density. The promoters of Actb,
Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new
expression vectors for the production of recombinant proteins in
mammalian cells. The in silico-derived promoter regions were
experimentally verified and the majority showed transcriptional activity
comparable or higher than CMV. Also, stable expression following a
reduction of culture temperature was investigated. The characterized
endogenous promoters (excluding Rps18) constitute a promising
alternative to CMV promoter due to their high strength, long-term
expression stability and integration into the regulatory network of the
host cell. These promoters may also comprise an initial panel for
designing cell engineering strategies and synthetic promoters, as well
as for industrial cell line development.%
\end{abstract}%



\sloppy


\textbf{SCREENING OF CHO-K1 ENDOGENOUS PROMOTERS FOR EXPRESSING
RECOMBINANT PROTEINS IN MAMMALIAN CELL CULTURES}

Ileana Tossolini\textsuperscript{1,a,b}, Agustina
Gugliotta\textsuperscript{1,a}, Fernando L\selectlanguage{ngerman}ópez Díaz\textsuperscript{4},
Ricardo Kratje\textsuperscript{1}, Claudio Prieto\textsuperscript{2,3}

\textsuperscript{1} UNL, CONICET, FBCB (School of Biochemistry and
Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad
Universitaria, Ruta Nacional 168 Km 472.4, C.C. 242. (S3000ZAA), Santa
Fe, Argentina.

\textsuperscript{2} UNL, FBCB (School of Biochemistry and Biological
Sciences), CBL (Biotechnological Center of Litoral), Ciudad
Universitaria, Ruta Nacional 168 Km 472.4, C.C. 242. (S3000ZAA), Santa
Fe, Argentina.

\textsuperscript{3} Cellargen Biotech S.R.L., Antonia Godoy 6369, CP
3000, Santa Fe, Argentina.

\textsuperscript{4} Regulatory Biology Laboratory, Salk Institute for
Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037.

\textsuperscript{a} These authors should be considered joint first
author.

\textsuperscript{b} Present address: Instituto de Agrobiotecnología del
Litoral (CONICET-UNL), Cátedra de Biología Celular y Molecular, Facultad
de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral,
3000 Santa Fe, Argentina.

\textbf{Corresponding Author:}

Claudio Prieto. UNL, FBCB (School of Biochemistry and Biological
Sciences), CBL (Biotechnological Center of Litoral). Ciudad
Universitaria, Ruta Nacional 168 Km 472.4, C.C. 242. (S3000ZAA), Santa
Fe, Argentina.

Tel./Fax: +54 342 4552928. E-mail: cprieto@fbcb.unl.edu.ar

\textbf{Keywords}

CHO cells; Endogenous promoters; Mammalian cells; Transcriptional
activity.

\textbf{Abbreviations}

Actb: Actin beta; ANOVA: Analysis of variance; AP-1: Activator protein
1; AP-2 alpha: Activating enhancer binding protein 2 alpha;
\textbar{}BHK: Baby hamster kidney; bp: base pair; C/EBP alpha:
CCAAT/enhancer binding protein alpha; CHO: Chinese hamster ovary; CpG:
5\selectlanguage{english}'-cytosine-phosphate-guanine-3\selectlanguage{english}'; Ctsz: Cathepsin Z; DMEM: Dulbecco's
Modified Eagle Medium; E-box: Enhancer box; EF1-alpha: Elongation factor
1-alpha~; ELISA: Enzyme-Linked ImmunoSorbent Assay; FAU: fluorescence
arbitrary units; FBS: fetal bovine serum; FPKM: Fragments per kilo base
per million mapped reads; hCMV: Human cytomegalovirus; HEK: Human
embryonic kidney; Hmox1: Heme oxygenase 1~; Hspa5: Heat shock protein
family A (Hsp70) member 5; IFN\selectlanguage{greek}α: \selectlanguage{english}interferon alpha; MEM: Minimum
Essential Medium ; MFI: mean fluorescence intensity; NEO: Neomycin; NF1:
nuclear factor 1; pBKS: pBluescript II KS plasmid; PCR: Polymerase chain
reaction; RNA-seq: RNA sequencing; Rps18: Ribosomal protein S18; SP1:
Specificity protein 1; SV40: Simian virus 40~; TBP: TATA-binding
protein; TF: Transcription factor; TFBS: Transcription factor binding
site ; TSS: Transcription start site; Vim: Vimentin; ZsGreen1: Zoanthus
sp. green fluorescent protein.

\textbf{Abstract}

For the production of recombinant protein therapeutics in mammalian
cells, a high rate of gene expression is desired and hence strong
viral-derived promoters are commonly used. However, they usually induce
cellular stress and can be susceptible to epigenetic silencing.
Endogenous promoters, which coordinates their activity with cellular and
bioprocess dynamics while at the same time they maintain high expression
levels, may help to avoid such drawbacks. In this work, endogenous
promoters were identified based on high expression levels in RNA-seq
data of CHO-K1 cells cultured in high density. The promoters of Actb,
Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new
expression vectors for the production of recombinant proteins in
mammalian cells. The \emph{in silico} -derived promoter regions were
experimentally verified and the majority showed transcriptional activity
comparable or higher than CMV. Also, stable expression following a
reduction of culture temperature was investigated. The characterized
endogenous promoters (excluding Rps18) constitute a promising
alternative to CMV promoter due to their high strength, long-term
expression stability and integration into the regulatory network of the
host cell. These promoters may also comprise an initial panel for
designing cell engineering strategies and synthetic promoters, as well
as for industrial cell line development.

\textbf{Introduction}

The production of recombinant proteins is one of the most important
areas in the biopharmaceutical industry. Chinese hamster ovary (CHO)
cells are the most widely used platform for the industrial production of
therapeutic proteins. \textsuperscript{{[}1{]}--{[}3{]}} Other mammalian
cell lines thar are commonly used include BHK-21 cells and HEK293T
cells. \textsuperscript{{[}2{]}, {[}4{]}} Cell line development is
continuously improving towards increased productivities, faster
production, and process development. But, the generation of
high-producing cell lines is still a laborious and multi-step
process.\textsuperscript{{[}5{]}} Many technologies for optimization of
heterologous protein production have been developed. However, the most
pivotal control mechanism, the transgene expression promoter, has often
been ignored.

The choice of a suitable promoter is a crucial stage to achieve very
high and stable transgene expression in mammalian cell
cultures.\textsuperscript{{[}6{]}} Today, viral-derived promoters, such
as human cytomegalovirus (hCMV) or simian virus 40 (SV40), are
frequently used for constitutive expression of recombinant proteins in
mammalian cells. On one hand these promoters allow high expression
rates, but on the other hand they can lead to constitutive
overexpression of the transgene causing a permanent stress on the cell
\textsuperscript{{[}6{]}}. In addition, viral promoters show cell-cycle
dependency \textsuperscript{{[}7{]}, {[}8{]}}. The CMV, for example, is
active primarily in the S phase and presents very low activity in G0/G1
\textsuperscript{{[}8{]}}. In a previous work, we found a G0/G1
synchronization mainly in late exponential and stationary growth phases
in CHO-K1 cells cultured in high density.\textsuperscript{{[}9{]}} This
is particularly important because the G1-phase is considered the
appropriate stage for increasing the production of recombinant proteins.
\textsuperscript{{[}10{]}} Moreover, several studies showed that CMV
promoter could be susceptible to histone modifications
\textsuperscript{{[}11{]}, {[}12{]}} or DNA
methylation,\textsuperscript{{[}13{]}--{[}18{]}} linking these
epigenetic modifications to production instability in recombinant CHO
cells.

A solution to such drawbacks would be the use of endogenous promoters
which function in a coordinated manner with cellular and bioprocess
characteristics. Some cellular promoters have been investigated for this
purpose in the last few years, for example, by using libraries with
several genomic fragments, or flanking sequences of highly expressed
housekeeping genes, by analyzing transcriptome microarray or RNA-seq
data. \textsuperscript{{[}19{]}--{[}24{]}} However, few analyses have
been done considering the expression stability during long-term culture
of those promoters.

In this study, endogenous promoters were identified from RNA-Seq data of
CHO-K1 cells cultured in high density.
\textsuperscript{{[}9{]}}Promoters of genes with high levels of
expression in both, late exponential and stationary growth phases, were
selected as potential candidates for industrial application. The
promoter sequences of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were
used to generate new expression vectors. These vectors proved to be
useful for high-level protein expression not only in CHO, but also in
BHK and HEK cells. The majority of these endogenous promoters constitute
an alternative to CMV promoter, and they are meant to achieve high
expression level, long-term expression stability and improve production
process efficiency.

\begin{enumerate}
\tightlist
\item
  \textbf{Materials and Methods}
\item
  \textbf{Screening of novel-promoters activity in mammalian cells}
\item
  \textbf{Selection of candidate endogenous promoters and Generation of
  expression vectors}
\end{enumerate}

Candidate promoters were selected based on RNA-seq data of CHO-K1
suspension-adapted cells cultured in 1 L bioreactors under a temperature
gradient from 37 \selectlanguage{ngerman}°C to 31 °C. \textsuperscript{{[}9{]}} Genes with high
levels of expression in both exponential and stationary phases were
chosen based on their FPKM values. DNA regions upstream of highly
expressed genes were visualized on IGV tool
\textsuperscript{{[}25{]}}using CHO-K1 genome reference and mapped
reads, to detect potential transcription start sites (TSS), promoter
location and enhancer regions. Also, a comparison with promoters of
homologous genes (\emph{M.~musculus, R.~norvegicus} and \emph{H.
sapiens} ) was performed. These sequences were retrieved from Eukaryotic
Promoter Database (EPD).\textsuperscript{{[}26{]}} A subset of 50 genes
that occurred in all datasets was then selected to form a `high
transcriptional activity group' (Table S1, Supporting Information). From
that group, six potential promoter candidates were randomly selected for
subsequent cloning, characterization and testing for promoter
functionality\emph{in vitro} .

The endogenous promoters were generated by PCR from genomic DNA of
suspension adapted-CHO-K1 cells. Primers were designed using
Primer3\textsuperscript{{[}27{]}} and primer-BLAST
\textsuperscript{{[}28{]}}in order to amplify 900-1000 bp upstream the
TSS (Table S2, Supporting Information). To achieve directional cloning,
SacI and KpnI restriction sites were introduced at 5' ends of primers
forward and reverse, respectively. PCR products were digested with SacI
and KpnI (Promega) and ligated in the pZsGreen1-1 plasmid, using a
standard~T4 DNA ligase~protocol (Invitrogen). The pZsGreen1-1 vector
lacks eukaryotic promoter and enhancer sequences, and carries the coding
sequence of the green fluorescent protein ZsGreen1, derived from
\emph{Zoanthus sp} . Also, it has a cassette~containing a~neomycin
resistance gene, for stable integration in mammalian cells. The
sequences of all plasmid constructs were confirmed by dideoxy DNA
sequencing (Macrogen).

\textbf{Cell cultures}

CHO-K1 cells were cultured in adherent conditions in chemically defined
medium DMEM-F12 (Gibco), supplemented with 5\% (v/v) of fetal bovine
serum (FBS) (PAA~Laboratories), 200 mM L-glutamine (Sigma\selectlanguage{english}-Aldrich),
2.441 g/L NaHCO\textsubscript{3} (Gibco) and 0.05 mg/mL gentamicin
sulfate (Gibco). HEK293T cells were maintained in 13.37 g/L DMEM medium
(Gibco), supplemented with 10\% (v/v) of FBS (PAA~Laboratories), 1.5 g/L
NaHCO\textsubscript{3} (Gibco), 0.05 g/L gentamicin sulfate (Gibco) and
0.11 g/L sodium pyruvate. BHK-21 cells were cultured in MEM medium
(Gibco), supplemented with 10\% (v/v) FBS (PAA Laboratories), 2.2 g/L
NaHCO\textsubscript{3} (Gibco) and 0.05 g/L gentamicin sulfate (Gibco).
Cells lines were cultured in a humidified atmosphere at 37 degC and 5\%
CO\textsubscript{2}.

\textbf{Transient expression}

CHO-K1, BHK-21 and HEK293T cells were transfected with 1 \selectlanguage{greek}µ\selectlanguage{english}g of novel
vectors containing the endogenous promoters. The pCMV-ZsGreen1-1 was
used as control to compare the transcriptional activity of endogenous
promoters. Co-transfection of each plasmid with pCIneo-CMV-IFN\selectlanguage{greek}α
\selectlanguage{english}vector\textsuperscript{{[}29{]}} was performed to evaluate transfection
efficiency, determined by quantification of interferon alpha (IFN\selectlanguage{greek}α\selectlanguage{english}) in
culture supernatants. At the same time, novel vectors were
co-transfected with pBKS plasmid used as a carrier to equalize DNA load
across all transfections. Lipofectamine\emph{\textsuperscript{TM}} 2000
Transfection Reagent (Invitrogen) was used, according to the
manufacturer's instructions.

After 48 h, transfection efficiency was analyzed by assessing
fluorescence intensity of ZsGreen1 in transfected cells by fluorescence
microscopy (Eclipse Ti-S, Nikon Instruments Inc) and flow cytometry
(Guava EasyCyte, Merck Millipore). The percentage of ZsGreen1-expressing
cells (\%ZsGreen) and ZsGreen1 mean fluorescence intensity (MFI) of each
sample were analyzed using Guava ExpressPlus software (Merck Millipore)
and MFI was expressed as fluorescence arbitrary units (FAU).
Additionally, the culture supernatant of pCIneo-CMV-IFN\selectlanguage{greek}α \selectlanguage{english}co-transfected
cells was collected and analyzed by ELISA sandwich using the protocol
described by Ceaglio \emph{et al.} (2008). \textsuperscript{{[}29{]}}

\textbf{Generation of stable pools}

CHO-K1 and BHK-21 cells were transfected with plasmid constructions (as
described above) and then cultured under neomycin (NEO) (Gibco)
selection pressure. Resistant cells were grown in culture medium without
selection pressure to investigate the stability of the transgene. The
stably transfected cells were screened for ZsGreen1 expression during
several generations. Analyses were performed by flow cytometry.

\textbf{Effect of temperature shift on levels of stable ZsGreen1
expression in CHO-K1 cells}

CHO-K1 stable cell lines were cultured in two replicates in 24-well
plates with a cell density of 2.0\selectlanguage{ngerman}×10\textsuperscript{5} cells/mL at
generation 25. The plates were maintained for 10 days at 37 °C (control
cultures) and at 32 °C, in a humidified atmosphere and 5\%
CO\textsubscript{2}. ZsGreen1 expression was analyzed by flow cytometry
on days 3, 5, 7 and 10. The MFI of ZsGreen1 positive cells for each
promoter was plotted.

\textbf{Statistical analysis}

Transient transfections were performed in duplicates. Differences were
assessed using one-way ANOVA and two different post hoc tests: Tukey's
test (for multiple comparison) and Dunnett´s test (for comparing with a
control sample).

In expression stability studies, the results obtained during the whole
experiment were analyzed by one-way ANOVA followed by Tukey´s and
Dunnett's post hoc tests. The transcriptional activity of endogenous
promoters at 32 °C and 37 °C was analyzed at four different time points
(3, 5, 7 and 10 days) in duplicate. These results were analyzed by a
Student's t-test.

Parameters were considered significantly different when p \textless{}
0.05. GraphPad Prism version 6.00 (GraphPad Software Inc.) was used to
perform data analysis and graphing.

\textbf{In silico analysis of endogenous promoter sequences}

Potential transcription factor binding sites (TFBSs) were identified
using Alibaba2, \textsuperscript{{[}30{]}} applying a minimum matrix
conservation value of 80\%; Match, \textsuperscript{{[}31{]}} using a
vertebrate matrix and a cut-off value set to minimize false positive
matches; and GPMiner, \textsuperscript{{[}32{]}} setting default~values
against mouse database. A search for putative CpG islands was carried
out using the EMBOSS cpgplot software. \textsuperscript{{[}33{]}} In
addition, promoter conservation for orthologous genes
from\emph{M.~musculus, R.~norvegicus} and \emph{H. sapiens} , was
analyzed. Promoter sequences were compared by pairwise global sequence
alignment using the EMBOSS needle software.\textsuperscript{{[}33{]}}

\textbf{Results}

\par\null

\textbf{Selection of promoter regions based on RNA-seq of CHO-K1
suspension cell cultures}

Selected endogenous promoters were identi\selectlanguage{english}fied starting from RNA\selectlanguage{english}-Seq data
previously generated.\textsuperscript{{[}9{]}} These data were obtained
from three replicates of CHO-K1 suspension-adapted cells that were
cultured in 1 L bioreactors. Transcriptional levels of all genes were
ranked from high to low, based on their FPKM values. A subset of genes
from each phase was selected in order to identify those that were highly
active at late exponential and stationary growth phases. Finally, six
potential candidate promoters (not previously described in other
studies) were selected for subsequent cloning, characterization and
testing of promoter functionality \emph{in vitro} : Actb, Ctsz, Hmox1,
Hspa5, Rps18 and Vim.

\textbf{Screening of transcriptional activity of endogenous promoters in
mammalian cells}

Promoter regions of selected genes were PCR-amplified from genomic DNA
of suspension\selectlanguage{english}-adapted CHO\selectlanguage{english}-K1 cells and then cloned into pZsGreen1-1
vector.

Despite many authors have demonstrated that EF1-alpha is a useful
promoter for stable and transient transgene expression, some of them
evidenced no significant differences when comparing with
CMV.\textsuperscript{{[}34{]}, {[}35{]}} Particularly, Mufarrege
\emph{et al.} (2014) \textsuperscript{{[}36{]}} have shown that stable
CHO cell lines producing recombinant human Factor VIII, under the
control of either CMV or EF1-alpha, presented no significant differences
in their productivities. For this reason, we decided to employ the
pCMV\selectlanguage{english}-ZsGreen1\selectlanguage{english}-1 plasmid to compare the transcriptional activity of
endogenous promoters with CMV. This viral promoter presents strong
transcriptional activity in mammalian cells culture \emph{in vitro} that
allows high expression of transgenes. Such vector contains the CMV
sequence, the immediate-early enhancer/promoter region, a chimeric
intron and the coding sequence of ZsGreen1.

\textbf{Transfection efficiency and transient transgene expression}

Firstly, a transient transfection assay was performed to evaluate
transcriptional activity of isolated endogenous promoters, not only in
CHO-K1 but also in HEK293T and BHK-21 cells. Transient gene expression
is particularly useful in early research or preclinical studies when
several potential therapeutic candidates are needed for evaluation or
when a molecule is required in the short term.\textsuperscript{{[}37{]},
{[}38{]}}

After 48 h, transfection efficiency was analyzed by quantifying IFN\selectlanguage{greek}α
\selectlanguage{english}concentration using an ELISA sandwich (data not shown). No significant
differences between IFN concentrations among transfections was observed
for each tested cell line (ANOVA test, P [?] 0.05), thus transcriptional
activity of endogenous promoters was evaluated by assessing fluorescence
intensity of ZsGreen1 in transfected cells by fluorescence microscopy
(Figure 1.A) and flow cytometry (Figure~1.B-G and Figure S1, Supporting
Information). When comparing co-transfections of each plasmid containing
the novel promoter, either with pBKS or pCIneo-CMV-IFN\selectlanguage{greek}α, \selectlanguage{english}we could
observe that ZsGreen1 expression was practically the same (Figure~1.B
and C for CHO-K1 cells; Figure 1.D and E for HEK293T cells; Figure 1.F
and G for BHK-21 cells). No expression of ZsGreen1 was determined using
the wild type cell line (negative control) which was set as marker 1
(M1) in flow cytometry analysis. The expressing population was divided
as cells with moderate fluorescence intensity (marker 2, M2, MFI between
10\textsuperscript{1} and 10\textsuperscript{3}) and cells with high
fluorescence intensity (marker 3, M3, MFI higher than
10\textsuperscript{3}). Analyzing M3 is particularly interesting because
promoters with high transcriptional activity are attractive candidates
for driving transgene expression in mammalian cells.

Hamster-derived cell lines (BHK-21 and CHO-K1) exhibited enhanced
transient transgene expression, and, as expected, CHO-K1 showed the
highest ZsGreen1 expression levels. It is important to mention that
Rps18 showed the lowest transcriptional activity in all cell lines
(ANOVA test, P<\selectlanguage{english}0.05, Figure 1). For this reason, it was excluded from
the subsequent analysis. This endogenous promoter exhibited high
expression levels during suspension culture in bioreactor,
\textsuperscript{{[}9{]}} which might be attributed to the bioprocess
dynamic and the assistance of additional regulatory elements for gene
expression.

CHO-K1 cells transfected with all endogenous promoter-containing vectors
displayed ~80-90\% of ZsGreen1 positive cells. Vim activity was
comparable to CMV promoter since nearly 100\% of the cells expressed
ZsGreen1 (ANOVA test, P<\selectlanguage{english}0.05, Figure 1.B-C). In general, HEK293T cells
exhibited the lowest transcriptional activity (significant differences
were found when comparing transcriptional activity of each promoter in
human and hamster-derived cells, ANOVA test P<\selectlanguage{english}0.05). This can be
explained by the fact that promoters were isolated from a hamster
genome. Particularly, CMV and Hspa5 showed around 70\% of ZsGreen1
expressing cells (Figure 1.D-E), and the other promoters displayed
inferior transcriptional activity (~25-50\%).

In BHK-21 cells (Figure 1.F-G), CMV displayed the highest percentage of
Zsgreen1 positive cells (99\%), while endogenous promoters Actb, Hmox1,
Hspa5 and Vim showed ~70-85\% of cells expressing the transgene.
However, no significant differences between the transcriptional activity
of endogenous promoters and CMV were found. Ctzs exhibited ~60\%
ZsGreen1 positive cells which was significantly lower compared to CMV
(ANOVA test, P<\selectlanguage{english}0.05).

MFI analysis indicates that ZsGreen1 expression in CHO-K1 and BHK-21
cells was high (in M3) and practically constant (Figure S1.B, F,
Supporting Information), showing values around
3.0-4.0x10\textsuperscript{3} FAU (fluorescence arbitrary units). In
HEK293T cells, differences among MFI values were especially marked. In
particular, CMV, Actb, Ctsz and Hspa5 promoters reached MFI values
higher than 3.8x10\textsuperscript{3} FAU (Figure~S1.D, Supporting
Information).

\textbf{Stable levels of long-term transgene expression driven by
endogenous promoters}

Long-term stable expression of the transgene is crucial for the
industrial production of recombinant proteins. As transient expression
cannot completely reveal the function of different promoters, it is
important that vectors can be stably expressed in cells, therefore we
analyzed the stability of ZsGreen1 under the control of endogenous
promoters in hamster-derived cell lines.

For cell line generation, CHO-K1 and BHK-21 cells were transfected with
endogenous promoter-containing vectors or pCMV-ZsGreen1-1 plasmid, and
selective pressure was performed using increasing concentrations of NEO.
This process allowed the gradual selection of cells that expressed
increasing ZsGreen1 intensity. BHK-21 cells resisted to higher
concentrations of the antibiotic compared to CHO-K1 cells (5.0 vs. 4.0
mg/mL NEO, respectively). Cell lines selected with the maximum
concentrations of NEO exhibited the highest expression of ZsGreen1
(Figure S2.A-B, Supporting Information). CHO-K1 and BHK-21 cell lines
resistant to the maximum concentration of NEO were grown in culture
medium without antibiotic, in order to analyze ZsGreen1 expression
stability. After 120 h, the expression of the reporter protein remained
practically constant for all promoters in both cell lines
(Figure~S2.C-D, Supporting Information), meaning that NEO is not
necessary to maintain high expression levels.

The stably transfected cell lines were screened for ZsGreen1 expression
during several generations (Figure~2 and Figure S3, Supporting
Information). Figure 2.A shows the stability of ZsGreen1 expression in
CHO-K1 cells during 45 generations. The \%ZsGreen1 was notably higher
for cell lines containing endogenous promoters compared to CMV (ANOVA
test, P<\selectlanguage{english}0.05), except for Rps18 promoter that exhibited the lowest
expression (ANOVA test, P<\selectlanguage{english}0.05). From now onwards this promoter (Rps18)
will be excluded from the analysis. Cells carrying Hspa5 promoter showed
the highest \%ZsGreen1, which was nearly 100\% during all generations.
Actb, Vim and Hmox1 promoters exhibited similar transcriptional activity
to Hspa5 (no significant differences were observed after ANOVA test and
Dunnett\selectlanguage{ngerman}´s post hoc test, p\selectlanguage{english}<0.05). In general, transcriptional activity
of endogenous promoters remained relatively constant and stable during
the 45 generations, while CMV activity varied periodically over time,
from 40 to 80 \%ZsGreen1. Even though CMV promoter delivers high gene
expression, some works reported protein production
instability.\textsuperscript{{[}17{]}, {[}18{]}, {[}39{]}--{[}41{]}}
Gene silencing mechanism of this promoter is not clear, although DNA
methylation, histone hypoacetylation, activation of signaling molecules
and altered levels of transcriptional factors have been
suggested.\textsuperscript{{[}11{]}--{[}13{]}, {[}15{]}--{[}17{]},
{[}42{]}--{[}44{]}}

For CHO cell lines, the percentage of each population expressing
moderate (M2) and high (M3) levels of ZsGreen1 during 45 generations,
are shown in Figure S3.A-B (Supporting Information). The lowest \% of
cells expressing ZsGreen1 at high fluorescence intensity (M3) was
observed for CMV promoter, whereas the highest (~70\%) was seen for
Hspa5 promoter (ANOVA test, P<\selectlanguage{english}0.05). Actb and Vim promoters also
exhibited a relatively high proportion of cells expressing ZsGreen1 with
high intensity (~50-70\% in M3). However, no significant differences
were found between them (ANOVA test, P<\selectlanguage{english}0.05). Furthermore, MFI values
among generations were represented for each cell line, distinguishing
between cells expressing ZsGreen1 in M2 and M3 (Figure S4, Supporting
Information). These results demonstrated that all endogenous promoters
presented higher ZsGreen1 intensity in M2 compared to CMV (Figure~S4.A,
Supporting Information).

In Figure 2.C-F, MFI is expressed as fold change, taking CMV as control
(reference value as 1.0). In the case of CHO-K1 cell populations in M2
(Figure 2.C), Ctsz and Hmox1 showed the strongest promoter activity
(2.90-fold and 2.72-fold, respectively), followed by Vim (2.22-fold),
Actb (1.75-fold) and Hspa5 (1.47-fold). When analyzing CHO-K1 cells in
M3 (Figure S4.B, Supporting Information), it was observed that Hspa5,
Actb and Vim promoters drove ZsGreen1 expression with a clearly higher
fluorescence intensity compared to CMV promoter; exhibiting fold-change
values of 1.27, 1.20 and 1.15, respectively (Figure 2.D).

For BHK-21 cells, ZsGreen1 expression stability during 50 generations is
shown in Figure 2.B. In general, less stable and non-uniform
transcriptional activity was observed for BHK-21 cells compare to CHO-K1
cell line. Hspa5-ZsGreen1 cell line exhibited the highest percentage of
positive cells, approximately 100\% after 15 generations (ANOVA test,
P<\selectlanguage{english}0.05). Also, Hspa5 allowed ZsGreen1 expression in a more stable manner
than the rest of endogenous promoters. CMV activity exhibited a slight
variation over time, and the proportion of cells expressing ZsGreen1 was
near 50\% (Figure 2.B). ZsGreen1 expression under the control of Actb
and Hmox1 promoters notably decreased after 10 generations and in the
case of Vim promoter, after 15 generations. Ctsz transcriptional
activity was rather constant, but the percentage of positive cells was
lower than the observed for CMV-ZsGreen1 cell line (t-test, p=0.0006).

The percentage of cells in M3 was extremely low for all BHK cell lines
(Figure S3.D, Supporting Information). The only exception was
Hspa5-promoter containing up to 70\% of positive cells in M3. The
results shown in Figure S4.C (Supporting Information) highlight Hspa5 as
the promoter with the greatest activity in BHK-21 cells, since it
exhibited the highest values of MFI, in M2. In terms of MFI fold-change,
taking CMV as control with a reference value of 1.0 (Figure 2.E-F), only
Hspa5 and Vim showed increased MFI for cells expressing ZsGreen1 in M2
(2.32 and 1.43-fold, respectively). In the case of cells in M3 maker,
Hspa5 exhibited a fold change of 1.30.

\textbf{Effect of temperature shift on stable transgene expression}

A temperature reduction step is commonly used during production cell
culture in the biopharmaceutical
industry.\textsuperscript{{[}45{]}--{[}51{]}} The initial rapid cell
growth phase is performed at 37 \selectlanguage{ngerman}°C followed by a growth arrest phase
when the culture is shifted to a lower temperature (typical 30 - 32~°C).
This change in temperature is used for simultaneously inducing growth
arrest and extending culture viability thus increasing recombinant
protein productivity and yield.

ZsGreen1 stable expression, driven by endogenous promoters and CMV, was
examined in CHO-K1 cells at 32 °C and 37 °C during 10 days (Figure 3).
For temperature shift, 32~°C was selected because the endogenous
promoters were identified based on RNA-seq data of CHO-K1 cells cultured
at 33 - 31 °C during stationary phase.
\textsuperscript{{[}9{]}}Moreover, 32°C represents the average
temperature applied by other studies mentioned before. Figure 3 shows
that almost all promoters enhanced their activity at 32~°C. Student's
t\selectlanguage{english}-test revealed significant differences between ZsGreen1 positive cells
at 37 and 32 degC for the following promoters: Actb, Ctsz and Rps18
(p=0.04, p=0.014, and p=0.01, respectively). In the case of Vim and
Hmox1 promoters, significant differences were found when comparing \% of
cells in M3 (p=0.015 and p=0.014, respectively). No significant
differences were observed for Hspa5. However, it was expected since this
promoter evidenced more than 99\% positive cells.

In particular, CMV exhibited a marked increase in total ZsGreen1
positive cells at lower temperature (1.80\selectlanguage{english}-fold). However, it is
important to consider that this promoter showed expression instability
during several generations (Figure 2.A), which makes it difficult to
ensure that such increment is caused by the temperature reduction.
Interestingly, stable gene expression driven by different endogenous
promoters remained relatively constant or even increased after a
reduction of the culture temperature from 37 degC to 32 degC. Considering
that candidate promoters (except for Rps18) showed expression stability
during 45 generations (Figure 2.A), this is a promising indication of
their potential to be used during biphasic cultures for the industrial
production of recombinant proteins.

\textbf{Characterization of endogenous promoters by \emph{in
silico}analysis of their sequences}

Several factors contribute to the activity of a promoter, such as
genomic cis\selectlanguage{english}-acting sequences, type of vector, cell line and
sequence-specific transcription factors (TFs). These TFs are important
regulators of biological processes that work by binding to
transcriptional regulatory regions, such as promoters, to control the
expression of their target genes. Each TF usually recognizes a
collection of similar DNA sequences known as transcription factor
binding sites (TFBSs), which can be represented as motifs using models
like position weight matrices. \textsuperscript{{[}52{]}} In addition,
AT or GC rich sequences are often located in gene promoters and play a
key role in transcription initiation. Characterization of TFBSs and
other functional sequences is an important first step in understanding
promoter activity, thus we applied \emph{in silico} tools for a more
comprehensive description of endogenous promoters, including prediction
of the TSS, TATA box and putative TFBSs (Table 1). All promoters contain
several putative specificity protein 1 (Sp1) binding sites, and some of
them have other common motifs such as GC\selectlanguage{english}-box, E-box and AP-1. In 4 out
of 6 endogenous promoters (Actb, Hspa5, Rps18 and Vim), a putative
TATA\selectlanguage{english}-binding protein factor (TBP)\selectlanguage{english}-binding site (TATA box) was identified
at an expected distance from the TSS.

Most mammalian promoters (around 60\% of mouse and human promoters)
contain CpG islands, where methylation status of CG dinucleotides and
their open chromatin organization can work as transcriptional
regulators. \textsuperscript{{[}53{]}} Hartl \emph{et al.}
(2019)\textsuperscript{{[}54{]}} revealed that promoters having CpG
islands show higher transcriptional activity than non CpG island
promoters, and they tend to be active in different cell types. Also,
they proposed that high CG content in promoter sequences might support
transcriptional activity in an indirect manner, by increasing DNA
accessibility so facilitating TFs binding. In this study, a putative CpG
island region was found only in Hspa5 endogenous promoter, and in CMV
promoter. Furthermore, GC content of endogenous promoters is relatively
high, ranging from 49 to 60\%, compared to 46\% for CMV promoter and the
average GC content of 42\% of CHO-K1 genome. \textsuperscript{{[}55{]}}

We also found that endogenous promoters have numerous potential TFBSs
(between 23 and 36 sites). Comparatively, CMV has the highest amount of
putative TFBSs. However, no consistent pattern of TFBSs in endogenous
promoter sequences that characterizes their transcriptional activities
was detected. Sp1 was found several times in all promoters.
Transcription factor AP-2 alpha A and regulator C/EBP~alpha were
identified one or two times in 5 out of 6 endogenous promoters. Nuclear
factor 1 (NF1) binding motif was identified between 1 or 2 times in 4
out of 6 endogenous promoters. Particularly, CMV has 4 putative binding
sites for C/EBP~alpha and 7 for NF1. These TFs were analyzed for their
expression on RNA-seq data. \textsuperscript{{[}9{]}} NF1 was
differentially expressed (Benjamini--Hochberg adjusted p\selectlanguage{english}-value
\textless{}0.05), showing higher expression during stationary phase.
AP-2 alpha A has a moderately constant expression during exponential and
stationary stages, and Sp1 expression slightly decreased in stationary
phase.

We also examined sequence conservation of endogenous CHO-K1 promoters by
comparison to mouse, rat and human orthologous genes (Figure S5). As
expected, higher values of sequence similarity were observed between
promoters of CHO-K1, rat and mouse. In the particular case of Actb,
Hspa5 and Vim promoters, sequence similarity ranges between 70 and 80\%.
Hmox1 and Rps18 promoters showed weak correlation between species while
Ctsz exhibited an intermediate correlation, between 60 and 70\%. These
results could explain why in transient transfection assays endogenous
CHO-K1 promoters exhibited higher activity in BHK-21 cells than in
HEK293T cells. Moreover, Actb, Hspa5 and Vim promoters, which showed
high correlation between species, are among the strongest promoters in
CHO-K1, BHK-21 and HEK293T cells.

\textbf{Discussion}

For the industrial protein production a high rate of transgene
expression is desired and hence strong viral\selectlanguage{english}-derived promoters are
commonly used.\textsuperscript{{[}6{]}, {[}56{]}} Constitutive strong
overexpression of the transgene under the control of such promoters,
e.g. CMV, can cause a significant stress on the cell, upregulating the
unfolded protein response and even inducing premature apoptosis.
\textsuperscript{{[}20{]}} Additionally, CMV is susceptible to
epigenetic silencing due to DNA methylation and/or histone
modifications, which is linked to production
instability.\textsuperscript{{[}11{]}--{[}13{]}, {[}18{]}} Endogenous
promoters, which function in a coordinated manner with cellular and
bioprocess characteristics could help to avoid such drawbacks and, at
the same time, maintain high expression levels of the transgene.

In this study, promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18
genes were selected and isolated from CHO-K1 genome for the generation
of new expression vectors. Their smaller size (around 1000 bp), compared
to other CHO-K1 endogenous promoters previously described,
\textsuperscript{{[}24{]}} constitutes an important advantage. Transient
transfections demonstrated that these endogenous promoters exhibited
transcriptional activity not only in CHO-K1 cells but also in BHK-21 and
HEK293T cell lines. Even though other works have identified CHO
endogenous promoters as potential candidates for delivering recombinant
gene expression,\textsuperscript{{[}21{]}, {[}23{]}} here we proved
their potentiality not only in CHO but also in other commonly used cell
lines.

Not only the level of transcription, but also its long-term stability is
critical for recombinant protein production. Only a few studies of
endogenous promoters have shown results of stable cell line
generation;\textsuperscript{{[}19{]}, {[}23{]}} however, no analysis
with respect to expression stability over extended cultivation time was
performed. The analysis of CHO-K1 stable cell lines during several
generations that was performed in this work revealed that ZsGreen1
expression driven by endogenous promoters was markedly higher compared
to CMV, which contains the immediate-early enhancer/promoter region and
a chimeric intron. In BHK-21 cells, endogenous promoters exhibited less
expression stability and non-uniform transcriptional activity.
Particularly, Hspa5 promoter demonstrated to be a proper candidate for
CHO and BHK cell line generation.

Temperature reduction is commonly used during production in the
biopharmaceutical industry to improve protein production and extend
cultivation time. Some reports have studied cold-shock responsive
promoters upon temperature shift in transient
transfections.\textsuperscript{{[}21{]}, {[}22{]}} Here we analyzed
ZsGreen1 stable expression driven by endogenous promoters and CMV, after
a reduction of CHO-K1 culture temperature from 37~degC to 32~degC.
Temperature shift was beneficial for endogenous promoter activity since
it improved significantly the protein productivity, which highlights the
potential use of these promoters for industrial production.

Mammalian promoters have a highly complex structure, not completely
understood yet. Despite this great diversity, common features of all
selected endogenous promoters were identified by \emph{in
silico}analysis, including high GC content, putative TFBSs such as Sp1,
GC\selectlanguage{english}-box, E\selectlanguage{english}-box, AP-1. Hspa5, one of the strongest endogenous promoters,
that exhibited high transcriptional activity for transient and also
stable expression in all cell lines evaluated, contains a potential CpG
island region and numerous TFBSs, which may explain its high
transcriptional activity. Additionally, Hspa5 promoter revealed a strong
sequence correlation between species (mouse, rat and human). It was
observed also for other endogenous promoter candidates, such as Vim and
Actb. Both promoters demonstrated high transcriptional activity in
CHO-K1 and BHK\selectlanguage{english}-21 cells, but expression was stable only in the former
one.

The endogenous promoters characterized in this work were strategically
identified using the RNA\selectlanguage{english}-Seq data of CHO-K1 cells cultured under typical
bioprocess conditions for recombinant protein production (high density
followed by a temperature gradient), in order to search for the ones
with high transcriptional activity under such conditions. These
endogenous promoters constitute a promising alternative to CMV promoter
due to their high strength, long-term expression stability and
integration into the regulatory network of the host cell. These
promoters may also comprise an initial panel for designing cell
engineering strategies and synthetic promoters, as well as for
industrial cell line development.

\textbf{Conflict of interest}

The authors have no conflicts of interest to declare.

\textbf{Data availability}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

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\textbf{Tables}

\textbf{Table 1.} Potential functional promoter elements predicted
by\emph{in silico} analysis.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllll@{}}
\toprule
\textbf{Promoter} & \textbf{Size (bp)} & \textbf{TATA box} &
\textbf{Distance TATA to TSS} & \textbf{\%GC} & \textbf{CpG islands} &
\textbf{Sp1} & \textbf{GC-box} & \textbf{E-box} & \textbf{AP-1} &
\textbf{Total putative TFBS\textsuperscript{a}}\tabularnewline
\midrule
\endhead
\textbf{Actb} & 999 & Yes & 11 bp & 55 & - & 12x & 2x & 3x & - &
31\tabularnewline
\textbf{Ctsz} & 972 & No & - & 56 & - & 12x & 3x & 5x & - &
34\tabularnewline
\textbf{Hmox1} & 900 & No & - & 51 & - & 5x & 2x & 6x & - &
23\tabularnewline
\textbf{Hspa5} & 906 & Yes & 31 bp & 60 & 1x & 17x & 8x & 6x & 1x &
35\tabularnewline
\textbf{Rps18} & 1043 & Yes & 42 bp & 49 & - & 5x & - & 5x & 2x &
25\tabularnewline
\textbf{Vim} & 961 & Yes & 17 bp & 58 & - & 24x & 2x & - & 4x &
36\tabularnewline
\textbf{CMV\textsuperscript{b}} & 1101 & Yes & 374 bp & 46 & 1x & 9x &
2x & 5x & 2x & 59\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a)} TFBSs were predicted using Alibaba2 online
software.

\textsuperscript{b)} This sequence contains the human cytomegalovirus
(CMV) immediate-early enhancer/promoter region and a chimeric intron.

\textbf{Figure Legends}

\textbf{Figure 1.} Effect of different promoters on transient transgene
expression. pZsGreen1-1 vectors containing CMV, Actb, Ctsz, Hmox1,
Hspa5, Rps18 and Vim promoters were transfected into CHO, HEK293T and
BHK-21 cells. \textbf{(A)} ZsGreen1 fluorescence profile of transfected
cells (100x, 200 ms exposure time). \textbf{(B, D, F)} \%ZsGreen1 for
CHO-K1, HEK293T and BHK-21 cells, respectively, co-transfected with new
vectors carrying endogenous promoters and pBKS plasmid. \textbf{(C, E,
G)} \%ZsGreen1 for CHO-K1, HEK293T and BHK-21 cells, respectively,
co-transfected with new vectors carrying endogenous promoters and
pCIneo-CMV-IFN\selectlanguage{greek}α \selectlanguage{english}plasmid. \textbf{M2:} positive cells with moderate
fluorescence intensity, from 10\textsuperscript{1} to
10\textsuperscript{3} FAU (Fluorescence arbitrary units).
\textbf{M3:}positive cells with high fluorescence intensity, FAU values
higher than 10\textsuperscript{3}.

\textbf{Figure 2.} ZsGreen1 expression stability in CHO-K1 and BHK-21
cells lines grown until 45 and 50 generations, respectively, in the
absence of neomycin selection pressure. Total \%ZsGreen1 positive CHO-K1
and BHK-21 cells (\textbf{A} and \textbf{B} , respectively). Moderate
ZsGreen1 MFI (M2) fold change in CHO-K1 and BHK1-21 cells (\textbf{C}and
\textbf{E} , respectively). High ZsGreen1 MFI (M3) fold change in CHO-K1
and BHK-21 cells (\textbf{D} and \textbf{F} ,
respectively).\textbf{MFI:} Mean Fluorescence Intensity. \textbf{M2:}
positive cells with moderate fluorescence intensity, from
10\textsuperscript{1} to 10\textsuperscript{3} FAU (Fluorescence
arbitrary units). \textbf{M3:}positive cells with high fluorescence
intensity, FAU values higher than 10\textsuperscript{3}.

\textbf{Figure 3.} Impact of temperature shift on stable ZsGreen1
expression driven by endogenous promoters and CMV in CHO-K1 cells,
during ten days upon cultivation at 37 \selectlanguage{ngerman}°C or 32~°C. Bars show \% of
positive cells expressing moderate (black) and high (gray) levels of
ZsGreen1 from samples taken on days 3, 5, 7 and 10. \textbf{M2:}positive
cells with moderate fluorescence intensity, from 10\textsuperscript{1}
to 10\textsuperscript{3} FAU (Fluorescence arbitrary units).
\textbf{M3:} positive cells with high fluorescence intensity, FAU values
higher than 10\textsuperscript{3}.\selectlanguage{english}
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