# Patent

## Title
Cell transformed by human Wnt3a gene

## Abstract

Disclosed is a cell transformed by a vector containing human Wnt3a gene, wherein the cell is selected from a group consisting of hair follicle-derived cells and prostate cancer-derived cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage application of PCT/JP2009/050884, filed Jan. 21, 2009, which claims priority from Japanese application JP 2008-010823, filed Jan. 21, 2008.
TECHNICAL FIELD
The present invention provides a cell transformed with a vector containing human Wnt3a protein gene that demonstrates high expression efficiency of human Wnt3a protein.
BACKGROUND ART
Wnt protein is a secretory glycoprotein having a molecular weight of about 40,000 that is known to be an important intercellular signaling molecule for embryonic morphogenesis and hair follicle regeneration, and is involved in differentiation and functional maintenance of melanocytes. Nearly 20 types of vertebrate Wnt are known, these Wnt form subfamilies, and each binds to a seven-pass transmembrane receptor known as Frizzled and a single-pass transmembrane receptors known as LRP on the cell membrane. Wnt3a, which is a member of the Wnt family, has been clearly demonstrated on the basis of transplant studies in mice to maintain hair follicle formation induction activity of hair papilla by acting on hair papilla cells (Kishimoto, J. et al., Genes & Dev., 2000 May 15, 14(10), 1181-5), and considerable attention has recently been focused on its relationship with hair follicle induction. Research is being conducted on recombinant avian or mouse Wnt3a protein produced by avian Wnt3a or avian cells transformed with mouse Wnt3a gene by allowing this recombinant protein to act on mouse hair papilla cells.
When considering use in clinical studies applicable to humans, it is necessary to prepare large amounts of human Wnt3a recombinant protein instead of human or mouse protein. However, recombinant human Wnt3a has the problem of it being difficult to produce protein that retains adequate activity probably due to problems in translation and modification following transcription. Thus, selection of host cells for use in efficiently producing human Wnt3a recombinant protein that retains hair follicle induction activity is an important factor for elucidating the mechanisms of hair follicle formation and regeneration as well as screening and evaluating drugs that induce and accelerate hair follicle formation and regeneration.

    Non-Patent Document 1: Genes & Dev., 2000 May 15, 14(10), 1181-5


DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
An object of the present invention is to provide a cell capable of efficiently expressing human Wnt3a recombinant protein having activity that induces formation of hair follicles by hair papilla cells.
Means for Solving the Problems
As a result of conducting extensive studies, the inventors of the present invention found that hair follicle-derived cells and prostate cancer-derived cells are optimum as hosts for efficiently expressing human Wnt3a recombinant protein that retains activity, thereby leading to completion of the present invention.
Thus, the present application includes the following inventions:
(1) a cell transformed with a vector containing human Wnt3a gene, wherein the cell is selected from a group consisting of hair follicle-derived cells and prostate cancer-derived cells;
(2) the cell of (1), wherein the hair follicle-derived cells are IROS or IDP cells;
(3) the cell or (1), wherein the prostate cancer-derived cells are PC3 cells; and,
(4) the cell of any of (1) to (3), wherein the vector is a pTarget vector.
EFFECTS OF THE INVENTION
According to the present invention, human Wnt3a recombinant protein, which has activity that induces hair follicle formation by hair papilla cells, can be efficiently expressed, and elucidation of the mechanisms of hair follicle formation and regeneration, as well as evaluation of effective drugs for hair follicle regeneration, are expected to be carried out efficiently.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide sequence of a gene encoding Wnt3a (SEQ ID NO:1).
FIG. 2 shows Western blotting of cells containing Wnt3a.
FIG. 3 shows confirmation of functional expression of Wnt3a introduced by accelerated expression of Lef-1.



BEST MODE FOR CARRYING OUT THE INVENTION
Human Wnt3a is a protein comprised of 352 amino acid residues, and the nucleotide sequence of a gene encoding this protein is shown in SEQ ID NO:1 and FIG. 1. An expression vector containing Wnt3a gene can be produced by linking Wnt3a gene to a vector through a suitable restriction site to be placed under the control of a suitable promoter of the vector using a known method. There are no particular limitations on the type of vector, and all types of commercially available vectors can be used, including commercially available animal cell expression vectors such as pTarget vector (Promega), pBK-CMV vector or pBK-RSV vector (Stratagene). pTarget vector is particularly preferable.
There are no particular limitations on a preferable promoter used in the present invention provided it is a suitable promoter corresponding to the host used to express gene, examples of which include CMV promoter, SRα promoter, SV40 promoter, LTR promoter, HSV-TK promoter and β-actin promoter.
In addition, an enhancer, splicing signal, poly(A) addition signal, selection marker or SV40 replication origin and the like may also be contained in the expression vector as desired.
Hair follicle-derived cells, such as human immortalized outer root sheath cells (IORS) or human immortalized dermal papilla cells (IDP), or human prostate cancer-derived cells (PC3) can be used as host cells. IORS are particularly preferable.
In order to obtain human outer root sheath cells, human scalp is obtained as a by-product of, for example, plastic surgery procedures or surgery procedures, and hair follicle tissue is obtained from the scalp under a stereo microscope. Next, after treating the hair follicle tissue with a hydrolase solution, such as a mixed solution of collagenase and dispase, for 30 minutes at 37° C., the tissue is allowed to stand undisturbed in a collagen-coated culture dish followed by culturing in a low-calcium serum-free medium such as K-GM (Clonetics) or Keratinocyte-SFM (Gibco BRL) and replacing the medium every 2 weeks. After confirming that cells have proliferated, the cells adhered to the culture vessel are released and recovered by centrifugation followed by sub-culturing in serum-free medium.
Human dermal papilla cells are also obtained in the same manner as outer root sheath cells from human scalp obtained as by-products of plastic surgery procedures and surgery procedures under a stereo microscope. Next, the hair papilla are cultured in an ordinary animal cell culture medium such as Dulbecco's Modified Eagle Medium containing fetal calf serum and the medium is replaced every 2 days. After confirming that cells have proliferated, cells adhered to the culture vessel are released and recovered by centrifugation followed by sub-culturing in serum-free medium.
Immortalization of human outer root sheath cells or human dermal papilla cells obtained in this manner can be carried out by known methods. Preferably, the Large T antigen gene of SV40 virus deficient in a replication origin can be used as described in Japanese Unexamined Patent Publication No. 2000-89. This gene is normally used in a status being introduced in a plasmid or virus vector. This gene is widely used in the art as a typical gene for immortalizing animal cells and can be acquired easily. For example, a virus in which the EIA region in the adenovirus vector, ΔEI/X (Doren, et al., J. Virol., Vol. 50, 606-614 (1984)) is substituted with SV40 Large T antigen virus deficient in a replication starting point is described in Doren, et al., Mol. Cell. Biol., 1653-1656 (1984)). In addition, retrovirus vector pZITtsa (W. Filsell, et al., Journal of Cell Science, Vol. 107, 1761-1772 (1994)) can be used that is obtained by cleaving a 2491 by fragment containing a gene encoding temperature-sensitive polyomavirus Large T antigen (Plttsa) from pLTtsa (M. Rassoulzadegan, et al., Proc. Nat. Acad. Sci. USA, Vol. 80, 4354-4358 (1983) by use of BglI and HingII, and then inserting this fragment into a BamHI site of retrovirus vector pZIPNeoSV(X)l (C. L. Cepko, et al., Cell, Vol. 37, 1053-1062 (1984)). Moreover, pSV40ori-, obtained by cloning SV40 viral DNA deficient in a replication origin in pBR322, or pSHPV16s (Tissue Cult. Res. Commun., Vol. 11, 13-24 (1992)), obtained by cloning human papillomavirus type 16 DNA in pSV2neo vector, can also be used.
Prostate cancer cell line PC3 is a commercially available cell line that can be acquired from, for example, the American Type Culture Collection (ATCC) or DS Pharma Biomedical (formerly Dainippon Sumitomo Pharma).
Transformation of host cells can be carried out by a commonly known method such as the calcium chloride method, calcium phosphate coprecipitation, DEAE dextran method, lipofection, protoplast polyethylene fusion or electroporation, and a suitable method is selected according to the host cells to be used. In addition, a suitable commercially available kit can also be used to carry out transformation. Examples of such kits include Fugene® (Roche Applied Science), CombiMag (OZ Biosciences) and PolyMag (OZ Biosciences).
The resulting transformed host cells are cultured in a suitable culture medium, the cells are separated from the culture liquid by conventional means without limitation such as centrifugation or filtration, and cells are lysed as necessary, the protein component of the supernatant or filtrate is precipitated with a salt in the manner of ammonium sulfate, and the target protein can be recovered by carrying out various chromatographic techniques such as ion exchange chromatography or affinity chromatography.
The hair follicle formation induction activity of Wnt3a can be measured by using, for example, LEF-1 (lymphocyte enhancer factor 1), which is a factor for which gene expression is accelerated by the action of Wnt3a, as an indicator (Filali, M. et al., J. Biol. Chem., 277, 33398-33410, 2002). Wnt instructs cells so as not to decompose β-catenin ((3-CAT), and as a result, β-catenin binds with LEF-1 and similar proteins resulting in induction of hair follicle regeneration and hair growth. Confirmation of the expression of LEF-1 can be carried out by a commonly known method such as RT-PCR or Western blotting.
The following provides a more detailed explanation of the present invention through specific examples thereof. Furthermore, the present invention is not limited by these examples.
EXAMPLES
Construction of Wnt3a and Wnt7a Plasmids
The total length of human Wnt3a was amplified by PCR using cDNA synthesized from commercially available human placental total RNA (Clonetech) as a template and using a sense strand primer gatggccccactcggata (SEQ ID NO:2) and an antisense strand primer ggtgcctacttgcaggtgt (SEQ ID NO:3). Each amplified gene PCR product was purified with a commercially available kit (Promega), coupled to pTarget vector, and cloned using E. coli. After identifying the plasmids containing each gene based on an analysis of restrictase cleavage patterns, the nucleotide sequence of each inserted gene was determined using a DNA sequencer. The nucleotide sequence of the gene encoding Wnt3a is shown in FIG. 1.
Transfection into Cells
Each of the cells shown in the following Table 1 were seeded into a 6-well plate at 1 to 3×105 cells followed by culturing until the cells reached sub-confluency under conditions of 37° C. and 5% CO2. Next, the constructed pTarget vector of Wnt3a or a control was transfected into the cultured cells using commercially available gene transfection reagents (Fugene® (Roche Applied Science), CombiMag (OZ Biosciences) and PolyMag (OZ Biosciences)). Which of the gene transfection reagents were used for which cells are as shown in Table 1. The transfection reagents were used in accordance with the protocol provided with each reagent. In addition, gene transfer efficiency was calculated by introducing a plasmid in which Azami Green gene (Medical & Biological Laboratories) was coupled to pTarget, followed by counting the number of cells positive for Azami Green fluorescence using a flow cytometer (Beckman Coulter). The medium, gene transfection reagent and results for transfer efficiency used for each cell are shown in Table 1.











TABLE 1









Gene





Gene Transfer
Transfection


Cell Name
Species
Origin
Efficiency
Reagent



















IORS
Human
Immortalized
25
Fugene




outer root




sheath cells


hKC
Human
Epidermal
8
CombiMag




keratinocytes


CEF
Chicken
Fetal
10
PolyMag




fibroblasts


IDP
Human
Immortalized
18
PolyMag




dermal




papilla cells


HacaT
Human
Immortalized
7
PolyMag




keratinocytes


Pam212
Mouse
Squamous
32
PolyMag




epithelial




cells


PC3
Human
Prostate
46
Fugene




cancer cells


LNcap
Human
Prostate
10
PolyMag




cancer cells


hFB
Human
Fibroblasts
17
Fugene


MCF7
Human
Breast cancer
0.8
Fugene




cells


Hela
Human
Cervical
0.3
Fugene




cancer cells


mDC
Mouse
Dermal cells
0.4
Fugene









As a result, Wnt3a gene was found to be introduced at comparatively high efficiency in IORS, IDP and PC3 cells.
Confirmation of Hyperexpression of mRNA by Wnt3a as Determined by RT-PCR
The constructed pTarget vector of Wnt3a or the control was introduced into IORS cells, and total RNA was extracted 2 days later using Isogen (Nippon Gene) to synthesize cDNA. Using the resulting cDNA of IORS cells as a template, a PCR reaction was carried out on human Wnt3a using a sense primer caggaactacgtggagatca (SEQ ID NO:4) and an antisense primer ccatcccaccaaactcgatg (SEQ ID NO:5), and hyperexpression was observed when expression of Wnt3a by the introduced plasmid was investigated (data not shown).
Confirmation of Expression of Recombinant Wnt3a Protein by Western Blotting
Cells transfected with each of the genes shown in Table 1 were lysed and subjected to heat treatment in SDS-polyacrylamide electrophoresis (SDS-PAGE) sample buffer 2 days after gene transfection followed by application to SDS-PAGE. At that time, samples were prepared so that the number of cells measured prior to protein extraction was equal between cells transfected with Wnt plasmid and the control vector. Next, protein was transferred from the gel following SDS-PAGE to a membrane using a semi-dry method. After treating the membrane with a commercially available blocking solution, the membrane was allowed to react with Wnt3a antibody (Abcam) diluted by 500-fold as the primary antibody. After washing with PBS-T, the membrane was allowed to further react with HRP-labeled anti-rabbit IgG (GE Healthcare Life Sciences) diluted by 2000-fold as the secondary antibody. Moreover, after washing with PBS-T, detection was carried out using ECL Advance (GE Healthcare Life Sciences). Commercially available mouse Wnt3a protein (R&D System) was used as a positive control. The results are shown in FIG. 2. High expression of Wnt3a was observed in IORS and IDP cells, and expression was also observed in PC3 cells.
Confirmation of Functional Expression of Introduced Wnt3a by Accelerated Expression of Lef-1
Whether or not Wnt3a hyperexpressed by introduction of a constructed plasmid functions physiologically was investigated by using Lef-1, which is a factor in which gene expression is accelerated by the action of Wnt (Filali, M. et al., J. Biol. Chem., 277, 33398-33410, 2002), as an indicator. The pTarget vector of Wnt3a plasmid or the control was introduced into IORS cells and total RNA was extracted 2 days later using Isogen (Nippon Gene) to synthesize cDNA. Using the resulting cDNA of the IORS cells as a template, a PCR reaction was carried out on human Lef-1 using a sense primer cttccttggtgaacgagtctg (SEQ ID NO:6) and an antisense primer gtgttctctggccttgtcgt (SEQ ID NO:7).
The results are shown in FIG. 3. In the case of having introduced Wnt3a plasmid, acceleration expression of Lef-1 gene was observed as compared with the control, and the introduced Wnt3a was determined to function physiologically.

## Claims

1. A PC3 cell transformed with an expression vector containing human Wnt3a gene.

2. The cell according to claim 1, wherein the expression vector is a mammalian expression T-vector.