Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02334168 2001-O1-12
Inhibition of Alopecia
The present invention relates to a process for inhibiting
alopecia and a system of identifying alopecia-inhibiting
substances.
Alopecia is a wide-spread hair disease which may result in
the complete loss of the hair. The causes of alopecia are
not known. In so far it is not possible to influence this
disease in well-calculated fashion.
Therefore, it is the object of the present invention to
provide a product by means of which this can be achieved.
According to the invention, this is achieved by the subject
matters defined in the claims.
The present invention is based on the applicant's findings
that certain forms of alopecia are based on an unbalanced
keratinization of the hair. Furthermore, he has found that
in the case of alopecia the mRNA of various genes is
lacking, e.g. that of the Ha3 gene, or underrepresented,
a . g . those of Hal , Ha2 , and Ha4 genes ( cf . figs . 1 and 2 ) .
The gene products of Hal, Ha2 , Ha3 , and Ha4 genes are hair
keratins. The applicant has found that the expression of the
Ha3 gene is controlled by a gene product of the whn gene. In
particular, he has found that the expression of the Ha3 gene
can be induced by the expression of the whn gene (cf. fig.
3). He has also found that the expression of other hair
keratin genes is essentially influenced by the gene product
of the whn gene. The applicant has also found that the
expression of the whn gene varies in the course of the hair
cycle. In particular, he has found that the whn expression
in the telogen of the hair cycle drops to no longer
detectable levels. In addition, he has discovered that the
whn gene can be transcribed by two promoters. The applicant
has obtained his findings by means of naked mice and HeLa
cells.
CA 02334168 2001-O1-12
2
According to the invention the applicant's findings are used
for a process for inhibiting alopecia, which comprises the
increase in the cellular amount of hair keratins.
The expression "increase in the cellular amount of hair
keratins" refers to the fact that the amount of one or
several hair keratins, particularly of Hal, H2, Ha3 and Ha4,
which may be present in small amount or not at all, is
increased in cells. This can be achieved by common methods
and substances, respectively. For example, one or several
hair keratins, particularly Hal, Ha2, Ha3 and Ha4, may be
added to the cells as such or in the form of DNA encoding
the same. The DNA may be present in common expression
vectors. It is also possible to add substances which
activate the expression of one or several hair keratins,
particularly of Hal, Ha2, Ha3 and Ha4. Such substances are
e.g. the gene product of the whn gene or a DNA encoding the
same. It may be present in common expression vectors.
Moreover, substances may be added which activate the
expression of the whn gene. They may also be present as such
or in the form of DNA encoding the same, it being possible
for the latter to be also present in common expression
vectors. The expression "cells" comprises cells of any kind
and origin. In addition, it comprises tissues and organisms,
particularly animals and human beings.
Substances inhibiting alopecia can be administered as usual,
preferably locally. The substances may also be present in
common formulations. If the substances are administered
locally, e.g. creams, ointments, shampoos and hair tonics
will be suitable. The substances may also be present as
particles which are easily absorbed. Examples of such
particles are liposomes. A person skilled in the art knows
processes to discover the suitable formulations and forms of
administration, respectively, for the individual substances.
A further subject matter of the present invention relates to
a system of identifying substances which are suited to
inhibit alopecia. Such a system comprises the increase in
CA 02334168 2001-O1-12
3
the cellular amount of hair keratins and/or substances
activating the gene expression thereof. In particular, the
system comprises animals or cells, cells being preferred, in
which one or several expressible hair keratin genes and/or
one or several expressible genes, whose gene products
activate the gene expression of hair keratins, are present
each in fused form with a reporter gene. The hair keratin
genes may be particularly those of Hal, Ha2, Ha3 and Ha4.
Moreover, it is favorable for the substance activating the
gene expression of hair keratins to be a gene product of the
whn gene. In addition, the above genes may have a wild type
sequence or a modified sequence, it being possible for the
latter to differ from the wild type sequence by one or
several base pairs. The differences may exist in the form of
additions, deletions, substitutions and/or inversions of
base pairs. Besides, an above reporter gene may be any gene,
particularly it may code for an enzyme, e.g. alkaline
phosphatase, or a fluorescent protein, e.g. GFP. The fusion
genes may also be available in extrachromosomal fashion or
in the cell genome, particularly in place of one or both
allels of hair keratins and/or the genes whose gene products
activate the expression of hair keratins. Besides, the
system may contain substances which are suited to detect the
expressed hair keratins and/or substances activating the
gene expression thereof and the fusion genes, respectively.
Such substances may be suited for the detection on a nucleic
acid level and protein level, respectively.
By means of the present invention it is possible to inhibit
alopecia. It is also possible to diagnose alopecia by
determining e.g. the gene expression of hair keratins and/or
substances which activate it. Moreover, it is possible to
discover substances which are adapted to inhibit alopecia.
For this purpose, a system is provided which is suited for
the rapid and reliable screening of the most varying
substances. Thus, the present invention provides products
serving for diagnosing and treating a wide-spread hair
disease.
CA 02334168 2001-O1-12
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Brief description of the drawiags:
Fig. 1 shows an in situ RNA hybridization using a probe
for mHa3 in normal (whn +/+) and mutant (whn -/-)
mice. The transcripts for mHa3 (perceptible as
brown silver grains) cannot be detected in hair
follicles of naked mice. The line corresponds to
100 ~,m.
Fig. 2 shows the expression of whn and hair keratins in
the hair follicle of a mouse.
A. Northern filter hybridization with RNA from the
total skin of normal mice (whn +/+) and naked mice
(whn -/-) by means of probes for hprt and whn
genes as well as Hal, Ha3, Ha4 genes at three
times following the birth dP7, 7 days after the
birth, etc.).
B. In situ RNA hybridization in the skin from
normal (whn.+/+) and naked mice (whn -/-) with
probes for Hal, Ha3, and Ha4 genes. An
autoradiogram of skin cuts on day 7 after the
birth is shown.
Fig. 3 shows the control of keratin gene expression. HeLa
cells were transiently transfected with a whn
expression construct (+), and the presence of Ha3-
specific mRNA was detected by means of RT-PCR. The
molecular weight markers are given in bp.
The present invention is explained by the below examples:
Example 1: Detectioa of the loss of expression of the
Ha3 gene in mice suffering from alopecia
The "Representational Difference Analysis"
(RDA) method was carried out. This method
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comprises the isolation of mRNA from skin
cells of (whn +/+) mice and (whn -/-) mice
(mice suffering from alopecia and having no
expression of the whn gene), respectively,
the transcription of mRNA into cDNA, and the
differentiation of the cDNA, thereby
identifying the one underexpressed and
overexpressed, respectively, in (whn -/-)
mice.
A~ Sequence of the oligonucleotide adapters
The following oligonucleotide adapter pairs
were required for RDA:
R-Bgl-12: 5'-GATCTGCGGTGA-3'
R-Bgl-24: 5'-AGCACTCTCCAGCCTCTCACCGCA-3'
R-Bgl-12: 5'-GATCTGTTCATG-3'
R-Bgl-24: 5'-ACCGACGTCGACTATCCATGAACA-3'
R-Bgl-12: 5'-GATCTTCCCTCG-3'
R-Bgl-24: 5'-AGGCAACTGTGCTATCCGAGGGAA-3'
B) Production of poly A-mRNA from tissues
to be compared with one another
First, RNA was obtained from the skin of (whn
+/+) mice and (whn -/-) mice, respectively,
according to the "single step RNA extraction"
method (Chomczynski and Sacchi, 1987). The
poly A-mRNA fractions from both RNA
populations were then isolated by means of
dynabeads oligo(dT) according to the
corresponding protocol from the company of
Dynal.
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C) Synthesis of double-stranded cDNA
The "ribo clone cDNA synthesis kit" from the
company of Promega was used for the synthesis
of double-stranded (whn +/+) cDNA and (whn -
/-) cDNA, respectively. 4 ~.g poly A-mRNA were
used each to obtain about 2 ~.g cDNA.
D) Difference analysis
1. Restriction digestion of the double-
stranded cDNAs
a) About 2 ~,g of each cDNA were digested in
a 100 ~.1 reaction batch by the
restriction endonuclease DpnII at 37°C
for 2 h.
b) The reaction solutions were then
extracted twice using a phenol/chloro-
form mixture (1:1) and once using 100 0
chloroform.
c) The DNA included in the aqueous phases
of the two reaction batches was mixed
with 2 ~g glycogen, 50 ~,1 10 M ammonium
acetate, and 650 ~C1 100 o ethanol each
and precipitated on ice for 20 min.
Following 14 minutes of centrifugation
at 4°C and 14,000 rpm, the supernatant
was discarded and the DNA pellet was
washed with 70 % ethanol. After another
centrifugation and removal of the
alcoholic phase, the dried DNA was
resuspended in 20 ~.1 TE buffer.
2. Ligation of the cDNAs to the R-Bgl
oligonucleotide adapter pair
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a) A reaction vessel collected the
following:
20 ~,1 cut cDNA (total reaction batch
from item D) lc)
8 ~,g R-Bgl-24
4 ~.g R-Bgl-12
6 ~.1 10 x ligase buffer
x ,ul water
57 ~1 final volume
b) The reaction batch was heated in a
thermocycler (Peltier Thermocycler PTC-
200, MJ Research) to 50°C, kept at this
temperature for 1 min., and then cooled
again down to 10°C in the course of one
hour (ramp rate: 0.1°C/9 sec).
c) After adding 3 ~1 T4 DNA ligase (1
U/ul), the mixture was incubated at 16°C
overnight.
3. Synthesis of "representations" of the
cDNA populations to be compared with one
another
a) In order to generate what is called
"representations" of the ligated cDNAs,
the volume of the ligation batches from
item 2c) was initially completed by
adding 140 ~,1 water each to give 200
~,1 .
Then, 30 reactions of 200 ~,1 each were
prepared from this dilute solution per
cDNA population (whn +/+) skin and (whn
-/-) skin.
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The following reactants were added to
such a batch one after the other:
143 ~.1 water
20 ~.1 lOx PCR buffer
2 0 ~.1 2 mM dNTPs
10 ~.l 25 mM Mg chloride
2 ~,1 R-Bgl-24 (1 ~.g/~tl)
4 ~1 dilute ligation batch
b) PCR:
3 min.: 72°C
addition of 1 ~.1 Taq-DNA polymerase (5
U/~tl )
20 x: 5 min.: 95°C
3 min.: 72°C
finally: cooling to 4°C.
c) For preparing the reaction solutions, 4
reaction batches each were collected in
a vessel.
Extraction: 2 x with 700 ~.1
phenol/chloroform each
(1:1), 1 x with
chloroform 100 %;
Precipitation: addition of 75 ~.1 3 M Na
acetate solution (pH 5.3)
and 800 ~.1 2-propanol to
each reaction vessel, 20
min. on ice.
Centrifugation: 14 min., 14,000 rpm,
4°C.
Washing of the DNA pellets with ethanol
70 % and resuspension in such an amount
of water that a concentration of 0.5
~,g/~,1 resulted.
4. Restriction digestion of the
"representations"
CA 02334168 2001-O1-12
9
a) In order to remove the R-Bgl
oligonucleotide adapters, 300 ~.g of each
representation (whn +/+) skin and (whn -
/-) skin, respectively, were subjected
to restriction digestion. After adding
the following reactants, incubation was
carried out at 37°C for 4 hours:
600 gel cDNA representation (0.5 ~.g/ul)
140 ~1 10 x DpnII buffer
100 ~C1 DpnI I ( 10 U/~.1 )
560 ~.1 water.
b) The restriction digestion batch was
distributed to 2 vessels prior to its
preparation.
Extraction: 2 x phenol/chloroform
(1:1) , 1 x chloroform 100
o;
Precipitation: addition of 70 ~,l 3 M Na
acetate (pH 5.3), 700 ul
2-propanol to each ves-
sel, 20 min. on ice.
Centrifugation: 14 min., 14,000 rpm,
4°C.
The DNA pellet was washed with ethanol
70 % and resuspended in such an amount
of water that a concentration of 0.5
~.g/~1 resulted.
The resulting DpnII-digested (whn +/+)
skin cDNA representation represented the
driver DNA population to be used for the
subtractive hybridization.
5. Synthesis of the tester DNA population
a) 20 ~.g of the (whn -/-) skin cDNA
representation digested by DpnII (_
CA 02334168 2001-O1-12
10
tester DNA) was separated in a TAE gel
by means of electrophoresis:
40 ~Cl tester DNA ( 0 . 5 ~g/~,1 )
50 ~1 Te buffer
10 ~,1 10 x loading buffer
were placed on a 1.2 o agarose TAE gel.
A voltage was applied to the gel until
the bromophenol blue component of the
loading buffer had migrated about 2 cm.
b) Thereafter, the bands containing the
representation DNA were cut out off the
gel and eluted by means of the "agarose
gel DNA extraction kit" from the company
of Boehringer Mannheim.
The DNA extracts were collected, so that
a total of 60 ~.1 solution was obtained.
The concentration of this solution was
evaluated by electrophoresis of 5 ~.1 in
a 1 % agarose gel.
c) Eventually, the tester DNA was ligated
with the J-oligonucleotide pair:
2 ~,g tester DNA eluate
6 ~,1 10 x ligase buffer
4 ~.1 J-Bgl-24 (2 ~g/~,1)
4 ~,1 J-Bgl-12 (1 ~,g/~tl)
x ,u.l water
57 ~,1 final volume
d) Transferring the reaction batch to the
thermocycler:
1 min.: 50°C
cooling down to 10°C within 1 h (ramp
rate: 0.1°C/9 sec.)
CA 02334168 2001-O1-12
11
e) The addition of 3 ~.1 T4 DNA ligase (1
U/~,1) was followed by incubation at 16°C
overnight.
f) Adjustment of the concentration of the
tester DNA to about 10 ng/~1 by the
addition of 120 ~,1 water.
6. Subtractive hybridization
a) 80 ~.1 driver DNA (40 fig) from step 4.
and 40 ~,1 (0.4 fig) dilute tester DNA
from step 5., ligated with J-
oligonucleotides, were collected in a
reaction vessel and extracted 2 x with
phenol/chloroform (1:1) and once with
chloroform 100 s.
b) Precipitation by adding 30 ~cl 10 M
ammonium acetate, 380 ~1 ethanol 100 0,
-70°C for 10 min.
Centrifugation: 14 min., 14,000 rpm,
4°C
Thereafter: 2 x washing the
pellet with ethanol
70 %, short
centrifugation after
each wash step;
drying of the DNA
pellet.
c) The DNA was resuspended in 4 ~1 EE x3
buffer (30 mM EPPS, pH 8.0 at 20°C
(company of Sigma), 3 mM EDTA) - with
pipetting off and on for about 2 min.,
then heated to 37°C for 5 min., shortly
vortexed and eventually the solution
was collected again at the vessel bottom
CA 02334168 2001-O1-12
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by centrifugation. Finally, the solution
was coated with 35 ~,1 of mineral oil.
d) Transferring the reaction batch to the
thermocycler:
5 min.: 98°C,
cooling down to 67°C and immediate
addition of 1 ~.1 5 M NaCl to the DNA,
incubation at 67°C for 20 h.
7. Synthesis of the first difference
product
a) After removing the mineral oil as
completely as possible, the DNA was
diluted step-wise:
1. addition of 8 ~.1 TE (+ 5 ~.g/~.1
yeast RNA),
2. addition of 25 ~.1 TE - thereafter
thorough mixing,
3. addition of 362 ~,1 TE - vortex.
b) 4 PCRs were prepared for each
subtractive hybridization. Per reaction:
127 ~,1 water
20 ~.1 10 x buffer
20 ~.1 2 mM dNTPs
5 ~1 25 mM Mg chloride
20 ~1 dilute hybridization solution
(from step 7a) )
c) PCR program:
3 min.: 72°C
addition of 1 ~,1 Taq DNA polymerase (5
U/~,1 )
5 min.: 72°C
addition of 2 ~,l primer J-Bgl-24 (1
N~g/~l )
CA 02334168 2001-O1-12
13
10 x: 1 min.: 95°C
3 min.: 70°C
finally: 10 min.: 72°C, then cooling
down to room temperature.
d) The 4 reaction batches were collected in
a 1.5 ml vessel.
Extraction: 2 x phenol/chloroform (1:1),
1 x chloroform 100 %.
After the addition of 2 ~g glycogen
carrier:
Precipitation with 75 ~.1 3 M Na acetate
(pH 5.3), 800 ~1 2-propanol, 20 min. on
ice.
Centrifugation: 14 min., 14,000 rpm,
4°C.
Washing of the DNA pellet with ethanol
70°s .
After drying the DNA, resuspension in 40
~.1 water .
e) 20 ~.l of the re suspended DNA from d)
were subjected to mung bean nuclease
digestion (= MBN):
20 ~1 DNA
4 ~,l 10 x mung bean nuclease buffer
(company of NEB)
14 ~.1 water
2 ~.1 mung bean nuclease ( 10 U/~.1;
company of NEB)
35 min., 30°C.
The reaction was stopped by adding 160
~.1 of 50 mM Tris-HC1 (pH 8.9) and 5
minutes of incubation at 98°C.
Thereafter, the vessel was placed on ice
up to the next step.
CA 02334168 2001-O1-12
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f) During the MBN incubation, 4 further
PCRs were prepared on ice:
127 ~1 water
2 0 ~C1 2 mM dNTPs
10 ~,1 25 mM Mg chloride
2 ul J-Bgl-24 (1 ~g/~,1)
20 ~1 MBN-digested DNA.
g) PCR program:
1 min.: 95°C
allowing to cool down to 80°C, addition
of 1 ~.1 Taq DNA polymerase (5 U/~,1)
18 x: 1 min.: 95°C
3 min.: 70°C
finally: 10 min.: 72°C, allowing to cool
down to 4°C.
h) The 4 PCR batches were collected in a
vessel
Extraction: 2 x phenol/chlorofortn (1:1)
1 x chloroform 100 0.
Precipitation: 75 ~.1 3 M Na acetate (pH
5.3), 800 ~.l 2-propanol,
20 min. on ice.
Centrifugation: 14 min., 14,000 rpm,
4°C.
Washing of the DNA pellet with ethanol
70
o.
Re suspension of the DNA in 100 ail water
(resulting concentration: 0.5 ~.g/~,l);
the resulting solution represented the
first difference product.
8. Exchange of the oligonucleotide adapters
of the difference product
a) Removal of the oligonucleotide adapters
by restriction digestion using DpnII:
CA 02334168 2001-O1-12
15
40 ~,1 dif f erence product 1 ( 0 . 5 ~.g/~.1 )
30 ~,l 10 x DpnII buffer
15 ~C1 DpnI I ( 10 U/~C1 )
215 ~.1 water
37°C for 2 h.
b) Preparation of the reaction batch:
Extraction: 2 x phenol/chloroform
(1:1) , 1 x chloroform 100
o.
Precipitation: 33 ~1 3 M Na acetate (pH
5.3), 800 ~,l ethanol 100
%, -20°C for 20 min.
Centrifugation: 14 min., 14,000 rpm,
4°C.
Washing of the pellet in ethanol 70 %
and resuspension in 40 ~.1 water.
c) Ligation of the difference product to N-
Bgl oligonucleotide adapter pair
1 ~,1 of the prepared DNA solution from
step b) was diluted with 9 ~.1 water to
give a concentration of 50 ng/~,1; 4 ~1
of this solution were used in the
following reaction:
4 ~.1 DpnII-digested difference product 1
(200 ng)
6 ~.1 10 x ligase buffer
2.5 ~.1 N-Bgl-24 (3.5 ~,g/~tl)
2 ~tl N-Bgl-12 (2 ~g/~1)
42.5 ~,1 water.
d) After transferring the reaction batch to
the thermocycler:
1 min.: 50°C,
allowing to cool down to 10°C within one
hour (ramp rate: 0.1°C/9 sec.).
CA 02334168 2001-O1-12
16
e) After adding 3 ~cl T4 DNA ligase (1
~.g/~.1), incubation at 16°C overnight.
9. Synthesis of the 2nd difference product
By adding 100 ml water, the ligation
batch from step 8e) was diluted to a
concentration of 1.25 ng/~1. 40 ~.l of
this dilution (50 ng) were mixed with 80
~.1 driver DNA ( see item 4 . ) and treated
again according to steps 6. to 8. When
the oligonucleotide adapters (step 8.)
were exchanged, the J-Bgl
oligonucleotides were then ligated to
the newly formed difference product 2.
10. Synthesis of the 3rd difference product
The concentration of difference product
2 ligated with the J-Bgl oligos was
reduced to a concentration of 1 ng/~.1.
10 ~.1 of this solution were diluted
again with 990 ~1 water (+ 30 ~g yeast
RNA), so that the concentration was then
10 pg/~.1. The subtractive hybridization
was carried out with 100 pg (10 ~1) J-
ligated difference product 2 and 40 ~Cg
(80 ~.1) driver DNA from step 4. ) . As for
the rest, the same steps were carried
out as in the first and second
difference products according to steps
6. to 8. An exception was the PCR
following the MBN reaction (item 7.g) -
here only 18 instead of 22 cycles were
carried out.
11. Cloning of the 3rd difference product
CA 02334168 2001-O1-12
17
The 3rd difference product was initially
subjected to restriction digestion using
DpnII so as to remove the
oligonucleotide adapters. The reaction
product was then applied to a TAE gel
and separated by means of
electrophoresis. The separated DNA bands
were cut out off the gel, the DNA was
eluted and cloned into a vector (pBS
Not) cut by BamHI.
12. Characterization of the difference
products
In order to confirm that the cloned DNA
fragments were not method artifacts but
sequences which were actually included
in the investigated DNA representations,
Southern analyses were carried out in
which the investigated cDNA
representations were hybridized with the
radioactively labeled cloning products.
Thereafter, those DNA fragments which
had proved to be "real" difference
products in the Southern analysis were
investigated by means of Northern
hybridizations: RNAs were blotted from
the investigated tissues ((whn +/+) skin
cDNA and (whn -/-) skin cDNA) and
hybridized with the radioactively
labeled cloning products. By this, the
differential expression of these
sequences was confirmed in the
investigated tissues. An analysis of the
sequences led to the result that the Ha3
gene is not expressed in nu/nu mice
(mice suffering from alopecia) (cf. fig.
1) .
CA 02334168 2001-O1-12
18
Example 2: Expression of hair keratin aad wha genes in
normal mice and mice suffering from alopecia.
RNA was isolated from the skin of differently
old normal (whn +/+) and naked (whn -/-)
mice, separated electrophoretically in
agarose gels, transferred to filters and
hybridized with gene-specific probes.
The employed probes were as follows:
mHal:nucleotides 1331 - 1551; GeneBank,
accession No. M27734
mHa3:nucleotides 1007 - 1204; GeneBank,
accession No. X75650
mHa4:nucleotides 1303 1542, cf. Bertolino,
-
A.P. et al., J. Invest. Dermatol. 94, (1990),
297 303
-
whn: nucleotides 1141 - 1374; GeneBank,
accession No. X81593
It showed that hair keratin genes and whn
genes in mice suffering from alopecia are not
expressed and expressed only slightly,
respectively.
Example 3: Detection of the expression induction of the
Ha3 gene by the gene product of the whn gene.
A whn gene tagged at the N-terminal epitope
was inserted in the expression vector pTRE
(Clontech). The resulting DNA construct was
used for a transient transfection of the HeLa
Tet-On cell line (Clontech) by means of the
calcium phosphate coprecipitation method. The
cells were treated with 5 ~.g/ml docycline
directly afterwards. 1 mM sodium butyrate was
CA 02334168 2001-O1-12
19
added 24 h later. The cells were harvested 48
h after the transfection and subjected to a
RT-PCR method. The primers used in the PCR
method were as follows:
hHa3:
5'-CTGATCACCAACGTGGAGTC-3',
5'-TACCCAAAGGTGTTGCAAGG-3'.
The PCR method included 35 - 40 cycles each
of 30 sec. at 95°C, of 30 sec. at 58°C and of
1 min. at 72°C.
It showed that an expression of the Ha3 gene
was induced by the expression of the whn
gene. Parallel controls in which no
transfection was effected by means of the whn
gene, did not result in an induction of the
Ha3 gene expression.
Example 4: Preparation of the system according to the
invention
A BAC clone referred to as BAC whn, which
comprises the entire whn gene of a mouse, was
isolated from a BAC library of the company of
Genome Systems (St. Louis, Missouri, U.S.A.)
(cf. Schorpp, M. et al., Immunogenetics 46,
(1997), 509-515).
In addition, a shuttle vector referred to as
pMB096-whn-GFP was used, which included the
mouse whn gene which contained the reporter
gene GFP in exon 3 (cf. Nehls, M. et al.,
Science 272, (1996),886-889).
BAC-whn was used to transform the recA' E.
coli strain CBTS. The transformation was
carried out by means of electroporation.
CA 02334168 2001-O1-12
20
Clones were isolated and transformed by means
of pMB096-whn-GFP using electroporation. A
homologous recombination was made between the
BAC clone and the shuttle vector within the
range of the whn gene so as to obtain a
vector referred to as BAC-whn-GFP. It
included the reporter gene GFP in the whn
gene.
BAC-whn-GFP was used for the transfection of
COS cells. The transfection was carried out
by means of the calcium phosphate
coprecipitation method. COS cells were
obtained which coded for a fusion gene from
whn and GFP.
It showed that these cells were suited to
identify substances which could induce the
gene expression of whn. Such substances were
suited to inhibit alopecia.
CA 02334168 2001-O1-12
Sequence ~istina
<1J.0> Deutsches Krebsforschungszentrum
<120> Inhibition of aJ.opecia
<130> K 2920
<140> PCT/DE99/02185
<141> 1999-07-13
<150> DE 198 31 043.9
<151> 1998-07-13
<160> 8
<170> PatentIn Ver 2.1.
<210> 1
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of the Artificial Sequence:
oligonucleotide adaptor
<400> 1
gatctgcggt ga x2
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
CA 02334168 2001-O1-12
2
<220>
<?23> Descr~.ption of the Artificial Sequence
oligonucleotide adaptor
<400> 2
agcactctcc agcctctcac cgca 24
1d
<210> 3
<211> 12
<?12> DNA
<~;13> Artificial Sequence
<i'20>
<223> Description of the Artificial Sequence:
oligonucleotzde adaptor
<400> 3
gatctgttca tg 12
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of the Artificial Sequence:
oligonucleotzde adaptor
<400> 4
CA 02334168 2001-O1-12
3
ac.cgacgtcg actatccat~ aaca 24
<210~ 5
<211> ~.2
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of the Artificial Sequence:
oligonucleotide adaptor
<.~D05 5
gatcttccct cg 12
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of the Artificial Sequence:
ol,igonucleotide adaptor
<~00> 6
3Q
aggcaactgt gctatccgag ggaa 24
<210> 7
<211> 20
<212~ DNA
<213> Artificial Sequence
CA 02334168 2001-O1-12
4
<220>
<223> Description of the Artificial Sequence:
frimex'
<400> 7
ctgatcacca acgtggagtc 20
i0
<210> 8
~211> 20
<212> DNA
a213> Artificial Sequence
<220>
~223~ Description of the Artificial Sequence:
Primer
<c~Op> 8
tacccaaagg tgttgcaagg
