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J. Biol. Chem., Vol. 276, Issue 4, 2775-2779, January 26, 2001
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From the Departments of Medicine, Physiology, and
Pediatrics, Cardiovascular Research Institute, University of
California, San Francisco, California 94143-0521
Received for publication, September 7, 2000, and in revised form, October 3, 2000
Hereditary non-X-linked nephrogenic diabetes
insipidus (NDI) is caused by mutations in the aquaporin-2 (AQP2)
water channel. In transfected cells, the human disease-causing mutant
AQP2-T126M is retained at the endoplasmic reticulum (ER) where it is
functional and targetable to the plasma membrane with chemical
chaperones. A mouse knock-in model of NDI was generated by targeted
gene replacement using a Cre-loxP strategy. Along with T126M, mutations
H122S, N124S, and A125T were introduced to preserve the consensus
sequence for N-linked glycosylation found in human AQP2.
Breeding of heterozygous mice yielded the expected Mendelian
distribution with 26 homozygous mutant offspring of 99 live births. The
mutant mice appeared normal at 2-3 days after birth but failed to
thrive and generally died by day 6 if not given supplemental fluid.
Urine/serum analysis showed a urinary concentrating defect with serum
hyperosmolality and low urine osmolality that was not increased by a V2
vasopressin agonist. Northern blot analysis showed up-regulated
AQP2-T126M transcripts of identical size to wild-type AQP2. Immunoblots
showed complex glycosylation of wild-type AQP2 but mainly
endoglycosidase H-sensitive core glycosylation of AQP2-T126M indicating
ER-retention. Biochemical analysis revealed that the AQP2-T126M protein
was resistant to detergent solubilization. Kidneys from mutant mice showed collecting duct dilatation, papillary atrophy, and unexpectedly, some plasma membrane AQP2 staining. The severe phenotype of the AQP2
mutant mice compared with that of mice lacking kidney water channels
AQP1, AQP3, and AQP4 indicates a critical role for AQP2 in neonatal
renal function in mice. Our results establish a mouse model of human
autosomal NDI and provide the first in vivo biochemical data on a disease-causing AQP2 mutant.
The formation of concentrated urine by the kidney requires high
osmotic water permeability across the collecting duct epithelium. Collecting duct epithelial cells express aquaporin water channels AQP2,1 AQP3, and AQP4 (1-4).
AQP2 is the vasopressin (antidiuretic hormone)-regulated water channel
(5, 6). Vasopressin induces the fusion of intracellular vesicles
containing AQP2 with the apical plasma membrane resulting in increased
water permeability (7-9). AQP2 is of considerable clinical importance
in fluid and electrolyte balance. Mutations in human AQP2 cause
hereditary non-X-linked nephrogenic diabetes insipidus (10-13).
Down-regulation of AQP2 expression occurs in several forms of acquired
NDI (14), and AQP2 up-regulation is important in the pathophysiology of fluid-retaining states such as congestive heart failure (15, 16). AQP3
and AQP4 are expressed constitutively at the basolateral membrane of
collecting duct epithelial cells, with AQP3 mainly in cortical
collecting duct and AQP4 in inner medullary collecting duct. Transgenic
mice lacking AQP3 have low water permeability in cortical collecting
duct and manifest NDI with marked polyuria and decreased urine
osmolality (17). In contrast, mice lacking AQP4 show only a mild defect
in urinary concentrating ability (18) despite a 4-fold reduction in
transepithelial water permeability in inner medullary collecting duct
(19). Although animal models exist of NDI caused by abnormalities
in neurohypophysial vasopressin secretion, such as the Brattleboro rat
(20, 21), there are no animal models of renal AQP2
deficiency/mutation.
The AQP2 point mutation T126M was identified as a cause of non-X-linked
recessive NDI in humans (22). In transfected mammalian cells,
AQP2-T126M protein is retained at the endoplasmic reticulum (ER) and
degraded more rapidly than wild-type AQP2 (23). We also showed recently
that ER-retained AQP2-T126M is mildly misfolded but functional (24).
Treatment of transfected cells with chemical chaperones such as
glycerol resulted in relocation of AQP2-T126M to the cell plasma
membrane where it was able to function as a water channel and correct
the defective cell phenotype (23, 24). Chemical chaperones or other
agents that modify interactions with molecular chaperones were proposed
as possible therapies for NDI. However therapy development requires
demonstration that disease-causing AQP2 mutants are ER-retained in
native kidney cells and that chaperone-modifying strategies are
effective in vivo.
The purpose of this study was to generate and characterize a mouse
model of recessive NDI caused by AQP2 mutation. We previously isolated
and analyzed the mouse AQP2 cDNA and gene and showed that the mouse
AQP2-T126M ortholog behaved like human AQP2-T126M in its ER
localization, function, and correction by chemical chaperones (25). The
homozygous AQP2-T126M knock-in mice created here had NDI and expressed
the mutant AQP2 protein in collecting duct epithelial cells, as
engineered, but the mice had an unexpectedly severe phenotype with
neonatal mortality.
Generation of AQP2-T126M Knock-in Mutant Mice--
Based on the
reported mouse cDNA sequence and gene structure (25), a gene
replacement targeting vector was constructed to knock-in the T126M
mutation (see Fig. 1). The vector contained a 5-kb mouse genomic DNA
fragment containing AQP2 exons 1-3. The T126M
mutation was generated by site-directed mutagenesis. To retain the
human consensus sequence for N-linked glycosylation, mutations H122S, N124S, and A125T were also introduced into the targeting vector. A silent FseI restriction site was
engineered for mouse genotype analysis. A Pol2neobpA selection cassette
flanked by loxP sites was inserted into intron 2 for positive
selection, and a PGKtk cassette was inserted at the 3'-end of the AQP2
targeting sequence for negative selection. The vector was linearized at a unique downstream NotI site and electroporated into CB1-4
embryonic stem (ES) cells. Transfected ES cells were selected with G418 and FIAU for 7 days, yielding 2 targeted clones of 246 doubly resistant
colonies upon PCR screening using a sense primer specific for the neo
cassette (5'-CAGTTCACCGCTGAATATGCAT-3') and an antisense primer
specific for the AQP2 gene (5'-GCAAAGCTCTGAGAAAACCGC-3') located downstream from the 3'-end of the construct. Homologous recombination was confirmed by Southern blot analysis using as probe a
3.2-kb genomic fragment as indicated in Fig. 1. ES cells were injected
into PC 2.5-day 8-cell morula stage CD1 zygotes, cultured overnight to
blastocysts, and transferred to pseudopregnant B6D2 females. Offspring
were genotyped by PCR followed by Southern blot analysis as described
above. Heterozygous founder mice were bred to produce homozygous
AQP2 mutant mice. Heterozygous females were also mated with
males (mouse strain FVB/N) homozygous for Northern Blot Analysis--
RNA from kidney was isolated using
TRIzol reagent (Life Technologies, Inc.). RNAs (10 µg/lane) were
resolved on a 1.2% formaldehyde-agarose denaturing gel, transferred to
a Nylon+ membrane (Amersham Pharmacia Biotech) and hybridized at high
stringency with a 32P-labeled probe corresponding to the
mouse AQP2 cDNA coding sequence.
Cell Transfection--
Full-length cDNAs encoding wild-type
and mutant AQP2 were PCR-amplified from kidney cDNA of wild-type
and mutant mice, sequenced, and subcloned into pCDNA3 plasmid
(Invitrogen) for transfection in CHO cells as described previously
(25).
Immunocytochemistry--
Immunofluorescence localization of AQP2
protein in fixed frozen kidney sections was done using a rabbit
polyclonal antibody against the 30-amino acid C terminus of AQP2 as
described previously (19, 25).
Endoglycosidase Digestion--
100 µg of proteins from kidney
homogenates were suspended in 9 µl of incubation buffer (20 mM sodium phosphate pH 7.5, 50 mM EDTA,
0.02% sodium azide, 0.1% SDS, 50 mM
Detergent Extraction--
Membranes from kidney homogenates were
incubated with phosphate-buffered saline containing 1% CHAPS at
4 °C for 30 min. Detergent-insoluble proteins were pelleted at
100,000 × g for 45 min and resuspended in the buffer
containing 0.1% SDS, 1% deoxycholate, and 1% Triton X-100. Buffer
was also added to the supernatant to give a final concentration of
0.1% SDS, 1% deoxycholate, and 1% Triton X-100. Supernatant and
pelleted samples were then subjected to immunoblot analysis.
Immunoblot Analysis--
Equal amounts of protein (5 µg/lane)
were resolved on a 12% SDS-polyacrylamide gel and electroblotted to a
nitrocellulose membrane. Membranes were blocked with 5% nonfat dry
milk in 10 mM Tris, 150 mM NaCl, pH 7.4 (TBS)
for 1 h, followed by a 1-h incubation in anti-AQP2 polyclonal
antibody (1:500). Membranes were washed in TBS containing 0.05% Tween
and incubated with horseradish peroxidase-conjugated goat anti-rabbit
IgG. Bands were visualized by enhanced chemiluminescence (Amersham
Pharmacia Biotech).
Urine and Serum Osmolality--
Urine and serum osmolalities
were measured by freezing point depression osmometry (micro osmometer,
Precision Systems, Inc.). In some experiments mice were injected with
DDAVP (0.4 µg/kg) intraperitoneally at 60 min prior to
obtaining the urine sample.
Xenopus Oocyte Expression--
Full-length cDNAs encoding
wild-type and mutant AQP2 were subcloned into an oocyte expression
plasmid. Complementary RNA was transcribed in vitro using
SP6 polymerase. Stage V and VI oocytes from Xenopus laevis
were isolated, defolliculated with collagenase, and microinjected with
50-nl samples of cRNA (0-200 ng/µl). After incubation at 18 °C
for 24 h, osmotic water permeability (Pf) was measured from the time course of oocyte swelling at 10 °C in
response to a 5-fold dilution of the extracellular Barth's buffer with
distilled water.
Targeted gene replacement in embryonic stem cells was done to
replace wild-type AQP2 with AQP2-T126M. As shown in Fig.
1, a positive-negative selection strategy
was used in which a loxP-flanked polII-neo selection cassette was
introduced in intron 2. In addition to the T126M mutation, mutations
H122S, N124S, and A125T were engineered in exon 2 of the coding
sequence to preserve the consensus site for N-linked
glycosylation found in human AQP2. Two mutant mouse lines were
generated: a CD1 line in which the Pol2neobpA cassette was retained in
the genome (strain A) and a hybrid CD1/FVB/N line (strain B) in which
the Pol2neobpA cassette was deleted by breeding with mice expressing
cre-recombinase.
Southern blot analysis of strain A mouse liver genomic DNA digested
with ApaI and probed as indicated in Fig. 1 showed a smaller fragment at 3.2 kb corresponding to the replaced gene containing a
1.8-kb polII-neo selectable marker (Fig.
2A, left). In the
strain B mice, Southern blot analysis of genomic DNA digested with
ApaI/FseI showed a fragment at 3.5 kb indicating
the engineered FseI restriction site and deletion of
polII-neo cassette (Fig. 2A, right). Deletion of
polII-neo sequence was confirmed by sequence analysis. The genotype
distribution from breeding of heterozygous mice was 13 wild-type, 31 heterozygous, and 14 homozygous mutant mice (strain A) and 10 wild-type, 19 heterozygous, and 12 mutant mice (strain B), in agreement
with a 1:2:1 Mendelian distribution expected in the absence of prenatal
mortality. Northern blot analysis showed the same 1.7-kb transcripts in
kidneys of wild-type and mutant mice, but with marked up-regulation
(3.5-fold by densitometry) of the transcript corresponding to the
mutant AQP2-T126M (Fig. 2B). AQP2 transcript up-regulation
is known to occur with dehydration (27, 28). To confirm that AQP2 in
kidneys of mutant mice contained the engineered mutations, the
full-length AQP2 coding sequence was RT-PCR amplified from wild-type
and mutant mice. Sequence analysis of the full coding region confirmed
the four mutations for strain A and strain B mice (Fig.
2C).
Neonatal Mortality in an Aquaporin-2 Knock-in Mouse Model of
Recessive Nephrogenic Diabetes Insipidus*
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-actin-cre (26)
to remove the Pol2neobpA selection cassette flanked by loxP sites in
intron 2 of the mutant AQP2 gene. After confirming absence
of the Pol2neobpA sequence, the heterozygous mice were bred to produce
homozygous mutant mice.
-mercaptoethanol, 0.75% Nonidet P-40). Endoglycosidase H or
endoglycosidase F (0.5 units) was added, and mixtures were incubated at
37 °C for 2 h prior to immunoblot analysis.
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View larger version (28K):
[in a new window]
Fig. 1.
Targeting strategy for AQP2
gene replacement. Homologous recombination results in
replacement of the indicated segment (thick line) of the
AQP2 gene by a 1.8-kb Pol2neobpA selection cassette flanked
by loxP sites (arrowheads). Small arrows indicate
the mutant sites. The probe used for Southern blot analysis is
indicated probe, and the expected sizes of hybridized
fragments are shown by dashed lines.
View larger version (21K):
[in a new window]
Fig. 2.
Analysis of genotype and transcript in
transgenic mice. A, Southern blot of strain A mouse
liver genomic DNA (left) digested with
ApaI and probed as indicated in Fig. 1. Southern blot of
strain B mouse liver genomic DNA digested with
ApaI/FseI and hybridized with the same probes
used in strain A (right). B, Northern blot of
kidney probed with the mouse AQP2 coding sequence. C,
sequence analysis of mouse AQP2 (bases 364-387) generated by RT-PCR
using kidney cDNA from wild-type and homozygous mutant mice.
Mutated amino acids are underlined.
Functional analysis and cellular localization of the RT-PCR amplified
wild-type and mutant AQP2 sequences were done in heterologous expression systems. In Xenopus oocytes microinjected with
transcribed cRNAs, osmotic water permeability was increased by
expression of the mutant AQP2, though to a lesser extent than wild-type
AQP2 (Fig. 3A), in agreement
with previous results for human and mouse AQP2-T126M (23, 25).
Transient expression in CHO cells revealed an endosomal/plasma membrane
expression pattern for the wild-type AQP2, but an endoplasmic reticulum
(ER) expression pattern for the mutant AQP2 (Fig. 3B), in
agreement with results for human and mouse AQP2-T126M (23, 25).
Therefore the mutant AQP2 coding sequence expressed in mice has the
expected functionality and ER-localization when expressed in
heterologous systems.
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Although the homozygous mutant pups appeared grossly normal at 1-2
days after birth, by 3-4 days they were much smaller than litter mates
and failed to thrive (Fig.
4A). For the strain A mice,
averaged body weights at 5 days (in grams, mean ± S.E.) were
3.8 ± 0.3 (wild-type), 3.9 ± 0.3 (heterozygous), and
1.7 ± 0.2 (mutant). Most strain A mutant mice died at 5-6 days
after birth when no intervention was done to prolong survival. Survival of some pups was prolonged up to 8 days by separation of most wild-type
and heterozygous pups from the homozygous pups (to minimize competition
for milk) and by fluid supplementation by oral feeding (30-50 µl of
milk, four times per day) or subcutaneous fluid injections (50-100
µl of half-normal saline, three times per day). Interestingly, many
hybrid strain B mice were able to survive to day 8, although they were
remarkably smaller than wild-type litter mates from day 3 onward.
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Measurement of urine osmolality showed relatively hypotonic urine in the mutant mice that was not sensitive to DDAVP administration (Fig. 4B); however, the urine osmolality of 221 ± 9 mosM probably underestimates the severity of the nephrogenic diabetes insipidus because of serum hyperosmolality and progressive renal failure. (It was not possible to obtain urine from very young mutant mice.) Serum osmolality was remarkably elevated in the dehydrated mutant mice (Fig. 4C). Blood urea nitrogen of the mutant pups was ~7-fold greater than in wild-type pups (79 ± 15 mg/dl versus 11.6 ± 0.6 mg/dl). To determine whether the heterozygous mice manifest any abnormality in urinary concentrating ability, urine osmolalities were compared in adult (4-5 weeks of age) wild-type and heterozygous mice. Averaged urine osmolalities were 1544 ± 69 mosM (heterozygous) and 1585 ± 187 mosM (wild type), increasing after a 36-hour water deprivation to 3001 ± 126 mosM (heterozygous) and 3081 ± 291 mosM (wild type, differences not significant).
Immunoblot analysis of whole kidney homogenates showed that the
majority of immunoreactive protein in wild-type and heterozygous mice
migrated as a diffuse band of apparent molecular size 34-40 kDa (Fig.
5A), which represents fully
processed AQP2 with complex glycosylation. A band of lower intensity at
the predicted molecular size of nonglycosylated AQP2 was also seen. In
contrast, the mutant AQP2 protein migrated mainly as a single band at
~31 kDa, which from previous data in oocytes and transfected cells
probably represents an ER-retained form of AQP2 with core
glycosylation. Sometimes bands at higher molecular weights were seen
that probably represent AQP2 multimers.
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To confirm the interpretation of the glycosylated bands, immunoblot analysis was done on endoglycosidase-treated samples. Endoglycosidase F treatment, which cleaves all sugars, gave a single band at 29 kDa for all genotypes (Fig. 5B). Endoglycosidase H treatment, which cleaves high-mannose core sugars of proteins that have not exited the ER, did not affect wild-type AQP2 but digested most of the 31-kDa form of the mutant AQP2. We found previously that AQP2-T126M protein was resistant to detergent extraction in a transfected CHO cell model (24), suggesting a different folding/aggregation state than wild-type AQP2. To determine whether this difference in detergent solubility occurs in native kidney, membrane homogenates were treated with CHAPS for 30 min (as done in Ref. 24) and centrifuged to separate supernatant (soluble AQP2) and pellet (insoluble AQP2). Fig. 5C shows remarkably greater detergent sensitivity of wild-type versus mutant AQP2. The in vivo biochemical data thus confirm several of the biochemical properties of the mutant AQP2 established in cell culture models.
Last, renal morphology and AQP2 immunolocalization were studied in
kidneys of wild-type and mutant mice. Remarkably, kidneys from the
mutant mice showed signs of obstructive uropathy including dilatation
of collecting ducts and papillary atrophy (Fig.
6A, right). Many
kidneys of the mutant mice contained a large fluid-filled cavity where
the medulla should be. Immunofluorescence of kidneys from 5-day-old
wild-type mice showed AQP2 localization in medullary collecting duct,
but unlike adult mice, very little AQP2 was found in cortical
collecting duct (Fig. 6, B and C,
left). AQP2 was found at the apical membrane, in agreement
with previous results in rat and human kidney (27, 29, 30). At low
magnification, AQP2 immunostaining was also found mainly in the medulla
of kidneys from mutant mice, albeit at a substantially greater level
than in wild-type mice (Fig. 6B, right). At
higher magnification, AQP2 was seen both within cells, probably in the
ER, as well as at the plasma membrane in some tubules (Fig.
6C, right). Insets in Fig.
6C show higher magnification.
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The impaired neonatal survival of the mutant mice expressing AQP2-T126M indicates an important role for AQP2 in fluid and electrolyte balance early in life. The mutant mice had a urinary concentrating defect resulting in dehydration, failure to thrive, and death. Interestingly, mice lacking AQP1 (31), AQP3 (17), and AQP4 (18), which manifest urinary concentrating defects to differing extents, do not have reduced neonatal survival. The severe NDI in the AQP2 mutant mice supports the conclusion that transepithelial water permeability in collecting duct is rate-limited by apical membrane water permeability.
The neonatal mortality in the mutant mice was an unexpected finding, especially in view of the limited urinary concentrating ability of young normal mice. For several reasons, we believe that the early death is because of polyuria and its consequences, rather than to other causes related to expression of the mutated AQP2 protein. First, mice lacking an unrelated protein (the V2 receptor), which probably have comparable polyuria to the mutant AQP2 mice, also fail to thrive and die early (32). Other knockout mouse models (AQP3, NKCC2) having polyuria appear to develop kidney damage (33, 34). The mouse kidney is probably particularly sensitive to damage by polyuria compared with rat (e.g. Brattleboro rat) or human (e.g. subjects with NDI). We think it is unlikely that mutant AQP2 expression is itself damaging to the kidney, because very little AQP2 is expressed at the time of birth or before, and because the mutant mice appear grossly normal at 1-2 days after birth. Last, the low urine osmolality and high serum osmolality measured in the mutant mice indicates marked polyuria and probably underestimates the severity of the polyuria because the mice are in renal failure by the time they are large enough to permit collection of urine and serum samples.
Initial efforts to prolong the survival of the mutant mice by fluid supplementation were only partially successful. The improved survival of the strain B mice with the hybrid CD1 and FVB/N background is probably related strain differences, because the mutant AQP2 sequence is identical in both mouse strains, and the amount of expressed protein was comparable. More work involving fluid supplementation and transferring the mutant genotype to mice of different genetic backgrounds will be needed to determine whether survival of the mutant mice until adulthood is possible.
Northern blot analysis indicated increased mutant AQP2 expression in the homozygous mutant mice, which is probably related to dehydration and high serum vasopressin concentrations. The AQP2 promoter contains a cAMP response element that is responsible for up-regulation of AQP2 expression in states of high circulating vasopressin (35). Immunoblot analysis showed that the mutant AQP2 protein was core glycosylated as demonstrated by its size and sensitivity to endoglycosidase H. As found previously in transfected cell models, mutant AQP2 in kidney was also relatively resistant to detergent solubilization. However, immunofluorescence showed a significant fraction of the mutant AQP2 at the apical plasma membrane. It is possible that transfected cell cultures, where the mutant AQP2 is found exclusively at the ER, are not good models for the in vivo targeting of mutant AQP2. However, the grossly abnormal collecting duct morphology in mutant mice precludes such interpretations. The plasma membrane localization of some mutant AQP2 molecules might be a consequence of the many changes in epithelial cell physiology resulting from increased intraluminal pressure. Another interesting possibility is that the accumulation of osmolytes in renal medulla might facilitate the plasma membrane targeting of some mutant AQP2 molecules, as was found in cell culture models (23).
In summary, we have created and characterized the first mouse model of
human NDI. The AQP2-T126M-expressing mutant mice permit in
vivo studies of the biochemistry of a disease-causing AQP2 mutant.
Adult AQP2 mutant mice, if and when available, should be useful in
addressing fundamental issues about the pathophysiology of NDI caused
by AQP2 mutations, and for testing the efficacy of established and
novel therapies to improve urinary concentrating ability.
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ACKNOWLEDGEMENTS |
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We thank Mark Lewandoski and Gail R. Martin
for the -actin-cre mice and Liman Qian for mice breeding.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK35124, HL58198, HL60288, and HL51854, and Grant R613 from the National Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 1246 Health Sciences
E. Tower, Cardiovascular Research Inst., University of California, San
Francisco, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax:
415-665-3847; E-mail: verkman@itsa.ucsf.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008216200
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ABBREVIATIONS |
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The abbreviations used are: AQP, aquaporin; NDI, nephrogenic diabetes insipidus; ER, endoplasmic reticulum; kb, kilobases; tk, thymidine kinase; ES, embryonic stem cell; CHO, Chinese hamster ovary cell; RT-PCR, reverse transcription-polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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