KR102015172B1 - Composition for Identifying Undifferentiation Stage of Canine Spermatogonial Stem Cells - Google Patents

Abstract

The present invention relates to a composition for analyzing the degree of differentiation of dog garden stem cells, a composition for separating undifferentiated dog garden stem cells, a method for analyzing the degree of differentiation of dog garden stem cells, and a method for isolating undifferentiated dog garden stem cells using CXCR4 and PGP9 By measuring the expression of .5 at the mRNA level or protein level, can effectively analyze the degree of differentiation of dog garden stem cells, and can be used to isolate undifferentiated dog garden stem cells using antibodies specific to CXCR4 and PGP9.5. The undifferentiated dog garden stem cells isolated through the present invention can be used for the study of the production of transformed sperm and animal as well as the study of male infertility, spermatogenesis process mechanism related to germ cell differentiation.

Description

Composition for Identifying Undifferentiation Stage of Canine Spermatogonial Stem Cells}

The present invention relates to a composition for analyzing the degree of differentiation of dog garden stem cells.

Spermatogonial stem cells (SSCs) are present in the testis and play an important role in spermatogenesis. The name raises the possibility of their existence in female mammals and calls them “male germline stem cells” to distinguish them.

These cells have both the unique self-renewal ability and the differentiation ability of sperm cells into adult stem cells, but because of their very small number in the testes, they are difficult to isolate and clinically use. There is a problem of insufficient establishment.

Thus, continuous efforts have been made for efficient separation by discovering new markers of garden stem cells, optimal culture conditions, sufficient in vitro propagation and maintenance method, but sperm formation in vitro still has technical limitations.

Garden stem cells are difficult to cultivate because they have limited isolation and large numbers of garden stem cells die in culture, and the greatest difficulty is the absence of morphological and biochemical markers that can distinguish between garden stem cells and differentiated garden cells. (Nagano et al., 1998; van Pelt et al., 2002).

Related prior arts include Korean Patent No. 10-1027718 (male germ cell-specific nucleotide sequence) and Korean Patent No. 10-1173116 (new swine spermatogonia stem cell isolation and culture method).

Recently, since the production of chimeric mice through garden stem cells in mouse testis, research reports on transplantation using garden stem cells such as rats, cows, pigs, and chickens have increased rapidly.

However, research on the production of transgenic animals using garden stem cells of medium and large animals such as dogs is still insignificant. Especially, the size, life span, and genetics of dogs are more closely related to humans than mouse models. In spite of high research on dog garden stem cells, many studies have not been conducted until now.

Therefore, there is an urgent need for the development of marker systems through the discovery of surface antigens specific to dog garden stem cells and the development of genetically engineered transformation and disease model dog production techniques based on these techniques.

The present inventors earnestly researched to develop a novel biological marker that can confirm the degree of differentiation of dog garden stem cells. As a result, dog garden stem cells expressing CXCR4 and PGP9.5 simultaneously express only CXCR4 or PGP9.5. The present invention was completed by confirming that the cells have more undifferentiated characteristics than the cells, and analyzing the expression of CXCR4 and PGP9.5 to analyze the degree of undifferentiation of canine stem cells.

Accordingly, an object of the present invention is to provide a composition for analyzing the degree of differentiation of dog garden stem cells.

Another object of the present invention to provide a composition for separating undifferentiated dog garden stem cells.

Another object of the present invention to provide a method for analyzing the degree of differentiation of dog garden stem cells.

Another object of the present invention is to provide a method for separating undifferentiated dog garden stem cells.

One aspect of the invention is an agent for measuring the expression of chemokine receptor type 4 (CXCR4) at the mRNA level or protein level; And it relates to a composition for analyzing the degree of differentiation of dog garden stem cells comprising an agent for measuring the expression of PGP9.5 (protein gene product 9.5) at the mRNA level or protein level.

In the present invention, it is observed that there are cell populations expressing CXCR4 and PGP9.5 at the same time and cell populations expressing only PGP9.5 in the testes of dogs. Especially, cells expressing CXCR4 and PGP9.5 simultaneously express only PGP9.5. It was confirmed that it is present in the basement membrane region rather than cells. It is well known that spermatogenesis begins at the basement membrane site and that the cells at the basement membrane site have an undifferentiated nature (Phil. Trans. R. Soc. B (2010) 365, 1663-1678), and we have found that CXCR4 and PGP9.5 Simultaneously, the cells expressing PGP9.5 were identified as cells having an undifferentiated nature than cells expressing only PGP9.5.

The canine spermatogonial stem cell refers to a cell that is present in the testis and plays an important role in spermatogenesis.

According to an embodiment of the present invention, the dog garden stem cells may be garden stem cells in dog testis or dog garden stem cells cultured in vitro.

The composition for analyzing the degree of differentiation of dog garden stem cells is an agent that measures the expression of CXCR4 at the mRNA level or the protein level; And it includes an agent that measures the expression of PGP9.5 at the mRNA level or protein level, it can be used to analyze the degree of undifferentiation of dog garden stem cells.

The agent measuring the expression of CXCR4 at the mRNA level is a primer pair or probe specific for the CXCR4 gene, and the agent measuring the expression of the PGP9.5 at the mRNA level may be a primer pair or probe specific for the PGP9.5 gene. have.

The primers and probes each independently have a sequence complementary to the nucleotide sequence of CXCR4 or PGP9.5.

As used herein, the term "complementary" means having complementarity sufficient to selectively hybridize to the above-described nucleotide sequence under certain specific hybridization or annealing conditions. Thus, the term "complementary" has a different meaning from the term perfectly complementary, and the primers or probes of the present invention may be capable of selectively hybridizing to the above-described nucleotide sequence so long as one or more mismatches ( mismatch) may have a nucleotide sequence.

As used herein, the term “primer” refers to a single that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) in a suitable buffer at a suitable temperature. -Refers to stranded oligonucleotides.

Suitable lengths of the primers can be varied depending on various factors, such as temperature and the use of the primers.

In addition, the sequence of the primer need not have a sequence that is completely complementary to some sequences of the template, it is sufficient to have sufficient complementarity within the range that can be hybridized with the template to perform the primer-specific action.

Therefore, the primer of the present invention does not need to have a sequence that is perfectly complementary to the above-described nucleotide sequence as a template, and it is sufficient to have sufficient complementarity within a range capable of hybridizing to the sequence and acting as a primer.

Primers of the invention may additionally be labeled with radiolabels, fluorescent labels, enzyme labels, sequence tags, or biotin.

As used herein, the term “probe” refers to a linear oligomer of natural or modified monomers or linkages, includes deoxyribonucleotides and ribonucleotides, and can specifically hybridize to a target nucleotide sequence, naturally It may be present or artificially synthesized.

The probe is used as a hybridizable array element and can be immobilized on a substrate. Preferred gases are rigid or semi-rigid supports, which may include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. Can be.

The hybridization array element can be arranged and immobilized on the gas phase.

The immobilization can be effected by chemical bonding methods or by covalent bonding methods such as UV, for example, the hybridization array element can be bonded to a glass surface modified to contain an epoxy compound or an aldehyde group, and a polylysine coating It can be bound by UV at the surface and can be bound to the gas via linkers such as ethylene glycol oligomers and diamines.

According to one embodiment of the invention, the composition for analyzing the degree of differentiation of the dog garden stem cells may be a gene amplification composition.

The term "amplification" described herein means a reaction that amplifies a nucleic acid molecule.

Various amplification reactions have been reported in the art, which include polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329, 822), ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA, WO 88/10315), self-sustained sequence replication, WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) , U.S. Patent 4,437,975), optional priming Arbitrarily primed polymerase chain reaction (AP-PCR), US Pat. Nos. 5,413,909 and 5,861,245, Nucleic acid sequence based amplification (NASBA), US Pat. Nos. 5,130,238, 5,409,818 Nos. 5,554,517, and 6,063,603), strand displacement amplification and loop-mediated isothermal amplification; LAMP), but is not limited thereto.

When the composition for analyzing the degree of undifferentiation of canine stem cells using primers, gene amplification reaction can be performed to investigate the expression level of the CXCR4 and PGP9.5 genes.

The expression level of the CXCR4 and PGP9.5 genes can be determined by investigating the mRNA levels of the CXCR4 and PGP9.5 nucleotide sequences in the dog garden stem cells.

According to one embodiment of the invention, the composition for analyzing the degree of differentiation of the dog garden stem cells may be a microarray composition.

The probe may be immobilized on the solid surface of the microarray.

The sample DNA applied to the microarray may be hybridized with a probe immobilized on the microarray.

The conditions of the hybridization may vary, and the detection and analysis of the degree of hybridization may be variously performed according to the labeling substance.

The probe may be labeled.

Labeling of the probe may provide a signal to detect whether hybridization, which may be linked to oligonucleotides.

Suitable labels include fluorophores (eg, fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), chromophores, chemilumines, magnetic particles, radioisotopes Elements (P ³² and S ³⁵ ), mass labels, electron dense particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (eg gold) and antibodies, streptavidin , But may include, but are not limited to, hapten with specific binding partners such as biotin, digoxigenin and chelating groups.

The labeling can be carried out in various ways conventionally practiced in the art, for example, nick translation method, random priming method (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and chination method (Maxam & Gilbert, Methods in Enzymology, 65: 499 (1986)).

After the hybridization reaction, the hybridization signal coming out through the hybridization reaction is detected.

The hybridization signal may be carried out in various ways depending on the type of label bound to the probe. For example, when the probe is labeled by an enzyme, the substrate of the enzyme may be reacted with the hybridization reaction result to determine whether the hybridization signal occurs. .

Combinations of enzymes / substrates that can be used include peroxidase (eg horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium). Nitrate), resorphin benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), tetramethylbenzidine (TMB), ABTS (2 , 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; Alkaline phosphatase with bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF substrates; Glucose oxidase, t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate). When the probe is labeled with gold particles, it can be detected by silver dyeing using silver nitrate.

By analyzing the intensity of the hybridization signal by the hybridization process, it is possible to analyze the expression level of the CXCR4 and PGP9.5 genes.

The agent for measuring the expression of the CXCR4 at the protein level in the composition for analyzing the degree of differentiation of canine stem cells is an antibody that specifically binds to the CXCR4 protein, and the agent for measuring the expression of the PGP9.5 at the protein level is PGP9. 5 may be an antibody that specifically binds to a protein, and may be, for example, a monoclonal antibody, a polyclonal antibody, or a chimeric antibody.

The antibody is a method commonly practiced in the art, for example, a fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA method (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). .

According to one embodiment of the invention, the antibody specific for the CXCR4 protein may be CXCR-4 Antibody (H-118) of Santa Cruz Biotechnology.

According to one embodiment of the invention, the antibody specific for the PGP9.5 protein may be MOUSE ANTI HUMAN PROTEIN GENE PRODUCT 9.5 (7863-1004) of AbD Serotec.

According to one embodiment of the invention, the composition for analyzing the degree of differentiation of the dog garden stem cells may be a composition for immunoassay (immunoassay).

The immunoassay composition can be carried out by an antigen-antibody reaction method.

The immunoassay can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art.

In addition, the immunoassay may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, Immunofluorescent staining and immunoaffinity purification.

When the composition for immunoassay is performed according to a radioimmunoassay, an antibody labeled with a radioisotope (eg, C ¹⁴ , I ¹²⁵ , P ³² and S ³⁵ ) may be used.

When the immunoassay composition is carried out in an ELISA method, (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; () Reacting the cell lysate with an antibody specific for the CXCR4 or PGP9.5 protein, each independently as a primary antibody; () Reacting with the secondary antibody to which the enzyme is bound; And () measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescence, luminescence or infrared reaction, for example, alkaline phosphatase,? -Galactosidase, hose Radish peroxidase, luciferase and cytochrome P450.

When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as a substrate Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis) if colorimetric substrates such as -B1-phosphate) and enhanced chemifluorescence (ECF) are used, and horse radish peroxidase is used -N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS substrates such as (phenzaine methosulfate) may be used. All.

Measurement of the final enzyme activity or signal in the ELISA method can be carried out according to various methods known in the art.

Detection of this signal allows for qualitative or quantitative analysis of CXCR4 and PGP9.5 protein expression.

For example, signals can be easily detected with streptavidin when biotin is used as a label and luciferin when luciferase is used.

By analyzing the final signal intensity in the above-described immunoassay, the expression of CXCR4 and PGP9.5 proteins can be analyzed.

Another aspect of the present invention relates to a method for analyzing the degree of differentiation of dog garden stem cells by measuring the expression of CXCR4 and PGP9.5 at the mRNA level or the protein level.

When the dog garden stem cells express CXCR4 and PGP9.5 at the same time, the dog garden stem cells may be determined to have an undifferentiated nature than dog garden stem cells expressing CXCR4 or PGP9.5 independently.

The method for analyzing the degree of undifferentiation of the dog garden stem cells can be carried out using the composition for analyzing the degree of undifferentiation of the dog garden stem cells, so the common content between the two is to avoid excessive complexity of the present specification. The description is omitted.

Another aspect of the present invention relates to a composition for isolating undifferentiated dog garden stem cells comprising an antibody that specifically binds to the CXCR4 protein and an antibody that specifically binds to the PGP9.5 protein.

Another aspect of the invention relates to a method for isolating undifferentiated dog garden stem cells comprising the steps of isolating the testis of the dog and isolating cells expressing CXCR4 and PGP9.5 from the isolated testes.

The undifferentiated dog garden stem cells isolated by the method of the present invention can be used for the study of the production of transformed sperm and animal, as well as the study of male infertility, spermatogenesis process mechanism related to germ cell differentiation.

Testis isolated from the dog can be suspended at the single cell level through various methods known in the art.

Undifferentiated canine stem cells can be obtained by isolating cells expressing CXCR4 and PGP9.5 from the testis of dogs suspended at the single cell level.

The cells expressing CXCR4 and PGP9.5 are obtained by first separating cells expressing CXCR4, and then separating cells expressing PGP9.5 from the separated cells, or first separating cells expressing PGP9.5. Then, the cells expressing CXCR4 can be isolated from the isolated cells.

For example, by contacting the testis of dogs suspended at the single cell level with an antibody that specifically binds to the CXCR4 protein, the cells bound to the antibody that specifically binds to the CXCR4 protein are isolated, and then the PGP9. 5 can be obtained by contacting an antibody that specifically binds to a protein, and isolating cells to which the antibody that specifically binds to the PGP9.5 protein is bound.

Separation of the antibody-bound cells is performed using a fluorescence-activating cell sorter (FACS), a magnetic activated cell sorter (MACS) or complement-mediated lysis method. Can be carried out.

When the separation method of the undifferentiated canine stem cells of the present invention using MACS, it can be carried out by a direct labeling method and an indirect labeling method.

For example, when MACS is performed according to a direct labeling method, an antibody that specifically binds to CXCR4 protein bound to a magnetic particle is contacted with the testis of a dog suspended in a single cell level, and then to a magnetic separator. It can be applied to isolate the undifferentiated dog garden stem cells bound to the antibody specifically binding to the CXCR4 protein.

For example, when MACS is performed according to an indirect labeling method, an antibody that specifically binds a CXCR4 protein to which a suitable mediator is bound may be contacted with testes of dogs suspended at the single cell level.

The mediator may include a fluorescent substance, streptavidin, an antibody having specificity to the Fc region of the antibody specifically binding to the CXCR4 protein and avidin.

For example, the phosphors include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE), eosin, carboxyfluorescein, fluorescein amidaite, rose bengal, dilite fluorine, methylene blue, coumarin, and rhodamine It is not limited.

The antibody that specifically binds the CXCR4 protein to which the mediator is bound is contacted with the testes of dogs suspended at the single cell level, and then treated with an antibody bound to the magnetic particles and having specificity to the mediator. Undifferentiated dog garden stem cells to which antibodies that specifically bind to CXCR4 protein are bound can be isolated by applying to a separator having the same.

The MACS is basically based on an immunomagnetic separation process, and the general contents of the process are disclosed in US Pat. Nos. 5,385,707, 5,541,072, 5,646,001, 6,008,002 and 6,153,411, and the magnetic particles to be used. Microbeads and separators are readily available from Miltenyi Biotech (Germany).

The undifferentiated dog garden stem cells expressing both CXCR4 and PGP9.5 isolated by the above method are more undifferentiated than dog garden stem cells expressing CXCR4 or PGP9.5 independently.

The present invention relates to a composition for analyzing the degree of differentiation of dog garden stem cells, a composition for separating undifferentiated dog garden stem cells, a method for analyzing the degree of differentiation of dog garden stem cells, and a method for isolating undifferentiated dog garden stem cells using CXCR4 and PGP9 By measuring the expression of .5 at the mRNA level or protein level, can effectively analyze the degree of differentiation of dog garden stem cells, and can be used to isolate undifferentiated dog garden stem cells using antibodies specific to CXCR4 and PGP9.5. The undifferentiated dog garden stem cells isolated through the present invention can be used for the study of the production of transformed sperm and animal as well as the study of male infertility, spermatogenesis process mechanism related to germ cell differentiation.

Figure 1a is a result of confirming the immunohistochemical staining marker (CXCR4) for undifferentiated germ cells in each stage of development in 1 month old dog testis.
Figure 1b is the result of confirming the immunohistochemical staining marker (CXCR4) for undifferentiated germ cells in each stage of development in 4 months old dog testis.
Figure 1c is a result of confirming the immunohistochemical staining marker (CXCR4) for undifferentiated germ cells in each stage of development in 7 months old dog testis.
Arrows in each figure represent cells showing the expression of CXCR4 protein, black arrows represent spermatogenic cells, red arrows represent sertoli cells.
Figure 2a is a result confirmed by immunohistochemical staining markers (IBFBP3) for undifferentiated germ cells in each stage of development in the 1 month old dog testis.
Figure 2b shows the results of immunohistochemical staining of markers (IBFBP3) for undifferentiated germ cells in each stage of development at 4 months of age.
Figure 2c shows the results of immunohistochemical staining for markers (IBFBP3) for undifferentiated germ cells in each stage of development in 7 months old dog testis.
Arrows in each figure represent cells showing the expression of IBFBP3 protein, black arrows represent spermatogenic cells, red arrows represent sertoli cells.
Figure 3a is a result of confirming the immunohistochemical staining marker (LIN28) for undifferentiated germ cells in each stage of development in 1 month old dog testis.
Figure 3b is a result of confirming the immunohistochemical staining marker (LIN28) for undifferentiated germ cells in each stage of development in 4 months old dog testis.
Figure 3c is a result of confirming the immunohistochemical staining marker (LIN28) for undifferentiated germ cells in each stage of development in 7 months old dog testis.
Arrows in each figure represent cells showing the expression of LIN28 protein, black arrows represent spermatogenic cells and red arrows represent sertoli cells.
Figure 4a is the result of confirming the immunohistochemical staining markers (SALL4) for undifferentiated germ cells in each stage of development in 1 month old dog testis.
Figure 4b is a result confirmed by immunohistochemical staining markers (SALL4) for undifferentiated germ cells in each stage of development in 4 months old dog testis.
Figure 4c is a result of confirming the immunohistochemical staining marker (SALL4) for undifferentiated germ cells in each stage of development in 7 months old dog testis.
Arrows in each figure represent cells showing the expression of the SALL4 protein, black arrows represent spermatogenic cells, red arrows represent sertoli cells.
FIG. 5A is a fluorescence image of co-expression of PGP9.5 and CXCR4 in dog testes after maturation using a PGP9.5 antibody. FIG.
5B is a fluorescence image of co-expression of PGP9.5 and CXCR4 observed in dog testes after maturation using CXCR4 antibody.
Figure 5c is an image staining the cell nuclei with TO-PRO 3 co-expression of PGP9.5 and CXCR4 in dog testis after maturation.
5D is a merged image of target protein and nuclei for coexpression of PGP9.5 and CXCR4 in dog testes after maturation.
White arrows indicate cells expressing both proteins and yellow arrows indicate cells expressing only PGP9.5.
6A is a fluorescence image of co-expression of PGP9.5 and IGFBP3 observed in dog testes after maturation using a PGP9.5 antibody.
6B is a fluorescence image of co-expression of PGP9.5 and IGFBP3 observed in dog testes after maturation using IGFBP3 antibody.
Figure 6c is an image staining the cell nuclei with TO-PRO 3 co-expression of PGP9.5 and IGFBP3 in dog testis after maturation.
6D is a merged image of target protein and nucleus for coexpression of PGP9.5 and IGFBP3 in dog testes after maturation.
White arrows in each figure represent cells expressing both proteins.
FIG. 7A is a fluorescence image of co-expression of PGP9.5 and LIN28 in dog testes after maturation using a PGP9.5 antibody. FIG.
FIG. 7B is a fluorescence image of co-expression of PGP9.5 and LIN28 using LIN28 antibody in dog test post-maturation.
Figure 7c is an image staining the cell nuclei with TO-PRO 3 co-expression of PGP9.5 and LIN28 in dog testis after maturation.
7D is a merged image of the target protein and nucleus for coexpression of PGP9.5 and LIN28 in dog testes after maturation.
White arrows indicate cells expressing PGP9.5 and LIN28 proteins and yellow arrows indicate cells expressing LIN28.
8A is a fluorescence image of co-expression of acrosine and LIN28 in a dog test after maturation using an acrosine antibody.
8B is a fluorescence image of co-expression of acrosine and LIN28 in dog testes after maturation using LIN28 antibody.
Figure 8c is an image staining the nucleus with TO-PRO 3 co-expression of acrosine and LIN28 in dog testis after maturation.
8D is a merged image of the target protein and nucleus for coexpression of acrosine and LIN28 in dog testes after maturation.
White arrows indicate cells expressing acrosine and LIN28 protein, and yellow arrows indicate cells expressing LIN28.
FIG. 9A is a fluorescence image of co-expression of SALL4 and LIN28 in dog testes after maturation using SALL4 antibody. FIG.
9B is a fluorescence image of co-expression of SALL4 and LIN28 in the dog test after maturation using LIN28 antibody.
Figure 9c is an image staining the cell nuclei with TO-PRO 3 co-expression of SALL4 and LIN28 in dog testis after maturation.
9D is a merged image of target protein and nuclei for coexpression of SALL4 and LIN28 in dog testes after maturation.
White arrows indicate cells expressing each protein.
10A is a fluorescence image of co-expression of SALL4 and SCP3 observed with dog SALL4 antibodies in maturation post-implantation.
FIG. 10B is a fluorescent image of co-expression of SALL4 and SCP3 using SCP3 antibodies at maturation post- maturation.
Figure 10c is an image staining the cell nuclei with TO-PRO 3 co-expression of SALL4 and SCP3 in dog test post maturation.
FIG. 10D is a merged image of target protein and nuclei for coexpression of SALL4 and SCP3 in dog test post maturation.
White arrows indicate cells expressing each protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Experiment method

1. Experimental design

Beagle's testicles were obtained surgically and the tissues were fixed within 12 hours. Testes were soaked in Bouin's solution and fixed overnight at 4 ° C. for experiments for each initiation stage (premature, 1 month old; early maturation, 4 months old; and post maturation, 7 months old; n = 3 each).

2. Immunohistochemistry

Chemokinine receptor type 4 (CXCR4), insulin-like growth factor-binding protein 3 (IGFBP3), LIN28, Sal-like protein 4 (SALL4), PGP9.5, synaptonemal complex protein 3 (SCP3) and acrosine Testis samples are washed with 70-100% (v / v) ethanol and xylene, embedded in paraffin, and microtome (Thermo Fisher Scientific, Waltham, Mass., USA) to identify cell locations expressing acrosin. Cut into 5 μm thick and mounted on a glass slide.

Mounted dog testis tissue was rehydrated with xylene and 100-0% ethanol. Antigens were recovered by boiling tissue for 30 minutes in Tris-EDTA solution (10 mM Tris base, 1 mM EDTA, and 0.05% Tween-20; pH 9). Antigen-repaired tissue membranes were subjected to membrane permeation for 10 minutes with PBS containing 0.2% Triton X-100. PBS containing 2% BSA (bovine serum albumin) was soaked at room temperature for 1 hour to block the binding of nonspecific proteins.

Tissues were reacted overnight at 4 ° C. with the following primary antibodies: CXCR4 (1:50 dilution; SC-9046; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), IGFBP3 (1:50 dilution; SC- 6004; Santa Cruz Biotechnology, Inc.), LIN28 (1:50 dilution; 11724-1-AP; Proteintech, Rosemont, IL, USA), SALL4 (1:50 dilution; Ab57577; Abcam, Cambridge, UK), PGP9. 5 (1: 1000 dilution; 7863-1004; AbD Serotec; Raleigh, NC, USA), SCP3 (1:50 dilution; SC-33195; Santa Cruz Biotechnology, Inc.), and acrosin (1:50 dilution; SC- 51504; Santa Cruz Biotechnology, Inc.). After washing three times with PBS, the secondary antibody was reacted at room temperature for 2 hours.

Peroxidase substrate detection kit (SK-4100; Vector Laboratories; Burlingame, Calif., USA) was used to detect dog germ cell markers. In order to confirm the coexpression of dog germ cell markers, a fluorescent antibody-bound secondary antibody was added and reacted at room temperature for 2 hours.

Cell nuclei were stained using TO-PRO? -3 (1: 1000 dilution; T3605; Thermo Fisher Scientific). Mounting solution (Dako, Carpinteria, CA, USA; S3025) was used to fix the immunostained dog testis tissue. Co-immunostained tissue was observed under confocal microscopy (Carl Zeiss, Oberkochen, Germany; LSM 700).

Experiment result

1. Expression and Location of Undifferentiated Garden Cell Markers in Dog Testis Tissue

Expression and location of expression of CXCR4, IGFBP3, LIN28 and SALL4 were identified in each developmental stage. Expression of germ cells in the lavage was confirmed by immunohistochemistry using antibodies specific for CXCR4, IGFBP3, LIN28 and SALL4, respectively. These markers for undifferentiated male germ cells showed similar levels of expression at different stages of development in different species.

Expression of CXCR4, IGFBP3, and LIN28 at premature stage (1 month old) was observed in the lavage lumen of undifferentiated ancestral cells (FIG. 1A, FIG. 2A, FIG. 3A). However, SALL4 was not observed in 1 month old dog testis (FIG. 4A).

In the early stages of maturation (4 months of age), cells expressing CXCR4, IGFBP3 and LIN28 were observed near the basement membrane of the lavage tube (FIGS. 5B, 6B and 3B), and no SALL4 was observed. SALL4 was observed in sertoli cells and spermatogonia in 4 month old dog testis (FIG. 4B). After maturation (7 months of age), cells expressing CXCR4 and IGFBP3 were observed near the basement membrane of the lavage tube (FIGS. 1C and 2C) and cells expressing LIN28 and SALL4 were observed in the lavage lumen (FIG. 3C, FIG. 3). 4c).

2. Undifferentiated Garden Cell With markers  After maturation in dog testicles CXCR4  And IGFBP3  Expression comparison

Co-immunohistochemical staining analysis was performed using antibodies against CXCR4, IGFBP3 and PGP9.5 to confirm expression of CXCR4 and IGFBP3 in adult canine testis (FIG. 5, FIG. 6). CXCR4, IGFBP3 and PGP9.5 were observed in undifferentiated garden cells near the basement membrane of the dog's lavage tube (FIG. 5). Most cells expressing IGFBP3 expressed PGP9.5 (FIG. 6). However, some PGP9.5 positive cells did not express CXCR4 (FIG. 5). Cells expressing CXCR4 and PGP9.5 simultaneously distributed more around the basement membrane than cells expressing PGP9.5 (FIGS. 5A, 5B, and 5C yellow arrows).

3. Expression of LIN28 in dog testis after maturation compared to canine germ cell markers

To confirm the expression of LIN28 in the testis, co-immunohistochemical staining analysis was performed using antibodies against LIN28, PGP9.5 and acrosine (FIG. 7 and FIG. 8). Most of LIN28 positive cells were distributed in the testicular lumen compared to PGP9.5 positive cells (FIG. 7). Cells co-expressing PGP9.5, acrosine and LIN28 were not observed. Cells expressing LIN28 were distributed between spermatogonia expressing PGP9.5 and spermatozoa and sperm expressing acrosin (Figs. 7 and 8).

4. Expression of SALL4 in dog testis after maturation compared to undifferentiated hair cell markers

Co-immunohistochemical staining analysis was performed using antibodies against SALL4, LIN28 and SCP3 to confirm the expression of SALL4 in the testis (Fig. 9, Fig. 10). Most of the SALL4 positive cells were distributed in the testicular lumen. Cells co-expressing PGP9.5, acrosine and LIN28 were not observed. Cells expressing LIN28 were distributed between spermatogonia expressing PGP9.5 and spermatozoa and sperm expressing acrosin (Figs. 7 and 8). Comparison of SALL4, LIN28 and SCP3 expression revealed that most SALL4 positive cells expressed LIN28 and SCP3 (Figure 9, Figure 10).

Claims (9)

delete delete delete delete delete Measuring the expression of CXCR4 and PGP9.5 in dog garden stem cells at the mRNA level or the protein level; And
When the dog garden stem cells express CXCR4 and PGP9.5 at the same time, determining that the dog garden stem cells have an undifferentiated nature than dog garden stem cells expressing each of CXCR4 or PGP9.5 independently
Undifferentiated degree analysis method of dog garden stem cells comprising a. The method according to claim 6, wherein the dog garden stem cells are garden stem cells in dog testis or dog garden stem cells cultured in vitro. delete Separating the garden stem cells from the testis of the dog; And
Separating the cells expressing CXCR4 and PGP9.5 from the isolated garden stem cells;
Separation method of the dog garden stem cells having a more undifferentiated nature than dog garden stem cells each independently expressing CXCR4 or PGP9.5.

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