Genomic integration and ligand-dependent activation of the human estrogen receptor α in the crustacean Daphnia magna
Genomic integration and ligand-dependent activation of the human estrogen receptor α in the crustacean Daphnia magna
Kerstin ToÈ rner 0 1
Tsuyoshi Nakanishi 1
Tomoaki Matsuura 0 1
Yasuhiko Kato 0 1
Hajime Watanabe 0 1
0 Department of Biotechnology, Graduate School of Engineering, Osaka University , Yamadaoka, Suita, Osaka , Japan , 2 Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu Pharmaceutical University , Daigaku-nishi, Gifu, Gifu , Japan , 3 Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University , Yamadaoka, Suita, Osaka , Japan
1 Editor: Toshi Shioda, Massachusetts General Hospital , UNITED STATES
The freshwater crustacean Daphnia have a long history in water quality assessments and now lend themselves to detection of targeted chemicals using genetically encoded reporter gene due to recent progress in the development of genome editing tools. By introducing human genes into Daphnia, we may be able to detect chemicals that affect the human system, or even apply it to screening potentially useful chemicals. Here, we aimed to develop a transgenic line of Daphnia magna that contains the human estrogen receptor alpha (hERα) and shows a fluorescence response to exposure of estrogens. We designed plasmids to express hERα in Daphnia (EF1α1:esr1) and to report estrogenic activity via red fluorescence (ERE:mcherry) under the control of estrogen response element (ERE). After confirmation of functionality of the plasmids by microinjection into embryos, the two plasmids were joined, a TALE site was added and integrated into the D. magna genome using TALEN. When the resulting transgenic Daphnia named the ES line was exposed to Diethylstilbestrol (DES) or 17β-Estradiol (E2), the ES line could reliably expressed red fluorescence derived from mCherry in a ligand-dependent manner, indicating that an estrogen-responsive line of D. magna was established. This is the first time a human gene was expressed in Daphnia, showcasing potential for further research.
Data Availability Statement; All relevant data are within the paper
Funding: One of the authors (YK) acknowledges
the support of the Frontier Research Base for
Global Young Researchers, Osaka University. This
work was supported by Japan Society for the
Promotion of Science KAKENHI Grant Number
JP26281027. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
The crustacean Daphnia, as keystone species of freshwater ecosystems, have been used in
water quality assessments for many decades [
]. High growth rate and high fecundity
combined with cheap and easy rearing make them an excellent organism for screening. Recently,
the sequencing of the Daphnia pulex and Daphnia magna genomes [
] has been completed
and different methods for genomic integration of foreign genes like CRISPR/Cas [
TALEN-mediated knockin [
] have been successfully adapted in D. magna. In order to
facilitate the expression of ectopic gene, an optimal mRNA structure in D. magna was also reported
. These progress of functional genomics allowed us for detecting one of arthropod
hormones, ecdysteroid using a GFP reporter gene under control of ecdysteroid responsive
elements in this species [
]. In addition, a candidate of an element responsive to another
arthropod hormone, juvenile hormone, was integrated together with its reporter GFP gene
into the genome [
]. These reporter constructs and transgenic lines have potential as tools to
detect agonists of ecdysteroids and juvenile hormones that are widely used as pesticides, which
can be used as a chemical sensor Daphnia.
Based on the progress of the development of chemical sensor Daphnia, we aimed to detect
chemicals that affect the human system [11±12] by making sensor Daphnia. In this study, we
aimed to develop estrogen sensor Daphnia by introducing human genes, as Daphnia hormonal
system is different from human and estrogen system does not exist in Daphnia. Estrogens and
estrogen like chemicals bind to estrogen receptors (ER), the resulting complex interacts with
Estrogen Response Elements (ERE) on the target gene of genome and activate transcription of
downstream genes [13±15]. In this study, we designed and integrated a plasmid to express the
human estrogen receptor alpha (hERα) (EF1α1:esr1) and to report estrogenic activity via red
fluorescence (ERE:mcherry) into the genome of D. magna using a set of previously established
]. The resulting estrogen sensor line (ES line) reliably indicates presence of
Diethylstilbestrol (DES), a synthetic estrogen, as well as that of 17β-Estradiol (E2), a natural estrogen.
To our knowledge this is the first time a human gene has been successfully expressed in
Daphnia, showcasing the potential to test the response of different human genes to environmental
stimuli relatively directly, and therefore determine potential health impacts in both animals
The genetic sequence for genomic integration was prepared on two separate DNA plasmids.
One expresses the human estrogen receptor α (hERα) (Fig 1A) ubiquitously. The other
plasmid (Fig 1B) contains 4x repeats of the Estrogen Response Element (ERE), mCherry as a visible
reporter, and the EF1α1 3'UTR truncated to 60 bp plus its poly(A) signal for higher RNA
stability and/or translation efficiency [
]. The sequence between the ERE repeats and the mCherry
start codon is the same as that between the EcRE (Ecdysteroid response element) repeats and
its reporter start codon in a previously established ecdysteroid reporter in this species [
4xEcRE reporter was used as a positive control in this experiment (Fig 1C).
To test the functionality of these plasmids, microinjection was conducted with wild-type
(NIES) Daphnia eggs. Fifty ng/μL pRC21-hERa plasmid, 50 ng/μL pRC21-ERE:mCherry
plasmid and 26.8 ng/μL (100 μM) DES were injected as single solution, in combinations of two or
all three together. When only 1/3rd (data not shown) or 2/3rd of the components were injected
(Fig 1E±1G), no red fluorescence could be detected after 18 h, similar to uninjected control
eggs (Fig 1D). When injecting the two plasmids together with an estrogenic compound, DES,
on the other hand, fluorescence could be detected (Fig 1H), as well as after injection of the
pRC21-EcRE:mCherry control plasmid that responds to endogenous ecdysteroids (Fig 1I).
Thus hERα is functional and active in an estrogen-dependent manner in D. magna embryos.
We also concluded that neither hERα nor ERE are activated by endogenous compounds in D.
magna eggs of this stage.
For genomic integration, we targeted the dsred2 locus of a previously generated transgenic
]. To make the donor plasmid DNA, the hERα expression cassette and ERE reporter
gene were cloned into a single plasmid with the target site of TALENs that cleave the dsRed2
]. Integration of the donor DNA into the DsRed2 locus causes the loss of red
fluorescence in absence of estrogenic compounds. We injected this donor plasmid (25 ng/μL)
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Fig 1. Structure of injected plasmids (A-C) and wild type Daphnia embryos injected with the plasmids at 50 ng/μL each and 26.8 ng/μL (100 μM)
DES, at 16 hpi (D-I). (A) pRC21-hERa. Human estrogen receptor α (esr1) with EF1α1 promoter and full length EF1α1 3'UTR. (B) pRC21-ERE:
mCherry. Mcherry with 4 x ERE repeats and a truncated version of the EF1α1 3'UTR. (C) pRC21-EcRE:mCherry. Mcherry with 4 x EcRE repeats and a
truncated version of the EF1α1 3'UTR. Lengths of the elements (not to scale) in bp are indicated underneath. (D) negative control (uninjected), (E)
injection of pRC21-hERa and pRC21-ERE:mCherry, (F) injection of pRC21-ERE:mCherry and DES, (G) injection of pRC21-hERa and DES, (H)
injection of pRC21-hERa, pRC21-ERE:mCherry and DES, (I) injection of control plasmid pRC21-EcRE:mCherry. Successful activation of the reporter is
marked with white arrowheads. Bar = 100 μm.
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together with in vitro synthesized mRNAs that code for DsRed2-targeting TALENs (250 ng/μL
each). Of the 106 embryos surviving at 1 hpi, one (0.94%) that showed a loss of red
fluorescence survived into adulthood. We named this transgenic animal ªES lineº.
We then exposed neonate Daphnia of this ES line (under 24 h old) to 2 mg/L DES for seven
days to find the optimal time-point for exposure evaluation. According to the values of total
fluorescence of the thoracic appendages, day four showed the highest difference between
control and exposed individuals (Fig 2) so all later exposures were conducted for four days. When
testing this line for sensitivity regarding DES and E2, we exposed them to 0.1 mg/L± 2.0 mg/L
DES and 0.1 mg/L± 4.0 mg/L E2. In these experiments, the detection threshold for DES was
0.5 mg/L and 4 mg/L for E2 (see Fig 3, Fig 4).
The new estrogen responsive line of transgenic ES D. magna could successfully detect both
DES and E2 at different concentrations and with different sensitivity, in accordance with their
different binding affinities to hERα [
]. There was no fluorescence response without any
exposure of estrogenic compounds even though the control EcRE-reporter gene [
detect endogenous ecdysteroids (Fig 1). The general background fluorescence in the transgenic
Daphnia was relatively low so it is sensitive enough to detect estrogenic chemicals four days
after exposure (Fig 2). This result is consistent with previous finding that Daphnia do not have
any endogenous estrogens nor any ERs [
]. This study is the pioneering demonstration that
hERα can function in Daphnia in vivo.
However it was notable that sensitivity of the transgenic Daphnia to estrogen was ranging
in the low mg/L. Compared to existing biosensors for estrogen like zebrafish and medaka
] or yeast, bacteria, and mammalian cell cultures [20±22], the sensitivity of this line was
quite low. One of the future perspective of the use of the estrogen sensing Daphna may be an
application to environment assessment or monitoring. In order to detect low concentration of
estrogens in the ng/L ranges as a biosensor, which is where serious environmental effects can
already occur [23±25], further improvement is necessary. This sensitivity could be improved
by improving several points. One of the major problem may be a cofactor which bridges
estrogen receptor and transcriptional machinery. In order to monitor Daphnia nuclear receptor
(ecdysone receptor) function in mammalian cells, we have introduced Taiman as a cofactor,
which suggested that direct interaction between daphnia nuclear receptor and mammalian
transcriptional machinery is weak. Similarly interaction between human nuclear receptor and
daphnia transcriptional machinery may be weak and introduction of a cofactor may improve
its sensitivity. It may be also useful for testing further iterations of the sequence between ERE
and the mCherry start codon , by optimizing codon usage for Daphnia [27±29], or by
integration into a different genetic location.
Red fluorescence was expected and found all over the body of the exposed waterfleas, with
stronger fluorescence in digestive tissues from feeding and the hepatopancreas which is
involved in sequestering hormones taken up by feeding [
]. Stronger fluorescence was also
detected in the ªjointsº of secondary antennae which are responsible for locomotion.
To express hERα we used the promoter of D. magna EF1a1 which produces highly
abundant mRNA in this species [
], which could easily lead to over-expression of hERα. And
indeed reproduction was slowed down in the ES line compared to both a wild-type strain of D.
magna (NIES) and the dsred2 line. Therefore particularly the EF1α1:esr1 sequence might need
to be adjusted to lower expression levels by truncating the promoter sequence or by using
promoters of the other ubiquitously expressed genes such as ribosomal protein L32 and β-actin,
which show moderate expression compare to EF1.
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Fig 2. Fluorescence of thoracic appendages of ES Daphnia exposed to 2 mg/L DES over seven days, pictures taken every 24h.
Normalized to fluorescence of control Daphnia of the same age, units are arbitrary (a.u.). Significant differences ( ) compared to control
(p<0.002) at day 4 and from day 7 onward.
Nevertheless, both hERα and ERE could be shown to be functional in Daphnia for the first
time, suggesting a huge potential for use of Daphnia to study the interaction of human genes
with environmental factors like the effect of EDCs in this study. This could be applied to
improved biomonitoring of water quality, or even to screen potentially useful chemicals.
Daphnia cells can conduct the post-translational modifications necessary for formation of
active hERα [
], as shown by the estrogen-dependent fluorescence response of ES line
Daphnia from both active hERα and its successful activation of the ERE. These results open up the
possibilities to test not only single receptors or other genes in Daphnia, but partial or even
whole pathways for environmental effects on them, especially with still improving methods for
genome editing in this species that allow for larger and larger sizes of inserted DNA [
It would be interesting to cross this line with other transgenic lines of Daphnia and visualize
more than one stressor at a time. And to increase ease of use, it would be beneficial to
implement a reporter that is not based in fluorescence but that can be detected by the naked eye or a
simple light microscope. Over-expression of hemoglobin and the subsequent redder color of
D. magna [
] is a promising approach, as is black color from expressing melanin or other
darker pigments which are usually only found in Daphnia species exposed to higher UV
radiation like Daphnia melanica [
In conclusion, for the first time, a human gene (esr1) was successfully and stably expressed
in the crustacean D. magna. The resulting new transgenic line of D. magna could indicate
presence of both DES and E2 after exposure in a dose-dependent manner.
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Fig 3. Concentration dependent response to DES. (A) ES Daphnia exposed to different concentrations of DES, pictures taken at day 4, bar = 100 μm. (B) Fluorescence
calculated from thoracic appendages, normalized to control Daphnia of the same age. Asterisks ( ) indicating p<0.002 compared to control. Dotted lines marking the
gut (autofluorescence from fed algae).
Materials and methods
A transgenic line of Daphnia magna containing a hemizygous DsRed2 gene under the control of the
D. magna EF1α-1 promoter was generated previously [
] from a D. magna NIES clone (obtained
from the National Institute for Environmental Studies, NIES; Tsukuba, Japan). This dsred2 line has
been maintained for more than 50 generations. It exhibits ubiquitous DsRed2 expression.
Daphnia culture conditions
Eighty neonates of dsred2 Daphnia (under 24 h old) were cultured in 5 L of the Aachener
Daphnien Medium (AdaM) [
] at 22±24ÊC under a light/dark cycle of 16/8 h. The culture
medium was renewed once a week. Daphniids were fed every day with 5 mg of Chlorella
vulgaris (Nikkai Center, Tokyo, Japan) during the first week. After maturation, offspring was
removed daily and adults were fed with 10 mg Chlorella per day.
After establishment of the estrogen responsive line (ES line), ES daphnia were cultured
under the same conditions with the exception of feeding and juvenile removal. ES daphniids
were fed every other day with 6 mg of Chlorella vulgaris (Nikkai Center, Tokyo, Japan) until
maturation. Then, offspring was removed twice a week and adults were fed with 10 mg
Chlorella every other day.
All methods regarding animal use were carried out in accordance with the relevant
guidelines and regulations.
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Fig 4. Concentration dependent response to E2. (A) ES Daphnia exposed to different concentrations of E2, pictures taken at day 4, bar = 100 μm. (B) Fluorescence
calculated from thoracic appendages, normalized to control Daphnia of the same age. Asterisks ( ) indicating p<0.002 compared to control. Dotted lines marking the
gut (autofluorescence from fed algae).
Microinjection was conducted according to established procedures [
]. In short, adult D.
magna (dsred2 line) with empty brood chambers were selected and observed until ovulation
started. Then D. magna were transferred to ice-chilled M4 medium [
] containing 80 mM
sucrose (M4-suc) and dissected to collect the eggs within the brood chamber. The eggs were
stored in ice-chilled M4-suc medium until injection to slow the hardening of the egg
membrane. Microinjection was performed within 1 hour post ovulation (hpi) for the same reason.
Successfully manipulated eggs were transferred to fresh medium and were cultured in a
96-well plate at 23 ± 1ÊC. From each clutch of eggs, 2±3 eggs that were not injected served as
control for development.
To generate the hERα expression plasmid, full length D. magna EF1α-1 promoter [
] and full
length human esr1 [
] were joined into a pRC21 backbone with full length EF1α-1 3'UTR via
InFusion (TAKARA, Kusatsu, Shiga, Japan); the resulting construct was termed pRC21-hERa.
To generate the EcRE reporter plasmid, full length mCherry and the first 60 bp of EF1α-1
] were joined into a pRC21 backbone containing the last 13bp of EF1α-1 3'UTR and
therefore providing a poly(A) signal via InFusion (TAKARA). A 4xEcRE promoter [
amplified via polymerase chain reaction (PCR) with primers introducing a restriction site for
MscI. Both the PCR fragment and the mCherry plasmid were digested with MscI and
EcoO109I (NewEngland BioLabs, Ipswitch, MA, USA) and joined via MightyMix ligation
(TAKARA); the resulting construct was termed pRC21-EcRE:mCherry.
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To generate the ERE reporter plasmid, the EcRE repeats were excised out of pRC21-EcRE:
mCherry with EcoO109I and XmaI (NewEngland BioLabs). A 4xERE sequence [
amplified with primers introducing a restriction site for XmaI, it contains an EcoO109I site.
This PCR fragment was also digested with EcoO109I and XmaI (NewEngland BioLabs) and
joined into the backbone plasmid via MightyMix ligation (TAKARA); the resulting construct
was termed pRC21-ERE:mCherry.
For genomic integration, pRC21-hERa as the backbone was digested with SalI and NdeI
(NewEngland BioLabs). pRC21-ERE:mCherry was digested with BssHII and NdeI
(NewEngland BioLabs). The dsred2 TALE site was amplified from genomic DNA with PCR primers
introducing digestion sites for SalI and BssHII. After digestion, all three fragments were joined
via MightyMix ligation (TAKARA); in the resulting construct the ERE reporter and the ER
halves face opposite directions, it was termed pRC21-estrogensensor.
All plasmids were transformed into XL10-GOLD E. coli after ligation, harvested with a
PureYield Miniprep kit (PROMEGA, Fitchburg, WI, USA), purified by phenol/chloroform
extraction followed by ethanol precipitation and their sequence was confirmed by sequence
In vitro RNA synthesis
For TALEN mRNA synthesis, left and right TALEN expression plasmids [
] were linearized
with Acc65I (NewEngland BioLabs), and purified using the QIAquick PCR purification kit
(QIAGEN GmbH, Hilden, Germany). Linearized DNA fragments were used for in vitro
transcription with the mMessage mMachine kit (Life Technologies, CA, USA). Poly(A) tails
were attached to TALEN RNAs using a Poly(A) Tailing Kit (Life Technologies), following
the manufacturer's instructions. The synthesized RNAs were column purified using the
Rneasy Mini Kit (QIAGEN GmbH, Hilden, Germany), followed by phenol/chloroform
extraction, ethanol precipitation, and resuspension in DNase/RNase-free ultra pure water
Diethylstilbestrol (DES) (Sigma Aldrich, St. Louis, MO USA) and 17β-Estradiol (E2) (Sigma
Aldrich) were dissolved in 100% N,N-Dimethylformamide (DMF) (Nacalai tesque, Kyoto,
Japan) to final concentrations of 10 mg/mL as stock solutions. For exposure, the stock was
diluted with AdaM [
] to final solvent concentrations of under 0.2%. For exposure, neonate
daphniids (under 24 h old) were kept in 24 well plates (Thermo Fisher Scientific, Waltham,
MA USA), one individual in 2 mL medium per well, at 23 ± 1ÊC under a light/dark cycle of 16/
8 h, with medium renewal every day.
Daphnia were partially immobilized in minimal amounts of medium on micro slide glasses
(Matsunami, Osaka, Japan). Red fluorescence intensity was recorded with a color digital
camera (Leica DC500) mounted on a Leica M165C fluorescence microscope (Leica
Microsystems Heidelberg GmbH, Mannheim, Germany) equipped with a 545 nm excitation and
a 620 nm barrier filter. The pictures were taken under 63 × magnification with 100%
aperture, 1 s exposure, 3.0 gain, 1.5 saturation, and 1.0 gamma. The red fluorescence intensity
of neonates was recorded every 24 h in the first exposure experiment, at day 4 of exposure
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Fluorescence intensity (fluo) was quantified using ImageJ software. Previously reported
] were adapted to reduce background interference. In this study, only the area of
thoracic appendages (thorap) was used for calculations with the following formula.
thorap total f luorescence of thoracic appendages
number of pixels of the selected area
mean of three background f luorescence measurements
This value was then normalized for exposed Daphnia by defining the value of unexposed
Daphnia of the same age as 1.
One of the authors (Y.K.) acknowledges the support of the Frontier Research Base for Global
Young Researchers, Osaka University. This work was supported by JSPS KAKENHI Grant
Funding acquisition: Hajime Watanabe.
Investigation: Kerstin ToÈrner, Hajime Watanabe.
Methodology: Yasuhiko Kato, Hajime Watanabe.
Project administration: Hajime Watanabe.
Resources: Tsuyoshi Nakanishi.
Supervision: Yasuhiko Kato, Hajime Watanabe.
Validation: Kerstin ToÈrner, Tomoaki Matsuura, Hajime Watanabe.
Writing ± original draft: Kerstin ToÈrner.
Writing ± review & editing: Yasuhiko Kato, Hajime Watanabe.
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1. Gersich FM , Blanchard FA , Applegath SL , Park CN . The precision of daphnid (Daphnia magna Straus, 1820) static acute toxicity tests . Arch Environ Contam Toxicol . 1986 ; 15 : 741 ± 749 . PMID: 3789811
2. Martins J , Oliva Teles L , Vasconcelos V . Assays with Daphnia magna and Danio rerio as alert systems in aquatic toxicology . Environ Int . 2007 ; 33 : 414 ± 25 . https://doi.org/10.1016/j.envint. 2006 . 12 .006 PMID: 17300839
3. Colbourne JK , Pfrender ME , Gilbert D , Thomas WK , Tucker A , Oakley TH , et al. The ecoresponsive genome of Daphnia pulex . Science . 2011 ; 331 : 555 ± 61 . Available: http://www.ncbi.nlm.nih.gov/ pubmed/21292972 https://doi.org/10.1126/science.1197761 PMID: 21292972
4. Orsini L , Decaestecker E , De Meester L , Pfrender ME , Colbourne JK . Genomics in the ecological arena . Biol Lett . 2011 ; 7: 2±3 . https://doi.org/10.1098/rsbl. 2010 .0629 PMID: 20702453
5. Nakanishi T , Kato Y , Matsuura T , Watanabe H. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna . PLoS One . 2014 ; 9 . https://doi.org/10.1371/journal.pone. 0098363 PMID: 24878568
6. Naitou A , Kato Y , Nakanishi T , Matsuura T , Watanabe H. Heterodimeric TALENs induce targeted heritable mutations in the crustacean Daphnia magna . Biol Open . 2015 ; 4 : 364 ± 369 . https://doi.org/10. 1242/bio.20149738 PMID: 25681393
7. Nakanishi T , Kato Y , Matsuura T , Watanabe H . TALEN-mediated homologous recombination in Daphnia magna . Sci Rep . 2015 ; 5 . https://doi.org/10.1038/srep18312 PMID: 26674741
8. ToÈrner K , Nakanishi T , Matsuura T , Kato Y , Watanabe H . Optimization of mRNA design for protein expression in the crustacean Daphnia magna . Mol Genet Genomics . 2014 ; 289 . https://doi.org/10.1007/ s00438-014 -0921-6 PMID: 25234163
9. Asada M , Kato Y , Matsuura T , Watanabe H . Visualization of ecdysteroid activity using a reporter gene in the crustacean , Daphnia. Mar Environ Res . 2014 ; 93 . https://doi.org/10.1016/j.marenvres. 2013 . 11 . 005 PMID: 24296240
10. Nakanishi T , Kato Y , Matsuura T , Watanabe H . TALEN-mediated knock-in via non-homologous end joining in the crustacean Daphnia magna . Sci Rep . Nature Publishing Group; 2016 ; 6 : 36252 . https:// doi.org/10.1038/srep36252 PMID: 27819301
11. Gunnarsson L , Jauhiainen A , Kristiansson E , Nerman O , Larsson DGJ ( 2008 ) Evolutionary conservation of human drug targets in organisms used for environmental risk assessments . Environ Sci Technol 42 : 5807 ± 5813 . https://doi.org/10.1021/es8005173 PMID: 18754513
12. Ma L ( 2009 ) Endocrine disruptors in female reproductive tract development and carcinogenesis . Trends Endocrinol Metab 20 : 357 ± 363 . https://doi.org/10.1016/j.tem. 2009 . 03 .009 PMID: 19709900
13. Eyster KM . The Estrogen Receptors: An Overview from Different Perspectives . Methods Mol Biol . 2016 ; 1366 : 1± 10 . https://doi.org/10.1007/978-1- 4939 -3127- 9 _1 PMID: 26585122
14. Klinge CM . Estrogen receptor interaction with estrogen response elements . 2001 ; 29 : 2905 ± 2919 . PMID: 11452016
15. Nilsson S , MaÈkelaÈ S , Treuter E , Tujague M , Thomsen J , Andersson G , et al. Mechanisms of Estrogen Action. Physiol Rev . 2001 ; 81 : 1535 ± 1565 . https://doi.org/10.1152/physrev. 2001 . 81 .4.1535 PMID: 11581496
16. Folmar LC , Hemmer MJ , Denslow ND , Kroll K , Chen J , Cheek A , et al. A comparison of the estrogenic potencies of estradiol, ethynylestradiol, diethylstilbestrol, nonylphenol and methoxychlor in vivo and in vitro . Aquat Toxicol . 2002 ; 60 : 101 ± 110 . PMID: 12204590
17. Baker ME . Trichoplax, the simplest known animal, contains an estrogen-related receptor but no estrogen receptor: Implications for estrogen receptor evolution . Biochem Biophys Res Commun . 2008 ; 375 : 623 ± 627 . https://doi.org/10.1016/j.bbrc. 2008 . 08 .047 PMID: 18722350
18. Kurauchi K , Nakaguchi Y , Tsutsumi M , Hori H , Kurihara R , Hashimoto S , et al. In vivo visual reporter system for detection of estrogen-like substances by transgenic medaka . Environ Sci Technol . 2005 ; 39 : 2762 ± 2768 . https://doi.org/10.1021/es0486465 PMID: 15884374
19. Gorelick DA , Halpern ME . Visualization of estrogen receptor transcriptional activation in zebrafish . Endocrinology . 2011 ; 152 : 2690 ± 703 . https://doi.org/10.1210/en.2010-1257 PMID: 21540282
20. Legler J , van den Brink CE , Brouwer A , Murk AJ , van der Saag PT , Vethaak AD , et al. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line . Toxicol Sci An Off J Soc Toxicol . 1999 ; 48 : 55 ± 66 .
21. Leskinen P , Michelini E , Picard D , Karp M , Virta M. Bioluminescent yeast assays for detecting estrogenic and androgenic activity in different matrices . Chemosphere . 2005 ; 61 : 259 ± 266 . https://doi.org/ 10.1016/j.chemosphere. 2005 . 01 .080 PMID: 16168749
22. Furst AL , Hoepker AC , Francis MB . Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor . ACS Cent Sci . 2017 ; 3 : 110 ± 116 . https://doi.org/10.1021/acscentsci.6b00322 PMID: 28280777
23. Kidd KA , Blanchfield PJ , Mills KH , Palace VP , Evans RE , Lazorchak JM , et al. Collapse of a fish population after exposure to a synthetic estrogen . 2007 ; 104 : 8897 ± 8901 . https://doi.org/10.1073/pnas. 0609568104 PMID: 17517636
24. Adeel M , Song X , Wang Y , Francis D , Yang Y . Environmental impact of estrogens on human, animal and plant life: A critical review . Environ Int . 2017 ; 99 : 107 ± 119 . https://doi.org/10.1016/j.envint. 2016 . 12 . 010 PMID: 28040262
25. Hanson AM , Ickstadt AT , Marquart DJ , Kittilson JD , Sheridan MA . Environmental estrogens inhibit mRNA and functional expression of growth hormone receptors as well as growth hormone signaling pathways in vitro in rainbow trout (Oncorhynchus mykiss) . Gen Comp Endocrinol . 2017 ; 246 : 120 ± 128 . https://doi.org/10.1016/j.ygcen. 2016 . 07 .002 PMID: 27388662
26. Nardulli AM , Romine LE , Carpo C , Greene GL , Rainish B . Estrogen receptor affinity and location of consensus and imperfect estrogen response elements influence transcription activation of simplified promoters . Mol Endocrinol . 1996 ; 10 : 694 ± 704 . https://doi.org/10.1210/mend.10.6.8776729 PMID: 8776729
27. Ayyar BV , Arora S , Ravi SS . Optimizing antibody expression: The nuts and bolts . Methods . 2017 ; 116 : 51 ± 62 . https://doi.org/10.1016/j.ymeth. 2017 . 01 .009 PMID: 28163103
28. Webster GR , Teh AY -H, Ma JK-C. Synthetic gene design-The rationale for codon optimization and implications for molecular pharming in plants . Biotechnol Bioeng . 2017 ; 114 : 492 ± 502 . https://doi.org/ 10.1002/bit.26183 PMID: 27618314
29. Comba S , Arabolaza A , Gramajo H . Emerging engineering principles for yield improvement in microbial cell design . Comput Struct Biotechnol J . 2012 ; 3: e201210016 . https://doi.org/10.5936/csbj.201210016 PMID: 24688676
30. Mykles DL . Ecdysteroid metabolism in crustaceans . J Steroid Biochem Mol Biol . 2011 ; 127 : 196 ± 203 . https://doi.org/10.1016/j.jsbmb. 2010 . 09 .001 PMID: 20837145
31. Kato Y , Matsuura T , Watanabe H . Genomic Integration and Germline Transmission of Plasmid Injected into Crustacean Daphnia magna Eggs . PLoS One . 2012 ; 7 . https://doi.org/10.1371/journal.pone. 0045318 PMID: 23028929
32. Leader JE , Wang C , Popov VM , Fu M , Pestell RG . Epigenetics and the estrogen receptor . Ann N Y Acad Sci . 2006 ; 1089 : 73 ± 87 . https://doi.org/10.1196/annals.1386.047 PMID: 17261756
33. Kumagai H , Nakanishi T , Matsuura T , Kato Y , Watanabe H. CRISPR /Cas-mediated knock-in via nonhomologous end-joining in the crustacean Daphnia magna . PLoS One. Public Library of Science; 2017 ; 12 : e0186112. https://doi.org/10.1371/journal.pone. 0186112 PMID: 29045453
34. Gorr TA , Cahn JD , Yamagata H , Bunn HF . Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent . J Biol Chem . 2004 ; 279 : 36038 ± 47 . https://doi. org/10.1074/jbc.M403981200 PMID: 15169764
35. Ha MH , Choi J. Effects of environmental contaminants on hemoglobin gene expression in Daphnia magna: a potential biomarker for freshwater quality monitoring . Arch Env Contam Toxicol . 2009 ; 57 : 330 ±7. https://doi.org/10.1007/s00244-007 -9079-0 PMID: 19471991
36. Rhode SC , Pawlowski M , Tollrian R. The impact of ultraviolet radiation on the vertical distribution of zooplankton of the genus Daphnia . Nature . 2001 ; 412 : 69 ± 72 . https://doi.org/10.1038/35083567 PMID: 11452307
37. Schumpert CA , Dudycha JL , Patel RC . Development of an efficient RNA interference method by feeding for the microcrustacean Daphnia . BMC Biotechnol . 2015 ; 15 : 91 . https://doi.org/10.1186/s12896- 015-0209 -x PMID : 26446824
38. KluÈttgen B , DuÈlmer U , Engels M , Ratte HT . ADaM, an artificial freshwater for the culture of zooplankton . Water Res . 1994 ; 28 : 743 ± 746 . https://doi.org/10.1016/ 0043 - 1354 ( 94 ) 90157 - 0
39. Kato Y , Shiga Y , Kobayashi K , Tokishita S , Yamagata H , Iguchi T , et al. Development of an RNA interference method in the cladoceran crustacean Daphnia magna . Dev Genes Evol . 2011 /02/18. 2011 ; 220 : 337 ± 345 . https://doi.org/10.1007/s00427-011 -0353-9 PMID: 21327957
40. Elendt B , Bias W. Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing . Effects of the optimization of culture conditions on life history parameters of D. magna . Water Res . 1990 ; 24 : 1157 ± 1167 . https://doi.org/10.1016/ 0043 - 1354
41. Zhang Z , Hu Y , Guo J , Yu T , Sun L , Xiao X , et al. Fluorene-9 -bisphenol is anti-oestrogenic and may cause adverse pregnancy outcomes in mice . Nat Commun . 2017 ; 8 : 14585 . https://doi.org/10.1038/ ncomms14585 PMID: 28248286
42. Yoshioka H , Hiromori Y , Aoki A , Kimura T , Fujii-Kuriyama Y , Nagase H , et al. Possible aryl hydrocarbon receptor-independent pathway of 2, 3 , 7 , 8 - tetrachlorodibenzo-p -dioxin-induced antiproliferative response in human breast cancer cells . Toxicol Lett . 2012 ; 211 : 257 ± 265 . https://doi.org/10.1016/j. toxlet. 2012 . 04 .005 PMID: 22521833
43. Gavet O , Pines J . Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis . Dev Cell . 2010 ; 18 : 533 ± 43 . https://doi.org/10.1016/j.devcel. 2010 . 02 .013 PMID: 20412769
44. Potapova TA , Sivakumar S , Flynn JN , Li R , Gorbsky GJ . Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited . Mol Biol Cell . 2011 ; 22 : 1191 ± 1206 . https://doi.org/10.1091/mbc.E10-07-0599 PMID: 21325631