KR20030067204A - Peroxiredoxin II-deficient mice and their production method - Google Patents

Abstract

PURPOSE: Provided are a peroxiredoxin II-deficient mice and a production method thereof which are useful for the study of oxidation or anti-oxidation mechanism in a blood cell and the development of a therapeutic agent for anemia. CONSTITUTION: A production method of a peroxiredoxin II-deficient mice is characterized by introducing a vector prxII-K.O which is deleted from peroxiredoxin II gene into a stem cell to give peroxiredoxin II deficient stem cell; micro-injecting the obtained peroxiredoxin II deficient stem cell into blastocoel to give a chimeric animal; and back crossing the chimeric animal.

Description

Peroxiredoxin II-deficient mice and their production method

The present invention relates to a peroxredoxin-II gene deficient mouse and a method for producing the same, and more particularly, by replacing a part of the peroxyredoxin-II gene in an antioxidant enzyme with a neo gene to peroxyredoxin- Ⅱ Gene Deletion Vectors are produced, and studies on the oxidation or antioxidant mechanisms of blood cells produced using them, as well as peroxredoxin-II gene deficient mice, which can be very usefully used for various stress-related disease model animals and their production It is about how to.

Peroxyredoxin is a type of antioxidant enzyme that transfers electrons to hydroperoxide, one of the metabolic intermediates of oxygen, and reduces it to water. The relevance has been reported in several areas.

Antioxidant deficient animals that have been developed to date in this field include Cu / Zn-superoxide dismutase and glutathione peroxidase 1. These antioxidant enzyme-deficient animals have been shown to respond sensitively to stimuli applied externally in common [Ohlemiller et al., Audiol. Neurootol. 4, 237, 1999; Haan et al., Journal of Biological Chemistry 35, 22528, 1998]. Therefore, there is an urgent need for studies on the relationship between the deletion of the antioxidant-related genes and the oxidative-related diseases and the study of their own phenotypes not by artificial treatment, but the model animal is not appropriate yet.

Therefore, the present inventors have made intensive researches according to the necessity of the development of the above-described genetically deficient mice. As a result, we have injected genetically modified embryonic stem cells in vitro into the blastocyst of the normally developing blastocyst. ) A chimeric mouse with blastocysts implanted into the uterus of a surrogate mother mouse was induced to continue development, and a chimeric mouse with embryonic stem cells participated in the development. The present invention was completed by confirming that peroxyredoxin-II was not expressed at all from gene-deficient mice.

Accordingly, an object of the present invention is to provide a mouse lacking the peroxyredoxin-II gene and a method of producing the same.

Figure 1a is a schematic diagram of the manufacturing process of the vector (prxII-K.O) for the peroxyredoxin-II gene deletion.

1B is a schematic diagram of the peroxyredoxin-II gene deletion vector (prxII-K.O).

2A is a schematic diagram of alleles with gene deletions.

Figure 2b is a picture confirmed by XL-PCR gene deletion in embryonic stem cells (embryonic stem) cells.

Figure 2c is a photograph confirmed by Southern blot analysis of gene defects in embryonic stem cells.

Figure 3 is a photograph of PCR results for analyzing the genotyping of peroxyredoxin-II gene deficient mice.

Figure 4 is a photograph of Western blot analysis (Western blot analysis) to confirm the expression of peroxredoxin-II protein in mice with a peroxredoxin-II gene deletion.

FIG. 5A is a photograph confirming splenomegaly in homozygous mice. FIG.

Figure 5b is a photograph of the spleen tissue of wild type (Wt, wild type), heterozygote and homozygous mice confirmed the increase of erythrocyte hemolysis by hemosiderin staining.

6A is a graph of hematological analysis of wild type and homozygous mice.

Figure 6b is a photograph confirming the Heinz body (heinz body) erythrocyte oxidation indicator in the blood collected from homozygous mice.

6C is a graph showing the proportion of erythrocytes containing Heinz bodies in homozygous mice between 1 and 12 weeks of age.

Figure 7 is a graph comparing the sensitivity to hydrogen peroxide of red blood cells collected from wild-type and homozygous mice.

The present invention is characterized by peroxyredoxin-II gene deletion vector (prxII-K.O) and peroxyredoxin gene-deficient mice.

In addition, a peroxyredoxin-II gene deletion vector (prxII-KO) is introduced into embryonic stem cells to produce embryonic stem cells deficient in the peroxyredoxin-II gene, and the blastocyst of the blastocyst is generated. After microinjection (blastocoel) to produce a chimeric mammal. And backcross it to produce peroxyredoxin gene deficient mice.

The present invention will be described in more detail as follows.

The present invention relates to a peroxredoxin-II gene deficient mouse and a method for producing the same, and more particularly, by replacing a part of the peroxyredoxin-II gene in an antioxidant enzyme with a neo gene to peroxyredoxin- Ⅱ Gene Deletion Vectors produced and produced using the hydroxyredoxin-II gene deficient mice can be very useful as a model of stress-related disease model animals, as well as studies on the oxidation or antioxidant mechanism of blood cells.

First, a gene deletion vector is produced in order to delete the peroxyredoxin-II gene. That is, a portion of the peroxredoxin-II gene and neo and cymidine kinase with antibiotic (G418 and gencyclovir) resistance are included in the vector for the deletion of the gene.

The vector is used to produce peroxyredoxin-II gene deficient mice. In other words, chimeric mice are produced by deleting the peroxyredoxin-II gene present in embryonic stem cells with the gene deletion vector and microinjecting the gene-deficient embryonic stem cells into the blastocyst of the normally developing blastocyst. do. The produced chimeric mice confirmed that embryonic stem cell-derived F1 mice could be born by backcrossing, and produced mice that were genetically deleted by crosses between F1 mice lacking one gene. Therefore, it is possible to obtain a gene-deficient mouse in which both of the peroxyredoxin-II genes are deleted and not expressed at all.

In addition, the present invention also includes a method for producing an antioxidant enzyme-deficient mouse comprising a series of processes, such as production of the gene-defective vector, production of gene-deficient embryonic stem cells, micro-injection, transplantation and development.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Construction of Peroxyredoxin Gene Deletion Vectors

The peroxyredoxin-II gene is a genome clone 3A [Lim et al., Gene of the peroxyredoxin-II gene isolated from mouse 129 / SvJ ( Mus musculus domesticus ) cultivar genome library. 216, 1998] was used to construct a gene deletion vector (prxII-KO).

The peroxyredoxin-II gene has a total of six exons, of which exons 2 to 5 are replaced by neo genes by genetic recombination to block the expression of mRNA and to be used as a selection marker. It is designed to be. Peroxyredoxin-II gene deletion vector production process is as follows (see Fig. 1a and 1b).

3.57 kb DNA containing exon 1 was added to the pBluescript II sk vector to make a 5'-end homology in genomic clone 3A of the peroxyredoxin-II gene.BamIt was cloned into the HI site and named prxII-5 '(3.57). Also, in this vectorXhoA PGK promoter cut with I and 1.6 kb DNA of the neo gene were inserted in the opposite direction of transcription to construct a prxII-BX vector that blocks mRNA expression and can be used as a selection marker. To make the 3 'end homology, 2.0 kb DNA containing 6 parts of exon, except for exons 2 to 5, followed by introns, was substituted for the pBluescript II sk vector.KpnI, HinIt was cloned into the dIII site and named prxII-3 '(2.0). Also, in this vectorHindIII andNotWith I The truncated mc1 promoter and thymidine kinase gene 2.3 kb DNA were inserted to reduce the probability of random insertion into the genome, which was named prxII-KN. In order to construct the prxII-BN vector by inserting the peroxyredoxin-II 3 ′ end of prxII-KN, the thymidine kinase gene and the mc1 promoter into the prxII-BX vector, and to further increase the homology of the 5 ′ end 5 4.23 kb of DNA was further added to the peroxyredoxin gene deletion vector (prxII-KO).

Example 2: Production of Peroxyredoxin-II Gene Defective Embryonic Stem Cells

After cutting the peroxyredoxin-II gene deletion vector (prxII-KO) prepared in Example 1 with restriction enzyme Not I, 15 to 20 µg was subjected to electroporation using 1 to 2 x 10 ^Seven embryonic stem cells were introduced. Electroporation was performed using the 260 ~ 270 volt and 500 μ F A Bio-Rad Gene Pulser (Biorad gene pulsor) [Biorad Co.] adjusted. After electroporation, G418 [Sigma Co., Ltd.] and GCV (gancyclovir) were used to select only embryonic stem cells in which the gene deletion vector was inserted into the genome of embryonic stem cells. Selected embryonic stem cells were identified for each colony, and whether the peroxyredoxin-II gene was correctly deleted was determined using XL-PCR (Perkin Elmer) method. Selective primers for the XL-PCR method were made as follows.

N-terminal primer (SEQ ID NO: 1)

5'-GTT-GGG-GCA-CAG-GTG-AAA-TCC-AAA-C-3 '

C-terminal primer (SEQ ID NO: 2)

5'-CTG-TCC-ATC-TGC-ACG-AGA-CTA-GTG-AG-3 '

The primers were recovered from each of the selected embryonic stem cells as a template by hybridization at the N-terminal and C-terminal in genomic DNA. XL-PCR was then subjected to PCR by the method provided by the manufacturer (Perkin Elmer), and electrophoresis was performed to confirm that the peroxyredoxin-II gene was correctly deleted from the resulting DNA product [ 2a and 2b]. Southern blot analysis was performed to confirm the results again (see FIGS. 2A and 2C). As shown in FIG. 2B, the positive band 3.1 Kb was detected in the same number as in the positive control group (P) in No. 3, and the Southern blot result confirmed the band that could be detected only when the gene was deleted in the same number. By identifying three clones in the same way, a total of four embryonic stem cell clones could be identified.

Example 3: Production of Peroxredoxin-II Gene Deficient Mice

In Example 2, four gene-deficient clones were microinjected into the blastocoel of the blastocyst recovered from 3.5 day-old C57BL / 6J female mice to produce chimera mice. The produced chimeric mice were again backcrossed with C57BL / 6J mice to confirm germ-line transmission to the germline. Mice translocated to the gonads produced by backcross were identified gene deletions using the primers described in Example 2 and the XL-PCR method, and both genes were completely deleted by crossing between mice in which gene deletions were identified. Homozygous mice could be produced. In order to confirm the deletion of both genes, genomic DNA was extracted by cutting tails of mice born after cross-crossing, and wild type and gene deletion alleles were identified by PCR using genomic DNA of these mice as a template. Selective primers for each were prepared as follows.

N-terminal primer of wild type allele (Wt allele) (SEQ ID NO: 3)

5'-ATG-GCC-TCC-GGC-AAC-GCG-CAA-ATC-G-3 '

C-terminal primer of wild type allele (SEQ ID NO: 4)

5'-GAT-GAT-CTC-CGT-GGG-GCA-AAC-AAA-AGT-GAA-G-3 '

N-terminal primer of the gene deletion allele (SEQ ID NO: 5)

5'-GCT-TGG-GTG-GAG-AGG-CTA-TTC-G-3 '

C-terminal primer of gene deletion allele (SEQ ID NO: 6)

5'-GTA-AAG-CAC-GAG-GAA-GCG-GTC-AGC-C-3 '

In addition, the primers were placed at each of the upstream and the downstream of the genomic DNA of the mouse mice, respectively, by hybridization, followed by PCR in a conventional manner, and the resulting DNA product. Electrophoresis was performed to identify wild-type and gene deletion alleles from [see FIG. 3]. As a result, it was possible to produce homozygous mice in which only a 700 bp gene deletion allele was detected without a 250 bp wild-type allele amplification product. Then, the 2-cell fertilized egg of the obtained peroxredoxin-II gene deficient mouse was deposited on September 4, 2001 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10063BP).

Experimental Example 1: Investigation of Peroxyredoxin-II Expression by Western Blot Analysis

In order to confirm the expression of peroxredoxin-II protein of the peroxyredoxin-II gene deficient homozygous mice obtained in Example 3, Western blot analysis was performed by a conventional method.

As in Example 1, only one allele produced by backcrossing hybridized male heterozygous male and female mice that had been deleted, and then isolated mouse embryonic fibroblasts from each fetus of 13.5 days of gestation. Genomic DNA and protein were extracted from each cell. The extracted genomic DNA was used to analyze each mouse fetal fibroblast genotype by the PCR method described in Example 3 above, and the isolated protein was used for western blot analysis in a conventional manner (see FIG. 4). That is, each mouse fetal fibroblast was lysed and centrifuged at 10,000 rpm to obtain crude protein (crude extract) of the upper layer. After quantifying the amount of each protein obtained, the same amount was loaded onto a 10% SDS-PAGE gel, respectively. The protein loaded on the gel is transferred to a nitrocellulose filter, first reacted with a monoclonal antibody against mouse peroxredoxin-II, and immunization of mice with alkaline phosphate (AP). Globurin (Ig-G) was reacted. Specifically bound proteins were identified by ECL (enzyme coupled immunoluminescence) [Amersham].

As a result, as shown in Figure 4, it was confirmed that the peroxyredoxin-II protein is not expressed at all in mouse fetal fibroblasts in which both alleles are deleted. However, the band in the heterozygotes was almost at the endogeneous level.

Experimental Example 2: Histological Analysis of Peroxyredoxin-II Gene Deficient Mice

When the wild type, heterozygous and homozygous mice obtained in Example 3 were slaughtered to confirm the weight and degree of pigmentation of the spleen extracted, splenomegaly was observed in homozygous mice (see FIG. 5A).

In addition, the extracted spleens were fixed with 10% formalin, inoculated in paraffin, and cut into 5 μm thicknesses. The hemolysis of erythrocytes was examined by hemosiderin staining, and the spleen of homozygous mice was the strongest. The staining reaction was observed, and the proliferation of red pulp was also observed (see FIG. 5B).

Therefore, it was confirmed that splenomegaly of homozygous mice is caused by increased hemolysis of red blood cells.

Experimental Example 3: Hematological Analysis of Peroxyredoxin-II Gene Deficient Mice

After separating blood from capillaries of 8-week-old wild-type and homozygous mice obtained in Example 3, using Hemavet 3500, mean corpuscular volume (MCV), HCT (hematocrit), and MCHC, which are the criteria for the degree of disease in the blood. (mean corpuscular hemoglobin concentration) was examined, the difference was not observed (see Fig. 6a).

In addition, wild type, heterozygous and homozygous mice obtained in Example 3 were collected from 1 week to 12 weeks of age to obtain Heinz body formation, which is an oxidation indicator of erythrocytes. It was confirmed by staining with [Merck]. As a result, the percentage of erythrocytes containing Heinz bodies, which are oxidative indicators of erythrocytes, remained below 3% in wild-type and heterozygous mice, while in homozygous mice it increased to 5 weeks of age and then about 30 after 6 weeks of age. It could be observed that the level was maintained at the level of% (see FIGS. 6B and 6C).

Therefore, it was found that red blood cells without peroxyredoxin-II protein began to accelerate oxidation after birth.

Experimental Example 4: Investigation of Sensitivity to Hydrogen Peroxide in Peroxyredoxin-II Gene Deficient Red Blood Cells

Blood was isolated from capillaries of 8-week-old wild-type, homozygous mice obtained in Example 3, and then the sensitivity of erythrocytes was examined by artificially administering hydrogen peroxide. That is, red blood cells recovered to investigate the hydrogen peroxide sensitivity were washed with PBS containing no calcium and magnesium, and then treated with 75, 100, 200, 300, and 400 mM hydrogen peroxide for 20 minutes at 37 ° C. The ratio of erythrocytes was converted by measuring the OD value. As a result, it was found that homozygotes exhibited about two times or more sensitivity to red blood cells of wild-type mice in the treatment groups of 100 mM or more (see FIG. 7).

As described above, a disease model mouse lacking the peroxyredoxin-II gene, which is one of the antioxidant enzymes according to the present invention, prevents the peroxyredoxin-II gene from being expressed so that the resistance of red blood cells to oxidation is reduced. Not only did this cause splenomegaly, but also the genotype is well transmitted to descendants, and it is very useful as a disease model animal that can be used to study the oxidation or antioxidant mechanism of blood cells and to develop anemia treatment. Do.

Claims (3)

Peroxiredoxin II gene deletion vector (prxII-KO), wherein exons 2 to 5 are substituted with neo genes. Peroxyredoxin-II gene deficient mice (KCTC 10063BP) transformed with a peroxiredoxin II gene deletion vector (prxII-K.O). Peroxiredoxin II gene deficient vector (prxII-KO) was introduced into embryonic stem cells to produce embryonic stem cells deficient in the peroxodidoxin-II gene, which were then generated in the blastocyst of the developing blastocyst. A method of producing peroxyredoxin-II gene deficient mice, characterized in that the microinjection produces chimeric mammals and then backcrosses them.