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Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- showed no marked change until 24 h,
whereas that of rat IL-6 and TGF- increased with time. We then
introduced and expressed the IL-6 mutant genes, which were designed to
behave as IL-6R antagonists, and found that their ability to induce
erythema was lower than that of the wild-type gene. Furthermore,
preintroduction of some mutant genes delayed the erythema induced by
postintroduction of the wild-type IL-6 gene, suggesting that the mutant
forms of IL-6 prevent wild-type IL-6 from binding to IL-6R. This
result indicates that keratinocyte gene therapy may be possible for
inflammatory skin diseases using IL-6 mutant
genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, s.c. injection of naked DNA has been shown to transfer genes efficiently and preferentially into keratinocytes in various species, including humans (11, 12, 13). This method is simple, does not require special equipment and can introduce DNA into keratinocytes of fully developed adult animals. It has also been suggested that gene therapy for somatic cells to treating human skin disease would be practical if prolonged, high level transgene expression could be achieved using this method (12). We have found that a promoter/enhancer cassette constructed with the ß-actin promoter plus the CMV enhancer and the 3'-flanking sequence of the ß-globin gene expresses the inserted gene very strongly in keratinocytes in vivo.3 Combination of the naked DNA method and this strong promoter/enhancer cassette enabled the IL-10 gene to be expressed so strongly in keratinocytes in vivo that the transgenic IL-10 overflowed into the bloodstream and exerted biological effects in distant areas of skin (14).
In the present study, we overexpressed IL-6 in keratinocytes using this
system in an attempt to clarify the biological functions of IL-6 in the
skin. In vivo transfer of the IL-6 gene to keratinocytes induced
keratinocyte proliferation and lymphocytic infiltration in the skin.
Our results suggest that IL-6 is involved in the pathogenesis of skin
inflammatory diseases, and furthermore that treatment of the diseases
may be possible by transfer of IL-6 mutant genes whose products act as
antagonists at the IL-6R.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To amplify the 639-bp coding region of human IL-6 cDNA by PCR, we synthesized two primers, 5'-GGGAAGCTTGCTATGAACTCCTCCTCCACA-3' and 5'-GGGGAATTCATGCTACATTTGCCGAAGAGC-3', based on the sequence of the human IL-6 gene (15). Also we synthesized two primers, 5'-GGGAAGCTTGCTATGAAGTTTCTCCTCCGCA-3' and 5'-GGGGAATTACACTAGGTAAGCCGAGTAGA-3', to amplify the 636-bp coding region of rat IL-6 cDNA (16). The primers contained restriction enzyme sites at the 5' and 3'-ends for subcloning. PCR was performed with oligo(dT)-primed human and rat keratinocyte cDNAs as templates. After sequence analysis of the DNA fragments, they were digested with EcoRI and HindIII, and subcloned into the pCY4B expression vector, which contains the CMV immediate early enhancer, the modified ß-actin promoter, and the 3'-flanking sequence of the ß-globin gene. The human and rat IL-6 expression vectors were designated phIL6 and prIL6, respectively. Plasmid pCY4B was constructed by deleting SV40 ori from pCAGGS, but their functional fragments were all the same in terms of gene expression (17). Plasmid pCAGGS-lacZ was constructed by inserting lacZ into pCAGGS. A plasmid, ph(-), containing the human IL-6 cDNA without any eukaryotic promoter, was used as a control.
In vivo DNA transfer to keratinocytes
The plasmid was injected into the back skin of hairless rats (14) that were anesthetized with 3.6% chloral hydrate at 1 ml/100 g body weight. Plasmid DNA was diluted in PBS(-) to various concentrations. The injection of DNA was performed using a 29-gauge needle. The injected volume was 40 µl per injection site, the maximum concentration of DNA was 0.2 µg/µl, and the maximum dose of DNA was 8 µg. When it was necessary to introduce more than 8 µg of DNA, we injected the DNA sample into multiple sites. Also, human rIL-6 (R&D Systems, Minneapolis, MN) was diluted with PBS(-) and injected in the same way as the DNA. Skin biopsies of the injection sites were performed at various time points after injection.
Assay for expressed human IL-6 mRNA
Eight micrograms of phIL6 was injected into the rat skin, and the injection site was biopsied 24 h later. Biopsy specimens were treated with dispase to obtain epidermal sheets (13). Total RNA was extracted from each epidermal sheet, oligo(dT)-primed keratinocyte cDNA was synthesized with reverse transcriptase (RT),4 and PCR was performed using the above primers for human IL-6 cDNA. To examine contaminating plasmid DNA, we carried out PCR amplification from RNA samples without the RT reaction. PCR products were fractionated by agarose gel electrophoresis.
Assay for mRNAs of rat IL-1, IL-6,
TNF-, and TGF- in keratinocytes
Eight micrograms of phIL6 was injected; total RNA was extracted
from the epidermal sheet immediately and at 3, 10, and 24 h after
the injection; 8 µg of ph(-) as control was also injected; and total
RNA was extracted just before, immediately, and at 24 h after the
injection. Oligo(dT)-primed keratinocyte cDNA was synthesized from RNA
samples. PCR was performed using the following primers:
5'-GACGGCTAAGTTTCAATCAGC-3' and 5'-TGGAAATCTATCATGGAGGGC-3' for the
543-bp rat IL-1 mRNA (18), 5'-ATGGCAATTCTGATTGTATGA-3'
and 5'-CTGACCACAGTGAGGAATGTC-3' for the 475-bp rat IL-6 mRNA
(16), 5'-TACTGAACTTCGGGGTGATTGGTCC-3' and
5'-CAGCCTTGTCCCTTGAAGAGAACC-3' for the 295-bp rat TNF-
mRNA (19), 5'-TGGTAGCTGTGTGTCAGGCT-3' and
5'-GGTTGGGCTGTCATCGGCCACCTG-3' for the 481-bp rat TGF- mRNA
(20), 5'-TTGTAACCAACTGGGACGATATGG-3', and
5'-GATCTTGATCTTCATGGTGCTAGG-3' for the 764-bp rat ß-actin mRNA
(21). We synthesized two or three forward and reverse
primers for each cytokine cDNA on the basis of computer and our
experience, and selected the best set of primers. We established PCR
conditions that resulted in exponential amplification, and PCR products
were fractionated by agarose gel electrophoresis.
ELISA for human IL-6
Skin biopsy specimens were taken from the treated sites. Epidermal sheets were prepared as described above, suspended in 0.25 M Tris (pH 7.8), lysed by three cycles of freeze-thawing, and centrifuged at 5000 x g. The concentration of human IL-6 in the supernatant was measured with a human IL-6 ELISA kit (Cytoscreen, Camarillo, CA) and expressed as picograms per nanogram of protein (Protein Assay Kit, Bio-Rad, Hercules, CA). The assay reactions were performed in triplicate.
Immunohistochemical staining
Skin biopsy specimens were taken from the treated sites. The specimens were embedded in OCT compound, and sections 10 µm thick were cut. The following Abs were used for experiments: anti-human IL-6 (AB206NA, R&D Systems) polyclonal Ab (22); anti-rat CD3 (G4.18, PharMingen, San Diego, CA); anti-rat IgM (MARM-4, ICN Pharmaceuticals, Aurora, OH), anti-rat monocyte/macrophage (ED-1, Chemicon, Temecula, CA); and anti-rat granulocytes/monocyte (OX-41, Chemicon) mAbs. Optimal concentrations of each Ab was predetermined by titration assay. Amplification of staining using diaminobenzidine was performed with a PAP Kit (Dako, Carpinteria, CA). The remaining operations were carried out according to the manufacturer’s instructions.
Mutagenesis of human IL-6 cDNA
Site-directed mutagenesis was performed using a Mutant-Super
Express Km Kit (Takara, Japan) (23). Briefly, human IL-6
cDNA was subcloned into the HindIII and EcoRI
sites in the multicloning site of plasmid pKF18kM, which contained dual
amber stop codons at the kanamycin resistance gene. Long PCR was
carried out with the mutagenic primer and amber-rescue primer using LA
taq polymerase (Takara). Each mutated IL-6 cDNA was
sequenced, cut out with HindIII and EcoRI, and
subcloned into pCY4B. The following mutagenic primers were used for the
experiments: 5'-CAAATTCGGGACATCCTCGACGAGATCTCAGCCCTGAGA-3' for
change from the wild type to 31D,
5'-GTCCAGATGCATACAAAAGACCTGATCCAGTTCCTGCAG-3' from 31D to 121D,
5'-AAAGATGGATGCTTCACGTGTGGATTCAATGAGGAGACT-3' from 121D to 75T,
5'-ATCCTCGACTTCATCTCAGCCCTGAGAAAG-3' from 31D to 35F, and
5'-GTCCAGATGCGTACAAAAGACCTGATC-3' from 35F to 118R (Table I
).
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Plasmids expressing mutant IL-6 were diluted in PBS(-) to various concentrations. The injection of DNA was performed with a 29-gauge needle. The volume injected at each site was constantly 40 µl, and the maximum DNA concentration was 0.2 µg/µl (the maximum dose; 8 µg). The injection sites were carefully examined, and the vertical and transverse diameters of the apparent erythema were measured by dial micrometer 48 h after injection. Erythema size was expressed as the mean of the diameters.
The suppressive effects of mutant IL-6 genes on the erythema induced by the wild-type IL-6 gene were also examined. Each mutant IL-6 gene was injected at the highest dose that did not induce visible erythema, and phIL6 (the wild-type IL-6 gene) at the dose of 0.5 µg was injected at the same site several times after introduction of mutant IL-6 gene. Plasmids pCAGGS-lacZ and ph(-), and PBS were also injected as negative controls for the mutant genes. The vertical and transverse diameters of the apparent erythema were measured 24 h and 48 h after injection of phIL6, erythema size was calculated, and the erythema size induced by each gene was expressed as a percentage relative to that induced by PBS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, to clarify whether keratinocytes expressed mRNA of human
IL-6 after introduction of the human IL-6 gene, plasmid phIL6 at a dose
of 40 µg (injection volume: 200 µl) was injected into the rat skin,
RNA was prepared from epidermal sheets of the treated areas 24 h
after introduction, and RT-PCR was performed. Fig. 1 shows that a positive 558-bp band was
observed in the sample from phIL6-transfected keratinocytes, whereas no
band was evident from ph(-)-transfected keratinocytes. Although we
performed PCR with a RNA sample to eliminate DNA contamination, no
amplification was observed (Fig. 1). To confirm the presence of
transgenic human IL-6 in the treated skin, phIL6 at doses of 8, 2, 0.5,
0.13, and 0.03 µg was injected, epidermal lysates were prepared
24 h later, and ELISA for human IL-6 was performed. Fig. 2A shows that a considerable
amount of human IL-6 was produced by the rat keratinocytes, and that
the amounts increased with the dose of phIL6 introduced. We also
examined the time course of the level of transgenic IL-6 in
keratinocytes, and found that it increased rapidly until 12 h
after injection and then gradually decreased (Fig. 2B).
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First we injected 8 µg of phIL6 and observed the treated area of the skin macroscopically. Erythema started to develop in the treated area 12 h after injection. The degree of erythema reached a maximum and slight elevation was observed 48 h after injection. Thereafter, the erythema gradually decreased and disappeared about 7 days after injection. We also injected 8 µg of prIL6 and observed the same inflammatory changes in the skin. Intradermal single injection of rIL-6 at doses of 500, 50, and 5 ng induced no inflammatory changes at the injection site, and injection of 500 ng of rIL-6 three times at 24-h intervals also did not induce erythematous reaction.
Induction of microscopic inflammatory changes
Biopsy specimens were obtained 12 h, 48 h, and 7 days
after the introduction of phIL6, and histological examination was
performed. Infiltration of a few lymphocytes in the upper dermis and
slight epidermal thickening were observed after 12 h. In the 48-h
specimen, we found thickening and hypergranulosis in the epidermis, and
lymphocytic infiltration and telangiectasia in the upper dermis (Fig. 3, A and B). Some
areas at the injection site showed marked lymphocytic infiltration,
which has resulted in epidermal trafficking of lymphocytes and slight
hydropic degeneration of the basal cells (Fig. 3A). These
changes lasted for about 7 days, corresponding to the period of evident
macroscopic erythema. Introduction of ph(-) did not induce any
particular changes in the treated skin (Fig. 3C). We also
introduced 8 µg of prIL6, and observed the same inflammatory changes
histologically (data not shown). The untreated skin was shown as
control (Fig. 3D).
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Biopsy specimens were obtained 12 h and 48 h after the
introduction of 8 µg phIL6, prIL6, and ph(-), and
immunohistochemical examination was performed using anti-human goat
IL-6 Ab. In the phIL6 specimen 12 h after transfer,
immunoreactivity was observed in the entire epidermis, whereas little
was evident in the dermis (Fig. 4C). Reactivity was also found
in the phIL6 specimen 48 h after transfer, and tended to be strong
in the area showing lymphocyte trafficking (Fig. 4A). The
prIL6 specimen also demonstrated epidermal thickening and lymphocytic
infiltration, but no immunoreactivity (Fig. 4B). The
manufacturer’s specifications for this Ab mention that it has no
reactivity with mouse IL-6, and the present findings also indicate that
it has little or no reactivity with rat IL-6. There was little or no
reactivity in the ph(-)-treated skin used as a control (Fig. 4F). Anti-human IgG goat polyclonal Ab used as an isotype
control showed no positive staining in any of the specimens (data not
shown).
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). These indicated that infiltrating
cells were predominantly T lymphocytes.
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Using RT-PCR, we examined the time course of rat IL-1, IL-6,
TNF-, and TGF- message levels until 24 h after injection of
8 µg phIL6. Fig. 6A showed
that the mRNA expression of IL-1 and TNF- mRNA showed no marked
change until 24 h, whereas that of IL-6 and TGF- increased with
time. Introduction of ph(-) did not change the mRNA expression of
cytokines (Fig. 6B). In RT-PCR, bands of these cytokines
were found in samples obtained from untreated epidermal sheets (just
before ph(-) injection). Expression of ß-actin mRNA as internal
control was constant throughout the experimental period.
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We next constructed vectors expressing mutant IL-6, introduced
each vector at doses of 8, 2, 0.5, 0.13, and 0.03 µg, and measured
the diameter of the observed erythema 48 h after gene transfer. We
substituted residues in in vitro mutagenesis (Table I
). The erythema
size induced by each construct decreased with the dose injected (Fig. 7), and was shown to be a good indicator
of erythema-inducing activity. The activities of the mutant forms of
IL-6 were lower than those of the human wild-type form; the 31D and
121D forms showed slightly decreased activity, the 35F and 118R forms
showed a decrease of about 16-fold, and 75T had no activity, even at a
dose of 8 µg. This further confirmed that IL-6 produced by the
gene-transfected keratinocytes was responsible for the erythematous
skin reaction.
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C), of 118R (Fig. 4D), and of 75T (Fig. 4E) 12 h after
phIL6 transfer. Suppression of IL-6-induced inflammation by mutant IL-6 gene transfer
On the basis of the results we had obtained in the above
experiments, we selected 35F, 118R, and 75T because of their low
erythema-inducing activities. Forms 35F and 118R at 0.5 µg and 75T at
8 µg, which were the maximum doses not causing erythema, were
used for transfection 24 h before administration of 0.5 µg of
wild-type phIL6, and then erythema size was measured 24 and 48
h after introduction of the wild-type phIL6. We injected 8 µg of
ph(-) and pCAGGSlacZ, and PBS as controls for the mutant genes.
Erythema size was expressed as a percentage relative to that at the
site treated with PBS. The results (Fig. 8A) obtained 24 h after
transfer showed that the percentage of erythema produced by 35F and
118R was significantly smaller than that in the controls, suggesting
that 35F and 118R had the ability to inhibit IL-6-induced skin
inflammation. On the other hand, 75T did not suppress the inflammation.
Significant inhibitory effects of 35F and 118R were not observed
48 h after transfer of wild-type phIL6 (Fig. 8A). Next,
contruct 118R at 0.5 µg, ph(-) at 8 µg, and PBS were injected, and
phIL6 at 0.5 µg was injected at the same site 48 h, 36 h,
24 h, 12 h, and just after 118R introduction. Erythema size
was measured 24 and 48 h after introduction of the wild-type
phIL-6. The result obtained 24 h after phIL6 transfer showed that
the inhibitory effect was found when 118R was injected 12, 24, and
36 h before phIL6 injection (Fig. 8B). The results
obtained 48 h after phIL6 transfer showed that no inhibitory
effect was observed in any cases (data not shown). Finally, 118R was
injected 24 h after phIL6 transfer and erythema size was observed
only 48 h after phIL6 transfer, but no inhibitory effect was found
(data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Also, an erythematous reaction
was observed after injection of phIL6 at doses of 8, 2, 0.5, and 0.13
µg, but not after that of 0.03 µg phIL6 (Fig. 7) and 500 ng rIL-6.
And, injection of 500 ng rIL-6 three times at 24-h intervals also did
not induce erythematous reaction. These suggested that IL-6
overexpression in the epidermis was critical for the erythematous
reaction and that the IL-6 minumum concentration in keratinocytes
necessary to induce a visible reaction was approximately 200 pg/ng
epidermal protein (Fig. 2A). Our study and previous ones
using transgenic mice (8) employed human and mouse IL-6
genes, respectively. Since different results between these two studies
suggested rodent IL-6 might lack the ability to induce a skin reaction,
we constructed a rat IL-6 expression vector (prIL6) and injected it
into the skin. It was found that prIL6 also induced erythematous change
in the treated skin. Our preliminary investigation had shown that a
pCAGGS or pCY4B cassette expressed the inserted gene about 25–50 times
more strongly than cassettes with keratin promoters, which had been
widely used for experiments with transgenic mice3 and that
IL-6 gene was expressed so strongly in keratinocytes in vivo that the
transgenic IL-6 overflowed into the bloodstream (X. Meng, D. Sawamura,
S. Ina, H. Narumi, H. Nakano, K. Tamai, K. Hanada, and I. Hashimoto,
unpublished data). In the present study, use of the naked DNA method
with this strong promoter/enhancer cassette enabled a considerable
amount of transgenic IL-6 to be expressed in keratinocytes, resulting
in an inflammatory skin reaction. This method may therefore be
extremely useful for studies of new biological functions of proteins in
keratinocytes and for basic research on keratinocyte gene therapy.
Histological examination of the skin reaction after introduction of
phIL6 showed epidermal thickening with hypergranulosis and lymphocytic
infiltration in the upper dermis. Epidermal thickening reflected data
in vitro indicating that IL-6 stimulates the proliferation of cultured
human keratinocytes IL-6 (6). Although IL-6 activates both
T and B lymphocytes, our immunohistochemical examination revealed that
the main infiltrating cells were CD3-positive T lymphocytes (Fig. 5).
Among inflammatory skin diseases showing T cell infiltration (24),
psoriasis and lichen planus are reported to show IL-6 overexpression by
keratinocytes (8, 9, 10). These suggested that IL-6 was
involved in pathogenesis of these two diseases.
IL-1 and TNF- are potent proinflammatory cytokines, and TGF-
is also a potent factor that controls the growth of keratinocytes. All
can be produced by keratinocytes. Although transfer of the IL-6 gene to
keratinocytes induced keratinocyte proliferation and lymphocytic
infiltration, these cytokines may have been stimulated by IL-6, and
consequently induced the skin changes. Using RT-PCR, we measured the
levels of rat IL-1, IL-6, TNF-, and TGF- mRNA in rat
keratinocytes 24 h after transfer. We did not examine these levels
beyond 24 h, since lymphocyte infiltration was marked after this
time, and keratinocyte RNA samples would have been contaminated with
lymphocyte RNA. The expression of IL-1 and TNF- mRNA did not show
marked change (Fig. 6), indicating that at least these cytokines were
not initially involved in the inflammatory changes. An increase in the
level of rat IL-6 mRNA level also suggested an auto- or paracrine
effect of transgenic human IL-6. Furthermore, we found an increase of
TGF- mRNA expression (Fig. 6), suggesting that it was induced by
transgenic IL-6. Recently, Aragane et al. have reported that TGF-
induces IL-6 in human keratinocytes, mainly by transcriptional
activation (25). Collectively these data suggest that IL-6
and TGF- cooperate to induce keratinocyte proliferation.
To further confirm that transgenic IL-6 released from keratinocytes
induced the changes in the skin, we constructed expression vectors of
mutant IL-6 and observed the changes in the skin after their transfer.
IL-6 has been shown to interact with two distinct receptor subunits,
IL-6R and gp130. IL-6R interacts specifically with IL-6 and keeps
it in a form available for interaction with gp130, which results in
activation of intercellular signaling. It has been proposed that IL-6
folds as a bundle of four -helices (A, B, C, and D)
(26), and recently it has been predicted to possess three
topologically distinct receptor-binding sites: site 1 for binding to
the subunit specific chain IL-6R and sites 2 and 3 for interaction
with two subunits of the signaling chain gp130 (27).
Mutation of residues Y31, G35, S118, and V121 at site 2 gave rise to
mutant IL-6 with no bioactivity but unimpaired binding to IL-6R, and
mutation of residues K70 and S60 at site 1 increased in the binding to
IL-6R (28, 29). In this study, we mutated residues
using in vitro mutagenesis as shown in Table I, expressed mutant IL-6
in keratinocytes, and observed the resulting skin reaction. Forms 31D
and 121D were found to show a slight decrease in activity, the 35F and
118R showed about a 16-fold decrease, and 75T had no activity, even at
a dose of 8 µg. These data provide further evidence that skin
inflammation after IL-6 gene introduction is caused by overexpression
of IL-6.
Cytokines, which are produced by various kinds of cells, possess
multiple biological properties and are now being used clinically for
treatment of many diseases. Since recent studies have shown that
keratinocytes can be a significant source for many of these cytokines,
keratinocyte gene therapy using cytokine genes has great potential for
intractable skin diseases. Furthermore, introduction of the cytokine
genes into keratinocytes in vivo can provide further understanding of
the biological roles of cytokines in both normal and abnormal skin
conditions. Hengge et al. succeeded in transfecting the IL-8 gene into
keratinocytes, and found that it was expressed and produced biological
activity in the treated skin (11). We tried to inhibit the
IL-6 gene-induced inflammation by transfecting keratinocytes with IL-6
mutant genes. An inhibitory effect was observed for 35F and 118R, which
were suggested to occupy the IL-6R of keratinocytes and prevent
wild-type IL-6 from binding to it. The significant inhibitory effects
of 35F and 118R would be marked at 24 h, and not be evident
48 h after phIL6 transfer (Fig. 8A). Next, phIL6 was
injected at the same site 48 h, 36 h, 24 h, 12 h,
and just after 118R introduction. Introduction of 118R delayed the
expression of phIL6 when 118R was injected 12, 24, and 36 h before
phIL6 injection (Fig. 8B). Since maximum expression of
transgenic IL-6 was found 12–24 h after introduction of the gene (Fig. 2B), no effect was thought to be detected when 118R was
injected 48 h before phIL6 injection (Fig. 8B). Even at
a dose of 8 µg, 75T did not induce inflammation and had no activity
to suppress inflammation, suggesting that further mutations in residues
75 and 76 (Table I) prevented 75T from binding to IL-6R.
It is expected that gene therapy will make it possible to treat intractable diseases, for which useful treatments have not yet been developed, and that keratinocytes gene therapy will be applicable to the treatment of genetic, neoplastic, and inflammatory skin diseases. Our findings show that it is possible to treat IL-6-related skin inflammation by introduction of IL-6 mutant genes into keratinocytes. Finally, this concept of gene therapy can be extended to other cytokines, and the systemic expression of transgenic cytokines using keratinocytes as a bioreactor will hopefully be applicable to systemic diseases (14).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. Daisuke Sawamura, Department of Dermatology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, 036 Japan. E-mail address:
3 Sawamura, D., X. Meng, S. Ina, H. Nakano, K. Tamai, K. Nomura, K. Hanada, J. Miyazaki, Y. Ohe, and I. Hashimoto. Promoter/enhancer cassettes for keratinocyte gene therapy using the naked DNA method: control transgene expression in keratinocyte in vivo. Submitted for publication.
4 Abbreviation used in this paper: RT, reverse transcriptase.
Received for publication February 17, 1998. Accepted for publication July 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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