KR100953759B1 - Transgenic rat expressing human selenoprotein M gene - Google Patents

Abstract

The present invention relates to a transgenic animal expressing human Selenoprotein (SelM) and a method of manufacturing the same, and more particularly, to the integration of a human Selenoprotein M gene functionally linked with a cytomegalovirus (CMV) promoter. The present invention relates to a transgenic animal prepared to have a chromosome, and an expression cassette, a recombinant plasmid, and a recombinant vector for producing such an animal model.

Selenium, human Selenoprotein M gene, transgenic animal

Description

Transgenic rat expressing human selenoprotein M gene

The present invention relates to a transgenic animal expressing human Selenoprotein (SelM) and a method of manufacturing the same, and more particularly, to the integration of a human Selenoprotein M gene functionally linked with a cytomegalovirus (CMV) promoter. The present invention relates to a transgenic animal prepared to have a chromosome, and an expression cassette, a recombinant plasmid, and a recombinant vector for producing such an animal model.

Selenoprotein is known as an enzyme with selenium in selenocysteine that has enzymatic activity. To date, selenoprotein residues encoding 25 genes have been found on the human genome (Kryukov GV, et al., 2003a, Characterization of mammalian selenoproteomes. Science. 300: 1439-1443). However, more than 30 selenoproteins have been identified in various tissues by the isolation and identification of rat radioisotope 75Se (Kyriakopoμlos A, et al., 2002, Selenium-containing proteins in mammals and other forms of life. Rev Physiol Biochem Pharmacol 145, 1-46). Furthermore, several studies have reported that selenoproteins are closely related to various chronic diseases. Glutathione peroxidase (GPX), thioredoxin reductase, methionine-sulfoxide-reductase and selenoprotein P have antioxidant defenses and intracellular redox activity. Via concomitant selenoproteins (Schweizer U, et al., 2004, Selenium and brain function: a poorly recognized liaison; Brain Res. Rev. 45 164-178; and Birringer M, et al., 2002 Trends in selenium biochemistry Nat.Prod.Rep. 19693-718). In addition, some selenoprotein activity occurs in the brains of humans and rodents. This activity alters neurological disorders associated with epilepsy, Parkinson's disease and Alzheimer's disease (Schweizer U, et al., 2004 Selenium and brain function: a poorly recognized liaison; Brain Res. Rev. 45 164-178; and Savaskan NE , et al, 2002, Impaired postnatal development of hippocampal neurons and axon projections in the Emx2-/-mutants. J. Neurochem. 83, 1196-1207). Induction of several types of cancer, including the prostate and breast, has been shown to be caused by a decrease in selenoproteins, especially in animals and humans (Diwadkar-Navsariwala V, et al., 2006, Selenoprotein deficiency accelerates prostate carcinogenesis in a transgenic model.Proc. Natl. Acad. Sci. USA 103, 8179-8184).

Selenoprotein M has been reported as the 0.7-Kb cDNA genetic code, a new selenoprotein identified from the mammalian EST database. The gene has a 145 amino acid reading frame from ATG codons in a favorable Kozak context and contains the TGA codon, a selenocysteine codon. Moreover, homologous proteins have been found in rats, zebrafish and other vertebrates and are homologous to selenocysteine (Korotkov KV, et al., 2002, Mammalian selenoprotein in which selenocysteine (Sec) incorporation is supported by a new form of Sec insertion sequence element.Mol Cell Biol. 22, 1402-1411). The previously reported eukaryotic selenoprotein gene has a selenium insertion array (SECIS) in the 3 'UTR. However, SECIS is not found in all human, mouse, or rat selenium protein M mRNA. In the motifs of mammalian selenium protein M SECIS (AAATGA_AA_GA) AA sequences are substituted with CC sequences. In addition, the AATGA-CC_GA sequence revealed a new selenoprotein O when screened (Kryukov GV, et al., 2005, Small fitness effect of mutations in highly conserved non-coding regions.Hum Mol Genet. 14 (15): 2221-2229). To date, studies by Mμller et al. (Mμller WE, et al., 2005, Selenium affects biosilica formation in the demosponge Suberites domuncμla.Effect on gene expression and spicμle formation.FEBS J. 272, 3838-3852) suggest that selenoprotein M It is a protein that plays an important role in the formation of spicules of demosponge suberies domuncμla. Hwang et al. (Hwang DY, et al., 2005, Differentially expressed genes in transgenic mice carrying human mutant presenilin-2 (N141I): correlation of selenoprotein M with Alzheimer's disease. Neurochem Res. 30, 1009-1019) M has been reported to play a pathologically inhibitory or protective role in Alzheimer's patients.

Until now, most of the functional studies on selenoprotein have been conducted in vitro, and some transgenic animals have been developed and reported. In particular, most studies have been carried out to develop transgenic animals and gene deletion animals using GPX proteins. Recently, gene-deletion mice for selenium protein P, which contains the highest amount of selenium, have been developed. It is known to be involved. However, rats expressing human selenium protein M under CMV (cytomegalovirus) promoter regulation to study the function of selenium protein M in vivo have not been reported so far.

Therefore, in the present invention, as a result of trying to develop a normal transgenic animal expressing human selenoprotein M in order to evaluate the antioxidant defenses and activity, ultimately a transformation comprising the expression cassette expressing human selenoprotein in the chromosome The present invention has been found to contribute to the development of therapeutic agents through the study of the function of selenoid proteins and the study of the correlation of diseases related to selenium by providing animals.

Accordingly, it is an object of the present invention to provide a novel antioxidant related transgenic animal into which the human selenoprotein M gene has been introduced.

It is also an object of the present invention to provide an expression cassette, a recombinant plasmid and a recombinant vector for expressing selenoprotein M.

In order to achieve the above object, in the present invention, the human selenoprotein M cDNA of SEQ ID NO: 1 is cloned into a pGEM T easy vector and then the pEGFP C-1 vector (BD bioscience, # 6084-1) The present invention provides a pCMV / EGFP-hSelM expression cassette prepared by binding to Sal I and Sal I sites and connecting to a CMV promoter to express human selenoprotein.

In another aspect, the present invention provides a method for preparing a rat pCMV / EGFP-hSelM expression cassette comprising the following steps and expressing human selenoprotein M:

1) amplifying the human selenoprotein nucleotide sequence according to claim 1 by PCR and cloning the pGEM T easy vector; And

2) separating the phSelM-T vector using SalI restriction enzyme and binding to the pEGFP C-1 vector to prepare a pCMV / EGFP-hSelM vector.

In addition, the rat pCMV / EGFP-hSelM expression cassette was used to insert a rat pCMV / EGFP-hSelM fusion gene into a chromosome to transform the rat fertilized eggs of the accession number KCTC 11203BP and the rat transplanted with normal rats. Transgenic rats produced by mating rats also fall within the scope of the present invention.

In the present invention, a transgenic animal expressing human selenoprotein can be prepared through a fertilized egg prepared using a rat pCMV / EGFP-hSelM expression cassette into which a human selenoprotein gene is introduced, and thus, in vivo. Has provided an important basis for studying the function of selenoid proteins and the interrelationship of diseases associated with selenium.

Hereinafter, the present invention will be described in more detail.

Selenoprotein M is a protein that is synthesized in various tissues and regulates the antioxidant status of cells. In particular, selenoprotein M is closely related to infectious diseases, cardiovascular diseases, cancer and external diseases. The nucleotide sequence of human selenoprotein M used in the present invention can easily obtain information on the sequence from an organ such as GENBANK (No .: NM_080430). In the present invention, the cassette was used to simultaneously perform the function of expressing human selenoprotein M using the above cassette (see FIG. 1).

In the transgenic animal of the present invention, a pCMV / EGFP-hSelM gene is integrated in a chromosome, and the gene is linked to a CMV promoter, in which a cassette functionally linked to enhanced green fluorescent protein (EGFP) and human selenoprotein M is linked. It is designed to induce antioxidant state by expressing human selenoprotein M.

The animal produced in the present invention is a new animal, which has not been produced previously, to increase the antioxidant status in animal tissues by overexpressing human selenoprotein M, thereby studying the function of selenoprotein M or infectious diseases, cardiovascular diseases, and cancer. And it is very useful in the process of analyzing and evaluating the usefulness of selenium as a new therapeutic agent in various diseases such as brain diseases.

The transgenic animal produced in the present invention is preferably a rat, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention, those skilled in the art to which the present invention belongs. Will be self-evident.

[ Example  1] person Selenoprotein  Securing base sequence of M and pCMV Of EGFP - hSelM  Expression cassette production

Human selenoprotein M cDNA was synthesized from human neuronal cell line SK-N-MC cell line using total RNA of RNAzol (Tel-Test Inc., CS104). RNAsol (Teltest) was added to cells cultured at 100 mm ² for RNA isolation, and then the cells were ground. The ground tissue was separated according to the company's recommended method, and the amount was measured at 260 nm absorbance and 5 μg was used for reverse transcription (hereinafter, referred to as “RT reaction”). The RT reaction was first treated with 0.5 μg oligo dT to 5 μg RNA and reacted at 70 ° C. for 10 minutes to bind oligo dT to RNA. To this was added 5x buffer, 10 mM dNTP, 0.1 M DTT, Superscript I (200 U / μl) and left for 10 minutes at room temperature and RT reaction was performed at 42 ℃ for 50 minutes. After the RT reaction, the RNA and cDNA were separated by treatment at 90 ° C. for 5 minutes and 3.2 U / μl of RNaseH was reacted at 37 ° C. for 20 minutes to decompose the RNA. These cDNAs were amplified by PCR in 30 cycles using human selenoprotein specific-primers, denatured for 45 seconds at 94 ° C., annealed for 30 seconds at 62 ° C., and extended for 45 seconds at 72 ° C. (FIG. 6A) (sense; 5'-ATG AGC CTC CTG TTG CCT CCG CTG-3 '(SEQ ID NO: 2), antisense; 5'-AGC TGG GGA AGG AAG AAA GTG G-3' (SEQ ID NO: 3). Selenoprotein cDNA was synthesized using RT-PCR and then electrophoresed to confirm that 606 bp of SEQ ID NO: 1 was generated (FIG. 2A).

[ Example  2] human Seleno  Gene acquisition and pCMV Of EGFP - hSelM  Preparation of Expression Cassette

The synthesized human selenoprotein cDNA was cloned into pGEM T easy vector and then digested with Sal I restriction enzyme to confirm gene insertion (FIG. 2B). Inserting the identified hSelM-T vector for separating a large amount of Sal I restriction enzyme cleavage by seleno protein vector for cDNA gene pEGFP-C 1 to the human (BD bioscience, # 6084-1) Sal I and S al site of the I Bound to the CMV promoter (FIG. 2C). The completed gene sequence was digested with restriction enzymes to confirm the insertion of the gene, and the nucleotide sequence was confirmed using BigDye Terminator Cycle Sequencing Ready Reaction Kits (PE Biosystems) (FIG. 2D).

Example 3 Transformation Rat Preparation

< Pure separation of genes and micro-injection into fertilized rats >

Recombinant genes were removed by cleaving and removing gene sites derived from bacteria, and only eukaryotic cell expression sites were harvested and dissolved in water. The purely isolated recombinant gene was diluted to a concentration of 4 ng / μl and injected into the fertilized eggs of rats (Wistar immamich). The injected embryos were delivered to SD pregnant rats of fertility to produce offspring.

< Confirmation of Transgenic Rat >

The produced pups were separated from genomic DNA by cutting the tail, and then confirmed the insertion of the gene through analysis by PCR using human selenoprotein gene specific primers (FIG. 3). At this time, PCR was carried out from these genomic DNAs by 25 cycles of denaturation at 94 ° C. for 45 seconds, annealing at 62 ° C. for 30 seconds, and expansion at 72 ° C. for 45 seconds using a selenoprotein specific-primer. (Sense; 5'-GGT TTT CCC AGT CAC GAC-3 '(SEQ ID NO: 4), antisense; 5'-GAG TTA GCT CAC TCA TAG GC-3' (SEQ ID NO: 5)). The identified pCMV / EGFP-hSelM transgenic rats confirmed that these genes were subsequently delivered through crosses with normal rats.

In FIG. 3, gene insertion was confirmed in the transformed chromosomes of two lines of transgenic rats (TG68, TG817), producing a 606 bp product.

The present inventors have inserted a fertilized egg of a transformed rat by inserting a pCMV / EGFP-hSelM fusion gene into an eukaryotic expression site, ie, a fertilized egg of a transformed rat by inserting a pCMV / EGFP-hSelM gene into a chromosome. It was deposited on September 18, 2007 by the Korean Collection for Type Cμltures, and was granted accession number KCTC 11203BP.

Hereinafter, the characteristics of the transgenic rats prepared in the present invention were analyzed through the following procedure.

[Test Example 1] CMV / GFP - hSelM particular organizational traits transfer protein gene expression of selenomethionine who switched from rats

       Brain, heart and lungs were expressed under CMV promoter control in all tissues to see the regulation of identified human selenoprotein electrons and in transgenic rats (Tg) and non-transgenic rats (Non-Tg) produced in Example 3 RT-PCR and Real time PCR were performed to see the expression of hSelM from various tissues including liver, kidney, intestine and muscle. RT-PCR was synthesized from the synthesized cDNA by PCR with 30 cycles of denaturation at 94 ° C. for 45 seconds, annealing at 62 ° C. for 30 seconds and extension at 72 ° C. for 45 seconds using a selenoprotein specific-primer. Amplification (sense; 5'-ATG AGC CTC CTG TTG CCT CCG CTG-3 '(SEQ ID NO: 6), antisense; 5'-AGC TGG GGA AGG AAG AAA GTG G-3' (SEQ ID NO: 7)). Real time PCR is performed by annealing once at 50 ° C. for 2 minutes using selenoprotein specific-primer from synthesized cDNA, and performing denaturation once at 95 ° C. for 10 minutes. The amplified signal was confirmed by performing 40 cycles of expansion for 15 seconds at 95 ° C. for 2 minutes at 62 ° C. (sense; 5′-ATG AGC CTC CTG TTG CCT CCG CTG-3 ′ (SEQ ID NO: 6), antisense; 5'-AGC TGG GGA AGG AAG AAA GTG G-3 '(SEQ ID NO: 7)). RT-PCR showed the highest expression of human selenoprotein in the kidney followed by brain, lung, heart, liver, muscle and intestine (FIG. 4A). However, their own expression of selenoprotein was not different in all tissues between non-transgenic and transgenic rats. In addition, real-time PCR revealed similar expression patterns of human selenoprotein transcripts and significantly higher human selenoprotein transcripts (RQ levels) in the kidney and brain of CMV / GFP-hSelM transgenic rats (FIG. 4B). ).

In addition, the expression of human selenoproteins in the kidneys of transgenic rats (Tg) and non-transformed rats (Non-Tg) was confirmed using Western blot and immunostaining method, and the results are shown in FIG. 5. . In FIG. 5A, GFP-hSelM fusion protein (52-KDa) was identified by Western blot using antibodies specific for rat / human selenoproteins in the kidney tissue of CMV / GFP-hSelM transgenic rats. In particular, expression of GFP-hSelM fusion protein was significantly higher in the height of transgenic rats than non-transgenic rats. In contrast, the low expression of selenoproteins it possessed was found in both non-transgenic and transgenic rats (16-KDa). In particular, it was analyzed in the kidney using an optical microscope to confirm the location and distribution of human selenoprotein in the tissue (Fig. 5B). Immunostaining of CMV / GFP-hSelM transgenic rats resulted in the spread of kidney kidney epithelial cells throughout the kidney. However, expression in nontransgenic rats is lower than in transgenic rats. These results suggest that selenoprotein expression and CMV / GFP-hSelM transgenic rats normally synthesize under CMV promoter regulation.

Test Example 2 SOD ( superoxide) in Various Tissues of CMV / GFP - hSelM Transgenic Rats dismutase ) and changes in GPX activity

BIOXYTECH SOD-525 and BIOXYTECH GPx and kits that act on specific substrates in all tissues of the CMV / GFP-hSelM transgenic rats produced in Example 3 to observe whether human selenoproteins induce changes in antioxidant enzyme activity Using the -340 kit (OxisResearch ^™ ; Portland USA), SOD and GPX activity were confirmed according to the reagents and recommended methods provided by the company, and the results are shown in Table 1.

group GPX (mU / ml) SOD (mU / ml) Nontransgenic Rats Transformed Rat Nontransgenic Rats Transformed Rat Brain (cortex) 15.4 ± 1.1 11.3 ± 0.8 * 1.67 ± 0.1 1.21 ± 0.1 * Brain (seahorse society) 13.3 ± 1.0 6.1 ± 0.4 * 1.67 ± 0.1 2.09 ± 0.1 * Heart 344.7 ± 25.2 331.3 ± 23.6 4.06 ± 0.3 1.94 ± 0.2 * lights 265.4 ± 20.3 383.5 ± 27.3 * 2.34 ± 0.2 2.41 ± 0.2 liver 653.3 ± 29.4 716.1 ± 31.3 * 2.91 ± 0.2 2.79 ± 0.2 kidney 712.0 ± 40.7 690.4 ± 33.5 4.24 ± 0.3 3.48 ± 0.3 * chapter 154.3 ± 9.6 238.7 ± 14.9 * 1.37 ± 0.1 1.98 ± 0.1 *

* Significant differences in GPX and SOD values in transgenic and nontransgenic rats

In the results of Table 1, the truncated rats according to the present invention showed the highest GPX activity in the kidney and liver, followed by heart, lung, intestine and brain. In particular, GPX activity in lungs, liver, and intestine was significantly increased compared to nontransgenic rats, while cortex and hippocampus of brain decreased compared to nontransgenic rats.

In addition, in the truncated rats according to the present invention, the highest SOD activity was observed in kidney and heart, followed by liver, lung, heart, brain and intestine. Moreover, SOD activity in cortex, hippocampus, and intestine was significantly increased compared to nontransformed rats, whereas decreased in heart and kidney than nontransformed rats.

These results suggest that overexpression of human selenoprotein regulates GPX and SOD activity changes differently in various tissues of transgenic rats.

Test Example 3 In CMV / GFP - hSelM Transgenic Rats H ₂ O ₂ and the influence of celecoxib no protein over-expression for the total antioxidant content

In previous studies, carbon radical formation was caused by 2,2'-azobis (2-amidinopropane) hydrochloride (hereinafter referred to as "AAPH"). Alkyperoxyl radicals and hydroperoxides are produced under aerobic conditions, resulting in modified lipids and DNA (Hiramoto K, et al., 1993, DNA breaking activity of the carbon-). centered radical generated from 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) .Free Radical Res.Commun. 19, 323-332; Lazar M, et al., 1989, in Free Radical in Chemistry and Biology, pp 10-16, CRC Pree, Boca Raton, FL; Ma YS, et al., 1994, The effects of vitamin C and urate on the oxidation kinetics of human low-density lipoprotein.Proc. Soc.Exp.Biol.Med. 2-6, 53-59; and Landi L, et al., 1997, DT-Diaphorase maintains the reduced state of ubiquinones in lipid vesicles promoting promoting th eir antioxidant function.Free Radical Biol.Med. 22, 329-335).

In the present invention, in order to observe the effect of human selenoprotein M overexpression on the potent free radicals, CMA / GFP-hSelM transgenic rats and non-transgenic rat serum produced in Example 3 after administration of AAPH using an ELISA kit. Total antioxidant and H ₂ O ₂ content were measured within and the results are shown in FIG. 6.

In the results of Figure 6, the antioxidant results showed that the total antioxidant content was significantly lower in the CMV / GFP-hSelM transgenic rats (Tg) of Example 3 than non-transgenic rats (Non-Tg). After administration of AAPH, both CMV / GFP-hSelM transgenic rats and non-transgenic rats increased significantly. However, the total antioxidant content in CMV / GFP-hSelM transgenic rats remained constant compared to transgenic rats. In particular, the increase in normal and AAPH-treated groups was very similar in CMV / GFP-hSelM transgenic rats and non-transgenic rats (FIG. 6A). Further increasing H ₂ O ₂ content pattern in transgenic rats from the H ₂ O ₂ results appear very similar to the total antioxidant measurements after administration of AAPH (Fig. 6B). These results suggest that selenoprotein M overexpressed under CMV promoter regulation increased antioxidant defense activity in CMV / GFP-hSelM transgenic rats.

Test Example 4 Changes in SOD and GPX Activity in Erythrocytes in CMV / GFP-hSelM Transgenic Rats

To determine if the total antioxidant and H ₂ O ₂ content in serum is associated with the disease, a kit is applied to the specific substrates in red blood cells of CMV / GFP-hSelM transgenic rats and non-transgenic rats produced in Example 3. SOD and GPX activity were measured and the results are shown in FIG. 7.

In the results of FIG. 7, SOD activity was slightly increased in CMV / GFP-hSelM transgenic rats (Tg) than non-transgenic rats (Non-Tg), and this aspect was observed to be maintained in the AAPH-administered population. In addition, compared with the non-transformed rats, CMV / GFP-hSelM transgenic rats were significantly increased by 3 to 4 times in red blood cells. However, GPX activity in the AAPH-administered group of CMV / GFP-hSelM transgenic rats was decreased compared to the normal population, while maintaining higher than the AAPH-treated group of non-transgenic rats. Therefore, these results suggest that increased SOD and GPX activity in erythrocytes of CMV / GFP-hSelM transgenic rats was shown by decreasing total antioxidant and H ₂ O ₂ content in serum.

Test Example 5 Changes in Leukocyte (WBC) Status in CMV / GFP - hSelM Transgenic Rats

       Selenoprotein and selenium are associated with the progression of infected disease (Birringer M, Pilawa S and Flohe L 2002 Trends in selenium biochemistry Nat. Prod. Rep. 19693-718) and the state of immune cells (Tapiero H, Townsend DM, Tew KD (2003). The antioxidant role of selenium and seleno-compound.Biomed.Pharmacother.57, 134-144). Both CMV / GFP-hSelM transgenic rats (Tg) and non-transgenic rats (Ng-Tg) produced in Example 3 after AAPH administration to examine the effects of overexpression of selenoproteins in immune-related cell populations. Hematological analysis was performed, and the results are shown in Table 2 and FIG. 8 (hematological analysis).

Item Nontransgenic Rats Transformed Rat (Example 3) AAPH- AAPH + AAPH- AAPH + Neutrophils 8.9 ± 0.7 48.0 ± 3.5 * 9.1 ± 0.9 76.2 ± 6.5 *, ** Lymphocyte 87.1 ± 7.3 37.9 ± 3.1 * 85.8 ± 7.8 16.8 ± 0.1 *, ** Monocytes 1.7 ± 0.1 11.2 ± 0.9 * 1.7 ± 0.1 4.9 ± 0.4 *, ** Eosinophils 0.4 ± 0.03 0.2 ± 0.01 * 0.5 ± 0.04 0.5 ± 0.05 ** Basophils 1.0 ± 0.1 1.6 ± 0.1 * 0.9 ± 0.07 0.8 ± 0.07 ** leukocyte 0.9 ± 0.08 1.0 ± 0.1 2.1 ± 0.2 0.7 ± 0.06 *, **

* Significant differences in GPX and SOD values in transgenic and nontransgenic rats

** Significant differences between AAPH- and AAPH-treated groups

In the results of Table 2, the leukocyte (WBC) composition among the hematological values in the normal population was not different in both CMV / GFP-hSelM transgenic rats and nontransgenic rats. However, after AAPH administration, the number of neutrophils was significantly increased in CMV / GFP-hSelM transgenic rats, whereas lymphocytes, monocytes and basophils were significantly reduced in transgenic rats. In particular, the ratios of neutrophil-to-lymphocyto (N / L) were not different between the CMV / GFP-hSelM transgenic rats and the transgenic rats. However, the N / L ratio in the AAPH-administered population increased 2-3 fold in CMV / GFP-hSelM transgenic rats compared to nontransgenic rats (FIG. 8). Thus, these results indicate that overexpression of selenoprotein induces immune cell changes in the AAPH response.

Test Example 6 In CMV / GFP - hSelM Transgenic Rats Changes in corticosterone (corticosterone)

Increased corticosteroids have been reported to increase the N / L ratio by lymphocytes destroyed in the thymus cortex or expanded neutrophil half-life (Davies KJ, et al., 1987, Protein damage and degradation by oxygen radicals. II.Modification of amino acids.J. Biol. Chem. 262, 9902-9907; and Kim CY, et al, 2005, Indirect indicator of transport stress in hematological values in newly acquired cynomolgus monkeys.J. Med. Primatol. 34, 183-192). Therefore, in order to determine the association between N / L ratio and corticosterone concentration, in CMV / GFP-hSelM transgenic rats and transgenic rats produced in Example 3 in serum using an ELISA kit for specific antibodies Corticosterone concentrations were measured and the results are shown in FIG. 9.

In the results of FIG. 9, corticosterone concentration in the AAPH treated group was significantly increased compared to the normal group in both CMV / GFP-hSelM transgenic and nontransgenic rats. However, there was no difference between CMV / GFP-hSelM transgenic rats and nontransgenic rats in increasing rates and patterns of corticosterone concentration (FIG. 9). These results indicate that corticosterone is not related to immune cell changes in selenoprotein M overexpressed in transgenic rats for AAPH response.

In the present invention, a transgenic model animal which induces an antioxidant state by simultaneously expressing a human selenoprotein expression cassette in each tissue of an animal was developed. The selenoprotein expression cassette developed in the present invention is a selenoprotein in cells and animals. It is likely to be used for the study of the function and the study of the mechanism of maintaining intracellular antioxidant.

In addition, the cassette expressing human selenoprotein M directly validates the metabolic effects of selenoprotein M in animals, and this animal model producing a new animal model is an infectious disease that is chronically diverse in modern people, It can be used as an important model for evaluating the usefulness of selenium in the development of cardiovascular disease, cancer and brain disease and evaluation of therapeutic agents.

1 is a schematic diagram showing a cloning process of a recombinant vector and a cassette expressing human selenoprotein M (pCMV / GFP-hSelM) linked to a CMV promoter.

Figure 2 is a restriction enzyme cleavage electrophoresis and sequencing pictures of pCMV / GFP-hSelM recombinant vector.

Figure 3 is an electrophoresis picture showing the genotype of pCMV / GFP-hSelM transgenic rats.

Figure 4 is a photograph confirming the expression of the hSelM gene at the RNA level in each tissue of pCMV / GFP-hSelM transgenic rats.

Figure 5 is a photograph confirming the expression of hSelM protein in the kidney tissue of pCMV / GFP-hSelM transgenic rats by Western blot and immunostaining.

Figure 6 is a graph showing the results of measuring the concentration of total antioxidants and the concentration of hydrogen peroxide in the serum of pCMV / GFP-hSelM transgenic rats.

Figure 7 is a graph showing the results of measuring the activity of GPX and SOD in the blood cells of pCMV / GFP-hSelM transgenic rats.

8 is a diagram showing the composition ratio in the blood cells of the pCMV / GFP-hSelM transgenic rats.

Figure 9 is a graph showing the results of measuring the N: L ratio and corticosterone concentration of pCMV / GFP-hSelM transgenic rats.

<110> KOREA FOOD and DRUG ADMINISTRATION <120> Transgenic rat expressing human selenoprotein M gene and the          method for preparing honey <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 739 <212> DNA <213> Human Sel protein M <400> 1 cgtggcgcag cgactcggag gttcgcctcc agcttgcgca tcatctgcgg ccgggtcccg 60 atgagcctcc tgttgcctcc gctggcgctg ctgctgcttc tcgcggcgct tgtggcccca 120 gccacagccg ccactgccta ccggccggac tggaaccgtc tgagcggcct aacccgcgcc 180 cgggtagaga cctgcggggg atgacagctg aaccgcctaa aggaggtgaa ggctttcgtc 240 acgcaggaca ttccattcta tcacaacctg gtgatgaaac acctccctgg ggccgaccct 300 gagctcgtgc tgctgggccg ccgctacgag gaactagagc gcatcccact cagtgaaatg 360 acccgcgaag agatcaatgc gctagtgcag gagctcggct tctaccgcaa ggcggcgccc 420 gacgcgcagg tgccccccga gtacgtgtgg gcgcccgcga agcccccaga ggaaacttcg 480 gaccacgctg acctgtaggt ccgggggcgc ggcggagctg ggacctacct gcctgagtcc 540 tggagacaga atgaagcgct cagcatcccg ggaatacttc tcttgctgag agccgatgcc 600 cgtccccggg ccagcaggga tggggttggg gaggttctcc caaccccact ttcttccttc 660 cccagctcca ctaaattccc tcctgcctta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 720 aaaaaaaaaa aaaaaaaaa 739 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 2 atgagcctcc tgttgcctcc gctg 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 3 agctggggaa ggaagaaagt gg 22 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 4 ggttttccca gtcacgac 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 5 gagttagctc actcataggc 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 6 atgagcctcc tgttgcctcc gctg 24 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 7 agctggggaa ggaagaaagt gg 22  

Claims (3)

Transformed fertilized egg of Accession No. KCTC 11203BP transformed with human selenoprotein M gene of SEQ ID NO: 1. A transgenic rat produced by the fertilized egg according to claim 1. delete

Images