KR100418187B1 - Transformed Caenorhabditis elegans Synthesizing Cholesterol in vivo - Google Patents

Abstract

The present invention relates to a transformed little nematode that synthesizes cholesterol in the body developed as an experimental animal for studying the metabolism of cholesterol. The transformed nematode prepared by the present invention is a transformant in which the human 7-dihydrocholesterol reductase gene is introduced into the nematode, and can be widely used as an experimental animal to study the effects of cholesterol in vivo. will be.

Description

Transformed Caenorhabditis elegans Synthesizing Cholesterol in vivo}

The present invention relates to a transformed little nematode that synthesizes cholesterol in the body. More specifically, the present invention relates to a transformed little nematode that synthesizes cholesterol developed in the body as an experimental animal for studying the metabolism of cholesterol. will be.

Cholesterol is a very important biological component in mammals. It is involved in the regulation of cell division, growth, development and differentiation, and is a precursor of various essential metabolites (e.g. hormones, bile acids, etc.). It is known to cause hyperlipidemia, including sclerosis. Recently, cholesterol and its precursors are known to play an important role in the early stages of development and in the aging process: SLO syndrome, in which the lack of cholesterol in the development process causes accumulation of 7-dihydrocholesterol, which causes problems in cerebral development. (Smith-Lemli-Opitz syndrome) develops. In addition, cholesterol is involved in signaling early in development, and lanosterol, an intermediate of cholesterol metabolism, activates meiosis. In addition, the sterol of the biofilm acts as an antioxidant against oxygen radicals and the like. Among the in vivo functions of such cholesterol and its precursor sterol compound, the field of sterol biosynthesis control and metabolism in animals is carried out using nematodes.

On the other hand, Caenorhabditis elegans is one of the nematodes that live freely in the soil, the adult length is about 1mm, the body is transparent and hermaphrodite, but there is also the male body. Since it was used as an experimental model in the 70's, it has been widely used because of its convenience, and the six chromosomal maps have been completed in 1999 and the genome project has been completed. The nematode becomes an adult through the egg stage and the larval stages of L1 to L4. Sometimes, when food is scarce, under stress or at high outside temperature, the nematode proceeds to the stage called Dauer in the L3 stage. In this state, metabolic activity decreases, external resistance increases, and lifespan increases. One hermaphrodite worm lays about 300 eggs, which can give birth to more eggs if sperm are supplied through it. In the laboratory, E. coli can be fed at 20 ° C and stored at -80 ° C except for some mutations. In addition, it is easy to artificially mutate or keep the trait pure, it is easy to observe the trait through a microscope.

Electric nematodes are used to synthesize some of the sterol compounds in the body or take external sterols and modify them.In the natural state, the sterols of the plant, such as stigmasterol or cytosterol, are transformed into 7-dihydrocholesterol and used in the body. It is known that dihydrocholesterol accounts for more than 50% of the sterols that make up the entire cell membrane. In addition, in the laboratory, the medium is usually cultured with cholesterol, and even in this case, 7-dihydrocholesterol is the main sterol form, which reduces the double bond of 7-dihydrocholesterol in the nematode to cholesterol. It is known to lack the activity of the converting 7-dihydrocholesterol reductase. In addition, the enzyme acts just before the production of cholesterol in the sterol biosynthesis stage, and it is known that in mammals, the deficiency of this enzyme activity causes SLO syndrome, a serious developmental malformation.

On the other hand, the nematodes, which have neurons close to 30% of about 1000 somatic cells, are known to be similar to humans in controlling cell suicide genes and other intracellular molecules, and efforts to use them as animal models continue to be made. However, no suitable transformants have been developed to study the regulation of cholesterol and sterol precursors in the body, and thus, the biosynthetic pathways of cholesterol, various enzyme expressions and their regulation, and the role of various sterol precursors in vivo are still unknown. .

Thus, there is a constant need to develop transformants suitable for studying the regulation of cholesterol and sterol precursors of the nematodes.

Accordingly, the present inventors have made intensive studies to develop a transformant suitable for studying the regulation of cholesterol and sterol precursors of the nematodes, and as a result, the micro-injection of the human 7-dihydrocholesterol reductase gene into the nematodes The transformant produces cholesterol in the body, and the characteristics of life span, aging, reproduction, and stress resistance of the electric transformant are changed, so that it can be used as an experimental animal to study the correlation between cholesterol and electrical properties. The present invention has been completed.

After all, the main object of the present invention is to provide a transformed little nematode that synthesizes cholesterol in the body.

1 is a genetic map showing a cloned vector.

2A is a chromatogram showing the composition of a sterol compound of N2, which is a normal nematode.

Figure 2b is a chromatogram showing the composition of the sterol compound of the transformant

3 is a graph showing the life expectancy of each nematode.

4 is a graph comparing the number of female bodies of the first generation of offspring.

5 is a graph comparing TS ₅₀ of each nematode.

6 is a graph showing the lifespan after irradiation of ultraviolet rays of each nematode.

The transformed nematodes, which synthesize the cholesterol of the present invention, are cloned by introducing a human 7-dihydrocholesterol reductase (7-DHCR) gene into a vector having a promoter of a heat shock protein. An expression vector which induces a roll of the vector and the nematode is a transformant introduced into the nematode.

The inventors note that the reason the nematodes store the sterol compound in the form of 7-dihydrocholesterol is the result of the lack of 7-DHCR which converts 7-dihydrocholesterol to cholesterol. A transformant was prepared by introducing a gene encoding. More specifically, the expression vector pRF4 which introduces a human 7-DHCR gene into a vector having a promoter of a heat shock protein, constructs a cloned transformed vector, and induces a roll of the nematode as a marker gene. After mixing, they were microinjected into the gonads of the nematodes, followed by culturing, and obtaining progeny to obtain progeny 1 generation (F1) exhibiting eddy traits, and then cultivating it again to produce progeny 2 generations of progeny ( F2) was prepared.

As a result of analyzing the sterol compound present in the body of the second generation of progeny produced, cholesterol accumulates in the body, the 7-DHCR expressed by the introduced gene was used to convert the 7-dihydrocholesterol of the nematode into cholesterol. It can be inferred that the genotyping of the electric transformants is consistently maintained in the offspring. As a result of examining the physiological characteristics of the second generation of progeny, it was found that the lifespan was increased, the mating activity was increased, and the heat resistance and the UV resistance were increased. Accordingly, the second generation of the offspring obtained was named `` Caenorhabditis elegans '' and deposited on April 16, 2001 as a deposit number 'KCTC 0996BP' to the International Bank for Biotechnology Research (KCTC). It was.

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 of ordinary skill 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. .

Example 1 Cultivation of Elfin Nematodes

NGM with normal nematode N2 and longevity strain age-1 (CGC No. TJ401) were obtained from the Caenorhabditis Genetic Center (CGC, Korea), and NGM with 1 ml of cholesterol dissolved in ethanol at a concentration of 5 mg / ml E. coli OP50 was fed in a solid medium (3 g / L sodium chloride, 17 g / L agar, 25 g / L bacterium peptone, 1 ml / L 1M magnesium sulfate, 25 ml / L 1M potassium phosphate, 1 ml / L 1M calcium chloride, pH 6,0) Each nematode which was obtained by feeding was incubated. In addition, inoculated with E. coli OP50 in S liquid medium (Bacterial tryptone 16g / L, Bacterial yeast extract 10g / L, sodium chloride 5g / L), incubated overnight, then centrifuged for 5 minutes at 4,000 × g to E. coli OP50 Obtained. Subsequently, three little nematodes and E. coli OP50 incubated with M9 buffer (potassium phosphate 3g / L, sodium phosphate 6g / L, sodium chloride 5g / L, 1M magnesium sulfate 1ml / L) were washed and suspended in M9 buffer solution. , Liquid S-medium with 1 ml of cholesterol dissolved in 5 mg / ml in ethanol (3.5 g / L sodium chloride, 10 ml / L trace metal solution, 3.24 g / L potassium citrate, 3 ml / L 1M magnesium sulfate, 30 ml 1M potassium phosphate) / L, 1M calcium chloride 3 ml / L, pH 6,0) each inoculated with each nematode and E. coli OP50, shake cultured at 20 ℃ for 4 days, and then centrifuged to obtain a mixture of the nematode and E. coli OP50 It was. The mixture was resuspended in M9 buffer, 70% (w / v) sucrose solution was added, followed by sucrose gradient centrifugation to obtain each nematode present in the supernatant and M9 buffer solution. Washed with.

Example 2 Preparation of Transformant

Cloning a human 7-DHCR gene labeled with 1.5 Kb of myc gene into pPD49.78 (Clontech, USA), a vector having a heat shock protein promoter, to produce an expression vector, and as a marker gene, a mutant mutant of a nematode (roll) ) Was mixed with the expression vector pRF4 to give a gene mixture to microinject the nematodes (see Figure 1). 1 is a genetic map showing a cloned vector. As shown in FIG. 1, the cloned vector has a human 7-DHCR gene located behind a heat shock protein promoter and a myc gene, and includes an ampicillin resistance gene as a selection marker.

The injection pad for microinjection is dipped in a 2% (w / v) agarose solution into a 50 x 75 mm cover glass, covered with the same size glass and hardened, and then the cover glass is removed and 1 at 65 ° C. It was left for hours and then stored at room temperature. The needle for micro-injection was produced by processing a capillary tube (Narishigi Co., Japan), and the mouse pipette was prepared by heating the middle portion of the capillary tube (Sigma Chem. Co., U.S.A.) with alcohol and then stretching it.

Insert the gene mixture to be injected into the nematode into the needle for microinjection under an anatomical microscope, mount it on the needle mounting site of the microinjector (Transjector 5246, Eppendorf, German), and then drop halocarbon oil on the injection pad. On top of that was placed a normal adult nematode N2. The electric injection pad was placed on the stage of the microinjector, placed under the Axiovert135 microscope so that the nematode's gonad was on the opposite side of the needle, and the clean button of the microinjector was pressed to inject the gene mixture into the gonad. The adult mixture injected with the gene mixture was transferred to the dissecting microscope, M9 buffer solution was added dropwise onto the injection pad, and the nematodes were removed from the bottom, and then inoculated into NGM solid medium containing E. coli OP50, and incubated at 20 ° C. The first generation of offspring showing roller phenomenon were selected. One generation of selected progeny was re-cultured under the same conditions to select two generations of progeny that exhibited eddy phenomena, named 'Caenorhabditis elegans', and as of April 16, 2001, the International Bank of Biotechnology Research Institute Gene Bank (KCTC). Deposited as 'KCTC 0996BP'.

Example 3 Lipid Extraction of Nematodes

Normal nematodes N2 and transformants were incubated in S liquid medium by the method of Example 1, purely separated, and placed in a homogenizer with M9 buffer solution, respectively, and homogenized to break the cuticle layer. Subsequently, 25% (w / v) potassium hydroxide solution dissolved in methanol was added to the same volume, heated at 80 ° C. for 10 minutes, saponified, and then an organic solvent was extracted with 8 times the volume of ether, followed by an 80 ° C. thermostat. After evaporating the ether while flowing nitrogen gas at, the solid component was dissolved in chloroform and analyzed using gas chromatography (GC 5890 II, Hewlett-Packard, USA) using 5-alpha cholesterone as standard. (See FIG. 2A, FIG. 2B). FIG. 2A is a chromatogram showing the composition of a sterol compound of N2, which is a normal nematode, and FIG. 2B is a chromatogram showing the composition of a sterol compound of a transformant. As shown in Figures 2A and 2B, normal nematodes N2 contain large amounts of 7-dihydrocholesterol and no cholesterol, but the transformants contain cholesterol and no 7-dihydrocholesterol Could know.

Example 4 Life Measurement

A 10-fold volume of aqueous hypochlorite solution (1% (w / v) sodium phosphate, 0.5 N sodium hydroxide) was mixed with each of the normal nematodes N2, the transformant, and the elastella of the longevity strain age-1, and stirred vigorously. Next, the supernatant was removed by centrifugation at 4,000 g for 1 minute, and the precipitate was washed with water and M9 buffer to obtain eggs of the nematodes. The eggs were incubated in NGM solid medium for 1 day to obtain 200 larvae of each hatched L1 stage, and 40 μM 5-fluoro-2-dioxyuridine was added to inhibit adult reproduction. Thereafter, 100 L1 larvae were inoculated into each of the NGM solid medium containing E. coli OP50 and cholesterol and the NGM solid medium containing only E. coli OP50, and cultured at 20 ° C., and the lifespan was measured (see FIG. 3). 3 is a graph showing the life expectancy of each nematode. As shown in Figure 3, the lifespan of the transformant was found to increase than the life of the normal nematode N2, even when cholesterol was not added or ephedra. However, compared to the lifespan of longevity age-1, the life span of the transformants was shorter than that of longevity age-1 when cholesterol was supplied, but the lifespan of longevity age-1 when cholesterol was not supplied. As it was found to increase, it was suggested that cholesterol in the body is related to life extension.

Example 5 Crossbreeding Activity of Male Body

In general, a nematode exists as a hermaphrodite, but may also exist in the form of a male body. The ratio of the electric body is about 5%, and if the temperature is increased at the time of making eggs, it occurs more. When the body and hermaphrodite are crossed, it is known that the body develops in the first generation of offspring.

Each L1 larvae of the normal nymph N2 and the transformant were cultured by the method of Example 4 to obtain L4 larvae, and females were selected from them. Subsequently, six L4 females and one L4 hermaphrodite of each nematode were inoculated into NGM medium, and after 24 hours at 20 ° C, the females were removed. The mating activity of the first generation of offspring was examined (see Fig. 4). 4 is a graph comparing the number of female bodies of the first generation of offspring. As shown in Figure 4, the number of females of the transformant increased by about 20% than the number of females of the normal nematode N2, it could be inferred that the breeding efficiency of the transformant is high.

Example 6 Thermal Stability Measurement

Normal nematodes N2 and transformants were cultured by the method of Example 1, and 40 animals were obtained. Each of them was inoculated in NGM solid medium maintained at 35 ° C. and incubated at 35 ° C., measuring the time until pumping of the pharynx stopped, and using 50% (TS ₅₀ ) thereof, the heat of each nematode Stability was compared (see FIG. 5). 5 is a graph comparing TS ₅₀ of each nematode. As shown in Figure 5, it was found that the thermal stability of the transformant is increased by about 18% than the thermal stability of the normal nematode N2.

Example 7 : UV resistance measurement

Thirty five-day adult hermaphrodite adults of normal nematodes N2, transformants, and longevity age-1 were inoculated into NGM medium and 20 J / m ² using an UV irradiator (UV Stratalinker, Stratagene Co., USA). After irradiating ultraviolet rays with a dose of / min, the life was measured while incubating at 20 ℃ (see Figure 6). Figure 6 is a graph showing the lifespan after irradiation of each nematode, (●) represents a normal nematode N2, (▼) represents a transformant, (○) represents longevity variance age-1. As shown in Figure 6, the resistance to ultraviolet light is almost similar to the three nymphs, but the UV resistance of the transformant was found to be more similar to the UV resistance of the longevity age-1 than the normal nymph N2.

As described and demonstrated in detail above, the present invention provides a transformed nematode that synthesizes cholesterol developed in the body as an experimental animal for studying the metabolism of cholesterol. The transformed nematode of the present invention is a transformant in which the human 7-dihydrocholesterol reductase gene is introduced into the nematode, and may be widely used as an experimental animal to study the effects of cholesterol in vivo.

Claims (1)

Human 7-dihydrocholesterol reductase (7-DHCR) gene is introduced into a vector having a promoter of heat shock protein to cause cloning of the transformed vector and the nematode mutant (roll) Vector pRF4 is simultaneously introduced into normal worms to transform cholesterol in the body, Caenorhabditis elegans (KCTC 0996BP).