KR100443425B1 - Transgenic animal expressing SRG3 antisense RNA fragment in thymocytes specifically and a method for preparing the transgenic animal - Google Patents

Abstract

The present invention relates to a transgenic animal expressing SRG3 antisense RNA fragments in a thymus-specific manner, and to a method for preparing the same. More specifically, the Lck proximal promoter and polyA sequence for inducing thymus specific gene expression. a fusion gene construct comprising a mouse SRG3 cDNA fragment inserted opposite to the promoter between human growth hormone gene fragments having a polyA sequence, containing the SRG3 antisense An expression vector capable of expressing an RNA fragment, a fertilized egg microinjected with a fusion gene construct in the vector, a transgenic animal obtained therefrom and a method for producing the same. The transgenic animals of the present invention have immature thymic cells that have decreased expression of apoptosis by glucocorticoids by inhibiting the expression of SRG3, which overexpresses SRG3 antisense RNA fragments in the thymus and induces apoptosis of thymic cells by glucocorticoids. It can be usefully used to study the regulation of cell differentiation and immune function.

Description

Transgenic animal expressing SRG3 antisense RNA fragment in thymocytes specifically and a method for preparing the transgenic animal}

The present invention relates to a transgenic animal expressing SRG3 antisense RNA fragments in a thymus-specific manner, and to a method for preparing the same. More specifically, the Lck proximal promoter and polyA sequence for inducing thymus specific gene expression. a fusion gene construct comprising a mouse SRG3 cDNA fragment inserted opposite to the promoter between human growth hormone gene fragments having a polyA sequence, containing the SRG3 antisense An expression vector capable of expressing an RNA fragment, a fertilized egg microinjected with a fusion gene construct in the vector, a transgenic animal obtained therefrom and a method for producing the same.

Immature thymus cells are differentiated into mature thymus cells with a self-major histocompatibility complex (MHC) restriction and self tolerance required for antigen recognition through an elaborate differentiation process in the thymus ( Von Boehmer H. et al., Cell 76, 219-228, 1994). MHC is a protein complex in which immune adjuvant cells or bone marrow cells express peptide antigens on the cell surface together with the peptide antigens in order to recognize the peptide antigens in thymic cells. TCR ") to stimulate thymic cells. Autologous MHC restriction of mature thymus cells is an essential ability for thymus cells to recognize only peptide antigens bound to autologous MHC and perform immunity. Self-tolerance does not recognize autologous peptide antigens linked to autologous MHC. It is an essential ability to protect autologous tissues from immunity.

To become mature thymic cells, immature thymic cells undergo a series of gene rearrangements to express α-TCR and β-TCR, and thymic cells expressing the appropriate TCR are selected to allow CD4 thymic cells or CD8 thymus Differentiate into cells. Specifically, when TCR is expressed on the cell surface of the thymus cells, the thymus cells recognize only autologous MHC to which peptide antigens are bound, and then through a positive selection process in which only thymus cells that can properly bind are selected. Differentiate into CD4 monopositive or CD8 monopositive thymic cells (Janeway CA Jr. et al., Immunity, 1, 3-6, 1994). In positive selection, CD4 / CD8 double-positive thymic cells are eliminated through apoptosis, a programmed apoptosis process, because apoptosis is induced because the double-positive thymic cells do not receive the appropriate survival signal mediated by TCR (Surh). CD and Sprent J. et al., Nature, 372, 100-103, 1994).

On the other hand, thymus cells that bind more strongly than necessary with autologous MHC bound to peptide antigens are removed through a negative selection process, whereby the death of the thymus cells removed is induced by apoptosis and survives. Cells have self-tolerance as a ratio to protect autologous tissues from immunity (Matzinger P. et al., Nature, 308, 738-741, 1984; Sprent J. et al., Immunol. Rev. , 101 , 173-190, 1988; Schwartz RH et al., Cell, 57, 1073-1081, 1989). Therefore, the binding force between TCR of thymic cells and autologous MHC bound to peptide antigens has been reported to play an important role in determining whether thymocytes in differentiation survive or die (Ashton-Rickardt PG et al., Cell 76). , 651-663, 1994; Sebzda E. et al., Immunol . 17, 829-874, 1999).

On the other hand, it is not yet known exactly how apoptosis does not occur when thymic cells are selected through a positive selection process, but glucocorticoids have been reported to play an important role in the positive selection process (Yang Y. and Ashwell JD). , J Clin. Immunol ., 19, 337-349, 1999). To date, studies on glucocorticoids are hormones produced in the adrenal gland and thymus, causing apoptosis of immature thymic cells that were not selected during the positive selection process. Thus, immature thymic cells are sensitive to apoptosis by the glucocorticoids, while mature mono-positive thymic cells are relatively resistant to apoptosis by glucocorticoids (Cohen J. et al ., Semin. Immunol ., 4, 363- 369, 1992). As described above, the molecular biological mechanisms that can clearly explain the difference in sensitivity to glucocorticoids between immature thymic cells and mature mono-positive thymic cells are not yet undertaken, so that immature thymic cells selected during the positive selection process are not present in the glucocorticoids. There is a need for a study on how to obtain resistance to apoptosis.

Based on the action of glucocorticoids in thymic cell differentiation as described above, dexamethasone or hydrocortisone, which are analogs of glucocorticoids, are commercially available as immunosuppressive agents. Glucocorticoid immune function is regulated through glucocorticoid receptors. As part of this study, SRG3 has recently been found to regulate the immune function of glucocorticoids.

SRG3 is a protein that plays a pivotal role in regulating apoptosis of immature thymic cells by glucocorticoids and is known to modulate the transcriptional activity of glucocorticoid receptors (Jeon SH et al., J. Exp. Med. 185, 1827-). 1836, 1997). SRG3 is much higher in immature thymus cells compared to mature thymic cells, whereas SRG3 expression in thymic cells selected by the positive selection process is reduced, which indicates that thymus cells are sensitive to apoptosis by glucocorticoids. Because it is determined. Specifically, when the expression level of SRG3 is high, SRG3 binds to the glucocorticoid receptor to accelerate the transcriptional activation of the receptor to induce apoptosis, and when the expression level of SRG3 is low, the number of SRG3 molecules that bind to the glucocorticoid receptor decreases, thereby reducing the transcriptional activity of the glucocorticoid receptor. Because it is lowered, apoptosis is not induced.

Recent studies on SRG3 have shown that Notch protein, an important function of thymic cell differentiation, inhibits the expression of SRG3 protein and inhibits apoptosis by glucocorticoids, thereby allowing immature thymic cells to differentiate into CD4 or CD8 monopositive thymic cells. It is known to affect (Deftos ML et al., Immunity, 9, 777-786, 1998). In particular, apoptosis by glucocorticoids is suppressed in mouse thymic cells expressing the internal domain of notch protein (hereinafter, abbreviated as "notch IC"), and overexpression of SRG3 in the mouse thymic cells results in thymic cell response to apoptosis by glucocorticoids. Sensitivity is partially restored, meaning that Notch I in Notch protein plays a key role in inhibiting SRG3 expression and inhibiting apoptosis of thymic cells by glucocorticoids (Deftos ML et al., Immunity, 9, 777). -786, 1998). Therefore, the expression level of SRG3 is precisely regulated by notch, thereby controlling apoptosis by glucocorticoids in thymic cells which are not selected during the differentiation of immature thymic cells.

Although the suppression of SRG3 expression by notch inhibits the apoptosis of immature thymic cells induced by glucocorticoids, the mechanism is still unknown. Therefore, in order to accurately control the regulation of apoptosis induced during thymic cell differentiation, active research on the mechanism by which glucocorticoid-induced apoptosis is suppressed by SRG3 expression inhibition is required.

Therefore, the present inventors are trying to elucidate the mechanism of regulating apoptosis of thymic cells by glucocorticoids, and expressing SRG3 protein that regulates apoptosis by glucocorticoids in thymic cells and thymic cancer cell lines using SRG3 antisense RNA fragment. Transgenic mice having thymic cells with reduced sensitivity to glucocorticoids were prepared, and overexpressed SRG3 antisense RNA fragments in the transgenic mice reduced expression of SRG3 protein, thereby reducing apoptosis sensitivity by glucocorticoids. The present invention was completed by confirming.

SUMMARY OF THE INVENTION An object of the present invention is to provide a transgenic animal having thymic cells with reduced apoptosis sensitivity to glucocorticoids and a method for producing the same, in order to provide a living animal model for studying the regulation of immune cell differentiation and immune function.

1 shows the results of electrophoresis confirming that 2.8 kb SRG3 cDNA fragment was inserted between the Lck proximity promoter and human growth hormone having a poly A sequence in the expression vector of the present invention.

Lane 1: DNA size marker

Lane 2: DNA fragment obtained by cleavage with restriction enzyme Not I

2 shows a cleavage map of the expression vector of the invention expressing SRG3 antisense RNA,

Restriction enzymes that may be used N: NotI, B: BamHI

hGH poly A: Human Growth Hormone Poly A

Figure 3 shows the process for producing a transgenic mouse of the present invention,

Figure 4a shows the result of performing a polymerase chain reaction (PCR) to select a transgenic mouse inserted with a fusion gene construct containing a mouse SRG3 cDNA fragment in the genome,

Lane 1: DNA size marker

Lane 2: SRG3 cDNA fragment obtained by PCR amplification of the tail DNA of the transgenic mouse

4B is a result of Southern blotting for selecting transgenic mice in which a fusion gene construct including a mouse SRG3 cDNA fragment is inserted in a genome.

Lane 1: control group FVB

Lanes 2 and 3: Southern blotting results of mice with two different lines

5 shows the results of Northern blotting using RNA isolated from each tissue in order to confirm the expression pattern of the SRG3 antisense RNA fragment in the transgenic mice of the present invention at the RNA level,

6 shows the results of Western blotting using thymic cell lysate in order to confirm the SRG3 expression level at the protein level in the transgenic mice of the present invention.

Lane 1: normal mice

Lane 2: transgenic mouse

Figure 7 shows the result of comparing the type of thymic cells and the number of thymic cells of the transgenic mice of the present invention with the number of thymic cells of the normal mouse by FACS (Fluorescence Activated Cell Sorter) as compared with normal mice In addition, CD4 + T cells and double negative T cell populations were increased in transgenic mice.

A: normal mice

B: transgenic mice

CD4: Thymic Cell Receptor

CD8: Thymic Cell Receptor

Figure 8 shows that immature thymic cells of the transgenic mice of the present invention is less sensitive to apoptosis by glucocorticoids than immature thymic cells of normal mice.

▲: normal mouse 10 ^-7 M concentration

●: Transgenic mouse 10 ^-7 M concentration

△: normal mouse 10 ^-8 M concentration

○: transgenic mouse 10 ^-8 M concentration

In order to achieve the above object, the present invention provides a fusion gene construct comprising a Lck proximity promoter, a mouse SRG3 cDNA fragment inserted in the opposite direction to the promoter and a human growth hormone to which the polyA sequence is bound.

In addition, the present invention provides an expression vector capable of expressing the SRG3 antisense RNA fragment by the thymus-specific containing the fusion gene construct.

In addition, the present invention provides a fertilized egg and a transgenic animal obtained by inserting the fusion gene construct of the present invention into the genome of the vector.

Finally, the present invention provides a method for producing the transgenic animal.

The present invention provides a fusion gene construct in which a mouse SRG3 cDNA fragment is inserted in a direction opposite to the promoter between a Lck proximity promoter and a human growth hormone gene fragment in which a polyA sequence is bound to thymus-specific gene expression. do.

The fusion gene construct specifically expresses the SRG3 cDNA fragment only in the thymus, including the Lck proximity promoter, which is activated only in the thymus, since SRG3 is a protein expressed only in the adrenal and thymus. The fusion gene construct of the present invention has a sense DNA strand encoding a protein from the mouse SRG3 cDNA fragment in the fusion gene construct of the present invention because the mouse SRG3 cDNA is inserted in an antiorientation with the Lck proximity promoter. Antisense DNA strands, which are complementary to the sense DNA strand, are transcribed to produce SRG3 anticess RNA fragments.

In addition, the human growth hormone gene fragment to which the polyA sequence is bound is attached to the 3'-terminus of SRG3 cDNA in the fusion gene construct of the present invention. It is because it is an essential sequence for expression. Accordingly, the fusion gene construct of the present invention is a fusion RNA with a human growth hormone RNA fragment attached to the polyA sequence at the 3'-end of the SRG3 antisense RNA fragment from the fusion gene construct under the control of the Lck proximity promoter. Thymic cells may be specifically expressed (see FIG. 1 ).

In addition, the present invention provides an expression vector capable of expressing the SRG3 antisense RNA fragment thymus-specifically, including the fusion gene construct.

The expression vector was prepared using a known vector p1017 (Chaffin KE et al., EMBO J. , 9, 3821-3829, 1990) comprising a human growth hormone gene fragment to which the Lck proximity promoter and polyA sequence were bound . .

To obtain an SRG3 cDNA fragment, a 2.8 kb SRG3 cDNA fragment was cleaved from a recombinant vector in which 2.9 kb of mouse SRG3 cDNA fragment was cloned, and then, between the Lck proximity promoter and the human growth hormone gene fragment to which the polyA sequence was bound in the vector p1017. Inserted in the opposite direction to the promoter to prepare an expression vector expressing the SRG3 antisense RNA fragment thymus specific (see Figure 2 ). When the expression vector of the present invention is transformed into thymic cells, the expression of the SRG3 antisense RNA fragment can be overexpressed by the regulation of the Lck proximity promoter, thereby suppressing the expression of the SRG3 protein.

Therefore, the expression vector of the present invention can be usefully used to understand the mechanism of action of glucocorticoids by reducing the expression level of SRG3 protein that regulates apoptosis by glucocorticoids in thymic cells and thymic cancer cell lines using SRG3 antisense RNA fragments.

In addition, the present invention is obtained by injecting a mouse fragment fertilized by the microinjection method of the expression vector Lck proximity promoter, the SRG3 cDNA fragment inserted in the opposite direction to the promoter, a human growth hormone gene fragment and a polyA sequence of the expression vector Transgenic Mouse Fertilized eggs and transgenic mice obtained therefrom are provided.

The DNA fragment was obtained by cutting the expression vector of the present invention with a restriction enzyme, and the transgenic mouse fertilized egg obtained by injecting the fertilized egg into the mouse fertilized egg by a microinjection method was deposited on March 16, 2001 to the Korea Biotechnology Research Institute Gene Bank ( Accession number: KCTC 0973BP) (see FIG. 3 ).

Specifically, the transgenic mouse fertilized egg of the present invention is a SRG3 cDNA fragment bound in the opposite direction to the thymus-specific promoter Lck proximity promoter and DNA fragments containing polyA sequences involved in gene expression in mammals, nucleus and nucleus It is secured by injecting into the unfused mouse fertilized egg by a microinjection method, the transgenic mouse fertilized egg contains the DNA fragment in all cells formed during cell division.

The transgenic mice can be obtained by overexpressing SRG3 antisense RNA fragments using the transgenic mouse embryos of the present invention.

The transgenic mice of the present invention implanted mouse fertilized mice with fertilized embryos injected with DNA fragments containing SRG3 cDNA fragments inserted opposite to the Lck proximity promoter and polyA sequences involved in gene expression in mammals. (See FIG. 3 ). The transgenic mouse has an SRG3 cDNA fragment inserted into the genome of all somatic and germ cells, so that the SRG3 cDNA fragment can be inherited through germ cells. In addition, the Lck proximity promoter is a thymus-specific promoter that induces gene expression only in thymic cells, and since the SRG3 cDNA fragment is inserted in the opposite direction to the promoter, the transgenic mice of the present invention are capable of generating SRG3 antisense RNA fragments only in thymic cells. Overexpress. The overexpressed SRG3 antisense RNA binds complementarily with SRG3 sense RNA and inhibits SRG3 protein production, thereby inhibiting apoptosis of thymic cells by glucoticoids induced during thymic cell differentiation.

Therefore, the transgenic mice of the present invention can be usefully used to understand the effect of desensitization of immature thymic cells to glucocorticoids on the differentiation process and subsequent immune response of glucocorticoids in the expression of SRG3 protein in vivo. have.

Finally, the present invention provides a method for producing the transgenic mouse.

The manufacturing method of the present invention

1) preparing an expression vector by inserting the SRG3 cDNA in the opposite direction to the promoter between the thymus specific promoter and the mammal-derived gene (step 1);

2) cutting the expression vector with a restriction enzyme to obtain a fusion gene construct composed of a thymus specific promoter, an SRG3 cDNA inserted in a direction opposite to the promoter and a mammalian-derived gene (step 2);

3) inserting the fusion gene construct into a mammalian fertilized egg to produce a transformed fertilized egg (step 3); And

4) implanting the transformed fertilized egg into the womb of the surrogate surrogate mother to obtain a transgenic animal (step 4).

Specifically, step 1, the thymus specific promoter can be selected from the group consisting of Lck proximity promoter, CD2 promoter, CD4 minigene promoter, TCR promoter and CD3 promoter. SRG3 cDNA is mouse SRG3 cDNA and all or part of the cDNA can be used. In the present invention, a mouse SRG3 cDNA part was used.

Mammalian-derived genes are fusion genes in which a polyA sequence is attached to all or a portion of cDNA of a gene expressed in a mammal. Preferably, the SRG3 antisense RNA has a length that does not interfere with binding to SRG3 mRNA (messenger RNA). cDNA fragment, and may be selected from the group consisting of human growth hormone (hGH PolyA), bovine growth hormone (bGH PolyA), human CD2 (hCD2 polyA), and SV40 (SV40 PolyA) to which a polyA sequence is bound. In the present invention, as a preferred embodiment, a fragment of the human growth hormone gene having a polyA sequence linked to the 3 'end was used.

Specifically describing step 2, the fusion gene construct of step 2 is a construct with the necessary components for generating RNA from a gene in a mammal. The thymus-specific promoter may be a Lck proximity promoter, a CD2 promoter, a CD4 minigene promoter, or a CD3 promoter as a promoter for inducing gene expression only in thymus cells. In the present invention, the Lck proximity promoter was used.

Specifically, in step 3, the mammalian fertilized egg is a one-celled fertilized egg in which the nucleus and the nucleus are not fused, and according to the transgenic animal to be prepared, mice, guinea pigs, rats, goats, A fertilized egg such as a cow may be used, and the transformed fertilized egg may be a microinjection method for microinjecting the fusion gene construct of step 2 into a mammalian fertilized egg of the first cell stage, and a liver cell into which the fusion gene construct is inserted. (Stem cell) can be prepared by a cell mixing method, such as by combining with a developing fertilized egg, in the present invention, the fusion gene construct was inserted into the mouse fertilized egg by microinjection.

Specifically describing step 4, the fertility surrogate mother is selected according to the transgenic fertilized eggs used and the female mammals fertilized by fertilizing the fertilized ducts with the resected male mammal, since the present invention uses a transgenic mouse fertilized egg. Fertility mouse surrogate mothers were selected. DNA analysis for screening transgenic animals is performed to identify transgenic first generation animals in which SRG3 cDNA is inserted in genomic DNA. Southern blotting and PCR using tail DNA can be used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of SRG3 Antisense RNA Fragment Expression Vector

To prepare an expression vector that overexpresses an SRG3 antisense RNA fragment only in thymus cells, we prepared a recombinant expression vector comprising an SRG3 cDNA fragment inserted in the opposite direction to the Lck promoter that specifically induces gene expression.

First, a cloned mouse SRG3 cDNA (Jeon, SH et al., J. Exp. Med ., 185, 1827-1836, 1997) was digested with restriction enzyme Xba I to cut a 2.9 kb SRG3 cDNA fragment. This was cloned into pBluescriptII KS (−) vector (Promega) to prepare a recombinant vector, and the recombinant vector was digested with restriction enzyme BamH I to identify 2.8 kb of SRG3 cDNA fragment. The 2.8 kb SRG3 cDNA fragment was reversed from the promoter between the Lck promoter and human growth hormone gene fragment to which the polyA sequence was bound in vector p1017 (Chaffin KE et al., EMBO J. , 9, 3821-3829, 1990). An expression vector capable of overexpressing SRG3 antisense RNA was inserted into the thymus and was named "pLck-αSRG3" ( FIG. 2 ).

In order to check whether the SRG3 cDNA fragment was cloned in the expression vector, the expression vector was digested with restriction enzyme Not I and electrophoresis was performed. As a result, it was confirmed that the SRG3 cDNA fragment was inserted into the expression vector pLck-αSRG3 by observing the 8.2 kb DNA band including the 2.9 kb DNA band and the 2.8 kb SRG3 cDNA fragment, which is the size of the vector itself ( FIG. 1 ).

Example 2 Preparation of Transgenic Mouse Embryos

In order to prepare a transgenic mouse embryo using a fusion gene construct in which an SRG3 cDNA fragment was inserted in the opposite direction to the promoter between the Lck proximity promoter and a human growth hormone gene fragment comprising a polyA sequence, The same experiment was carried out.

First, obtaining a fusion gene construct in which an SRG3 cDNA fragment is inserted in the opposite direction to the promoter between an Lck proximity promoter and a human growth hormone gene fragment comprising a polyA sequence from the expression vector pLck-αSRG3 prepared in Example 1 To this end, the expression vectors were digested and electrophoresed with restriction enzymes Not I and Apa I, respectively, and purified by dialysis from a gel of 8.2 kb containing the SRG3 cDNA fragment. The purified 8.2 kb fusion gene construct was microinjected into the fertilized embryos with no nucleus spermatozoa and the nucleus fused by a standard method to produce a transgenic mouse embryo, which was dated March 16, 2001 to the Korea Biotechnology Research Institute Gene Bank. (Accession: KCTC 0973BP) (Hogan, B. et al ., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).

Example 3 Preparation of Transgenic Mice

In order to prepare a transgenic mouse using the transgenic mouse fertilized egg prepared in Example 2, the inventors conducted the following experiment.

The transgenic mouse of Example 2 was implanted into the uterus of a fertility surrogate mouse (FVB strain, Macrogen Seoul National University Medical Research Institute, Cancer Institute). Separated. PCR was performed using a pair of primers based on the genomic DNA and described as SEQ ID NO: 1 and SEQ ID NO: 2, and specifically recognized the 3 'end of the SRG3 cDNA fragment. As a result, the first generation transgenic mice in which the 900 bp PCR product was detected by the PCR amplification were selected, and it was confirmed that 8.2 kb DNA fragments containing SRG3 cDNA fragments were inserted in their genomic DNA ( FIG. 4A ) . And FIG. 4B ).

Experimental Example 1 Analysis of SRG3 Antisense RNA Expression

In order to investigate the expression patterns of the SRG3 antisense RNA fragments of the transgenic mice selected in Example 3, the inventors used northern blotting using total RNA isolated from the tissues of normal mice and transgenic mice into which SRG3 cDNA was inserted. (Northern blotting) was performed to analyze the expression patterns of SRG3 antisense RNA fragments expressed from SRG3 cDNA fragments inserted into mouse genomic DNA.

First, using Chomczynski et al. (Chomczynski, P. et al., Anal. Biochem. 162, 156-159, 1987), the thymus of normal mice and the thymus, lymph nodes, spleen, brain, testis and liver of transgenic mice. After total RNA was purified from the tissue, 20 μg of total RNA was isolated by electrophoresis on 1.2% agarose gel. After electrophoresis, staining with EtBr (Ethidium bromide) confirmed 28S and 18S rRNA (ribosomal RNA) to confirm that the same amount of RNA was loaded, and then the RNA on the agarose gel was transferred to the nitrocellulose membrane. I was. The 2.9 kb Xba I SRG3 cDNA fragment of Example 1 was used as a template and a single-stranded SRG3 cDNA complementary to SRG3 antisense RNA was obtained by PCR using a T3 primer and then used as a probe. Blotting was performed.

As a result, no SRG3 antisense RNA fragment was observed in tissues other than the thymus of normal mice and the thymus of transgenic mice, but 3 kb of SRG3 antisense RNA fragment was identified in the thymus of transgenic mice. From the above results, it was confirmed that the SRG3 antisense RNA fragment was specifically overexpressed only in the thymus from the SRG3 cDNA fragment inserted into the genomic DNA of the transgenic mouse under the control of the Lck proximity promoter ( FIG. 5 ).

Experimental Example 2 Measurement of SRG3 Protein Expression

As confirmed by the northern blotting, in order to confirm that the transgenic mice of the present invention reduce the expression level of SRG3 protein due to the overexpression of SRG3 antisense RNA, the present inventors used western blotting in the following manner. blotting) was performed.

Cell lysates obtained by crushing thymocytes (10 ⁷ cells) of normal and transgenic mice were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and then proteins were transferred to nitrocellulose membranes. The skimmed milk was diluted in TBS / Tween-20 (hereinafter abbreviated as "TBST") to block the nitrocellulose membrane for 1 hour in a blocking solution made to a final concentration of 5% and then for SRG3, Glucocorticoid and Lck respectively. The primary antibody was added and reacted at room temperature for 3 hours. After completion of the reaction, the nitrocellulose membrane was washed three times with TBST for 15 minutes and the nitrocellulose membrane was reacted for 2 hours in TBST containing a secondary antibody conjugated with a horseradish peroxidase (HRP), followed by three times with TBST for 15 minutes. Nitrocellulose membranes were exposed to X-ray films using an ECL kit (Amersham Pharmacia, Sweden) to observe SRG3, glucocorticoids and Lck bands, followed by Bio-Rad Protein Assay. The amount of protein observed was determined to confirm that the same amount of protein was loaded, and the intensity of the protein band was determined using Gel-Pro Analyzer software, Media Cybernetics, USA. It was.

As a result, the glucocorticoid and Lck bands of the non-transformed normal mice and the glucocorticoid and Lck bands of the transgenic mice were found to be almost the same intensity, but the SRG3 band of the transgenic mice was detected at a weaker intensity than the SRG3 band of the normal mice. It was confirmed that the amount of SRG3 expression in the transgenic mice ( FIG. 6 ). From the above results, it was confirmed that the SRG3 antisense RNA fragment overexpressed in the thymus of the transgenic mouse of the present invention binds complementarily with SRG3 mRNA (messenger RNA) to inhibit SRG3 protein expression from SRG3 mRNA.

Experimental Example 3 Comparison of Thymic Cell Types and Thymic Cell Numbers in Transgenic and Normal Mice

In order to compare thymic cell types and thymic cell numbers of transgenic mice and normal mice, the present inventors performed flow cytometry to investigate the thymic cell types and thymic cell numbers of transgenic mice and normal mice. .

First, thymic cells isolated from 3 to 4 week old mice were suspended in single cells, washed once with PBS (phosphate buffered saline (PBS), and then with antibodies and biotin against CD4 labeled with Fluorescence isothiocyanate (FITC). The antibody against labeled CD8 was added and reacted for 10 minutes at room temperature. Thymus cells were washed with PBS, stained with streptavidin (PE) conjugated with PE (full name), and then washed with PBS. The washed thymic cells were suspended in 1 ml of PBS containing 2% FBS (fetal bovine serum) and then thymic cell types with a FACStarp ^lus flow cytometer (Becton Dicknson) using CellQuest software. Thymic cell number was examined.

As a result, the distribution of CD4 thymus cells and CD8 thymic cells in transgenic mice was similar to that of normal mice, but the number of thymus cells in transgenic mice was increased ( Fig. 7 ). From these results, it was confirmed that SRG3 does not affect the type of thymic cells but induces apoptosis of immature thymic cells by glucocorticoids.

Experimental Example 4 Measurement of Sensitivity to Apoptosis of Transgenic Mouse Thymic Cells

In order to investigate the effect of SRG3 antisense RNA fragments overexpressed in thymus cells of transgenic mice on the susceptibility to apoptosis of immature thymic cells by glucocorticoids, the present inventors treated glucocorticoids to induce apoptosis. And the number of thymic cells killed by apoptosis in thymic cells of normal mice was measured.

First, thymocytes isolated from transgenic mice and normal mice were cultured in medium without glucocorticoid and medium with glucocorticoid added at concentrations of 10 ⁻⁸ M and 10 ⁻⁷ M, respectively. After reacting with an antibody against CD8 conjugated with an antibody against and FITC, the number of surviving thymic cells was measured using a Fluorescence Activated Cell Sorter (FACS), and apoptosis incidence was calculated according to Equation 1 below.

<Equation 1>

Incidence of apoptosis in bipositive thymic cells (%) = 100 X (A-B) / (100-B)

A:% incidence of apoptosis in glucocorticoid added medium

B:% incidence of apoptosis in medium without glucocorticoids

As a result, when apoptosis was induced by glucocorticoids, the thymic cells of the transgenic mice of the present invention were different at different concentrations of 10 ^-8 M and 10 ^-7 M over time than those of normal mice. The incidence of apoptosis was shown ( FIG. 8 ). From the above results, SRG3 antisense RNA fragment overexpressed in thymic cells of transgenic mice of the present invention inhibits SRG3 expression inducing apoptosis by glucocorticoids, thereby reducing the sensitivity of immature thymic cells to apoptosis, thereby transforming Glucocorticoid apoptosis was inhibited in immature thymic cells of mice.

As described above, the transgenic mice of the present invention have immature thymic cells with overexpression of SRG3 antisense RNA fragments in the thymus and reduced apoptosis susceptibility by glucocorticoids, which is useful for studying the differentiation of immune cells and the regulation of immune function. Can be used.

<110> SEONG, Rho Hyun JANG, Jae Jin KIM, Kil Soo Bio Genomics. INC. <120> Transgenic animal expressing SRG3 antisense RNA fragment in thymocytes specifically and a method for preparing the transgenic animal <130> 0p-06-63 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 gcatctgcat gaacatactt cttg 24 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 ctagctcagc gtgtgctcat ctg 23

Claims (11)

A thymus-specific promoter is a fusion gene construct comprising a nucleic acid sequence consisting of a Lck proximity promoter, a mouse SRG3 antisense cDNA, and a sequence of human growth hormone genes linked to a polyA sequence. Transformed fertilized eggs obtained by injection into. delete delete The transformed fertilized egg of claim 1, wherein the fusion gene construct is obtained from an expression vector pLck-αSRG3. The transformed fertilized egg of claim 1, wherein the fusion gene construct is injected by a microinjection method. The transgenic mouse fertilized egg according to claim 1, wherein the mammalian fertilized egg is a mouse fertilized egg (Accession Number: KCTC 0973BP). A transgenic animal obtained by implanting the transformed fertilized egg of claim 1 into the uterus of a surrogate mother except for humans. 8. The transgenic mouse of claim 7, wherein the surrogate mother is a fertility female mouse. 1) preparing an expression vector by inserting the SRG3 cDNA in the opposite direction to the promoter between the thymus specific promoter and the mammal-derived gene (step 1); 2) cutting the expression vector with a restriction enzyme to obtain a fusion gene construct composed of a thymus specific promoter, an SRG3 cDNA inserted in a direction opposite to the promoter and a mammalian-derived gene (step 2); 3) preparing a transgenic fertilized egg by inserting the fusion gene construct into a mammalian fertilized egg other than human (step 3); And 4) The method of producing a transgenic animal according to claim 7, characterized in that the transgenic fertilized egg is implanted in the womb of a fertility surrogate mother except for humans to obtain a transgenic animal (step 4). 10. The method of claim 9, wherein the thymus specific promoter of step 1 is selected from the group consisting of Lck proximity promoter, CD2 promoter, CD4 minigene promoter, TCR promoter and CD3 promoter. The method of claim 9, wherein the mammalian-derived gene of step 1 is composed of human growth hormone (hGH PolyA), phytotrophic hormone (bGH PolyA), human CD2 (hCD2 polyA), and SV40 (SV40 PolyA) to which polyA sequences are bound The manufacturing method characterized in that it is selected from the group consisting of.