KR100442997B1 - Hypoxia-responsive promoter, method for the mass expression of a desired gene by using the same and method for selectively inducing rna interference to a desired gene thereby - Google Patents

Abstract

The present invention provides a method for expressing a large amount of a gene of interest using a hypoxia-responsive promoter described in SEQ ID NO: 2 , an expression vector linked to a gene of interest after the promoter, and introduced into the organism by the method. A method for selectively inducing RNA interference in a gene of interest, wherein the hypoxic-reactive promoter of the present invention promotes the transcription of the gene of interest under hypoxic conditions instead of heat shock, thereby inducing a secondary defect in the entire subject without causing collateral defects. By selectively inducing RNA interference in vivo, it can be useful not only for analyzing gene function but also for mass production of useful proteins.

Description

HYPOXIA-RESPONSIVE PROMOTER, METHOD FOR THE MASS EXPRESSION OF A DESIRED GENE BY USING THE SAME AND METHOD FOR SELECTIVELY INDUCING RNA INTERFERENCE TO A DESIRED GENE THEREBY}

The present invention provides a method for expressing a large amount of a gene of interest using a hypoxia-responsive promoter described in SEQ ID NO: 2 , an expression vector linked to a gene of interest after the promoter, and introduced into the organism by the method. A method of selectively inducing RNA interference in a gene of interest.

The process of obtaining mutants of specific genes through random mutagenesis to analyze the function of genes is necessary for the ultimate genetic study, but it takes considerable time and effort. Recently, methods have been developed to obtain phenotypes of gene expression suppression transiently but easily by RNA interference (RNA interference, RNAi) (Fire et al., Nature , 391: 806-811, 1998). RNAi experiments synthesize antisense RNA and sense RNA in vitro, microinject the mixture into nematodes, and then observe the phenotype of their genes under the inhibition of expression of specific genes. Expression of the gene is inhibited in adult progeny that have microinjected double-stranded RNA for the gene, and the resulting phenotype appears without altering the gene sequence.

RNAi is the first observed phenomenon in nematodes where double-stranded RNA introduced into cells hybridizes to endogenous mRNA, resulting in potent and specific inhibition of the corresponding gene, a gene specific loss of function. Not only does it quickly induce, but it also affects the offspring of animals that have had RNA interference. Thus, RNAi is currently the simplest and most effective way to inhibit genes in nematodes, resulting in selective and persistent interference effects. As a result, RNAi in gene expression using double-stranded RNA is a reverse-genetic study to characterize specific genes. Its importance is emerging as a tool for reverse genetic study.

The double stranded RNA produced by binding the complementary strands of RNA specific to RNAi has a very strong and selective interference with protein expression. The injection of double-stranded RNA was originally made in the gonads of adults, but it has been reported that double-stranded RNA injected in all regions other than the gonads can also induce mutant phenotypes in adults. Moreover, mutant phenotypes can be produced by feeding a nematode engineered to produce double stranded RNA or soaking the nematode in a double stranded RNA solution. Surprisingly, this phenomenon has the advantage of being transmitted to the offspring of adult nematodes, enabling the study of phenotypes throughout all stages of development.

The mechanism by which double-stranded RNA inhibits the expression of the gene of interest is not yet fully known. To date, studies show that the gene of interest does not appear to have been mutated by RNA interference, and transcription of the gene is not interrupted in the presence of double-stranded RNA, but mRNA produced by the gene of interest is not present in the cytoplasm. And decrease in the nucleus. These results indicate that double stranded RNA exerts an interfering effect during or following RNA processing, but not protein translation transfer.

Recently, RNAi for gene expression has been described as nematodes ( Caenorhabditis elegans ) (Fire, A., Xu, S., Montgomery, MK, Kostas, SA, Driver, SE & Mello, CC, Nature (London) 391, 806811, 1998 Montgomery, MK, Xu, S. & Fire, A., Proc. Natl. Acad. Sci. USA 95, 1550215507, 1998; Shi, Y. & Mello, C., Genes Dev. , 12, 943955, 1998; Plants (Voinnet), including Tabara, H., Grishok, A. & Mello, CC, Science 282, 430431, 1998; Timmons, L. & Fire, A., Nature (London) 395, 854 (lett.), 1998) , O., Vain, P., Angell, S. & Baulcombe, DC, Cell , 95, 177187, 1998; Waterhouse, PM, Graham, MW & Wang, M.-B., Proc. Natl. Acad. Sci. USA 95, 1395913964, 1998), fruit fly (Drosophila) (Kennerdell, JR & Carthew, RW, Cell, 95, 10171026, 1998;.... Misquitta, L. & Paterson, BM, Proc Natl Acad Sci USA 96, 14511456 , 1998), Triponizoma brucei (Ngo, H., Tschudi, C., Guli, K. & Ullu, E., Proc. Natl. Acad. Sci. USA 95, 1468714692, 1998) and Planarian (Alvarado, AS & Ne) wmark, PA, Proc. Natl. Acad. Sci. USA 96, 50495054, 1999).

As described above, the RNAi method, which has been widely used recently to clarify the function of a gene, has a strong advantage that it can be easily performed and a function can be inferred in a short time. have. Therefore, studies on methods to overcome these disadvantages have been actively conducted, and a method of using a heat shock promoter has been developed as a method for performing RNAi at a specific time point (Nektaris T. et al., Nature genetics , 24: 180-183, 2000). However, the method has a drawback of causing a secondary defect due to heat shock to the entire subject, the need for the development of a new in vivo RNAi method is increasing.

Therefore, the present inventors have made efforts to develop a genetic tool capable of analyzing gene function by selectively inducing RNA interference in vivo for a desired gene. As a result, the hsp16A gene, known as a heat shock protein gene, has only heat shock. In addition, it is found that the expression is rapidly increased even under hypoxia conditions, and a method for expressing a large amount of the gene of interest using the hypoxic-reactive promoter by analyzing the hypoxic-reactive promoter site controlling the same, and using the method The present invention has been completed by developing a method for selectively inducing RNA interference in a gene.

It is an object of the present invention to provide a hypoxic-reactive promoter capable of increasing the expression of genes in hypoxic conditions.

It is also an object of the present invention to provide a method for expressing a gene of interest in large quantities using the promoter.

In addition, it is an object of the present invention to provide a method for inducing RNA interference in vivo to a desired gene without causing an additional defect in the entire subject such as heat shock using the promoter.

1 shows the results of microarray analysis for finding genes whose transcription expression is changed in alcohol-exposed nematodes.

Figure 2 shows that the transcriptional regulation of the hsp16A gene of the present invention is regulated not only by heat shock but also by hypoxic shock,

Figure 3 shows a schematic diagram of various promoter analogs designed to determine the hypoxia-responsive element of a promoter involved in hypoxic-induced transcriptional regulation of the hsp16A gene in the present invention,

4 is a result confirming that the transcriptional regulation of other hsp16 genes in addition to the hsp16A gene of the present invention is regulated by hypoxic shock,

FIG. 5 shows the sense and antisense constructs of the hsp16A promoter fusion apm-1 constructed to verify RNAi assays in vivo using the hypoxic-reactive hsp16A promoter of the present invention.

Figure 6 shows the phenotype exhibited by RNAi in vivo of the apm-1 gene using the hypoxic-reactive hsp16A promoter of the present invention,

7A and 7B show possible schematics of promoter fusion constructs that can be used for in vivo RNAi assays using the low oxygen-reactive hsp16A promoter of the present invention.

In order to achieve the above object, the present invention provides a hypoxic-reactive promoter comprising a nucleotide sequence set forth in SEQ ID NO: 2 in which expression is induced by hypoxic shock.

The present invention also provides a method of expressing a large amount of a gene of interest using a hypoxic-reactive promoter of the hsp16A gene and a method of selectively inducing RNA interference to a gene of interest in vivo.

Hereinafter, the present invention will be described in detail.

The hypoxic-reactive promoter of the present invention comprises a -260 to -241 nucleotide region of the hsp16A gene promoter of a nematode having a cDNA nucleotide sequence of SEQ ID NO: 1 and has a nucleotide sequence of SEQ ID NO: 2 .

The present inventors found a gene whose expression is rapidly increased in hypoxia conditions and regulates its transcription in the process of finding genes whose transcription expression is changed in alcohol-exposed nematodes by microarray analysis. Reactive promoter sites were analyzed.

Microarray analysis of genes that change transcriptional expression in alcohol-exposed nematodes revealed that transcription was increased in the earliest time of exposure to ethanol and was not expressed in the control group. A significantly increased open reading frame (ORF) was discovered (see FIG. 1 ). The gene began to increase its transcription within 15 minutes and the extent of the increase continued after 6 hours of exposure. As a result of sequencing analysis, the gene has the nucleotide sequence set forth in SEQ ID NO: 1 and has the same nucleotide sequence as the previously reported hsp16A gene as one of heat shock proteins induced by heat shock. have.

The nematode was cultivated with or without alcohol in a minimal liquid medium to block normal oxygen supply, which in turn caused hypoxic shock to the nematode, resulting in a very rapid rate of the hsp16A gene, regardless of the presence of alcohol in the hypoxic state. It was confirmed that transcription was induced.

Accordingly, the present inventors attempted to compare the transcription induction of heat shock by measuring the responsiveness to the hypoxic condition of the heat shock protein gene hsp16A. First, a plasmid obtained by translating the GFP (Green fluorescent protein) at the back of the entire coding region including the entire regulatory element (340 bp) of hsp16A was prepared and the DNA extracted therefrom was nematode. Microinjection was made to transgenic animals. After the hypoxic condition or heat shock was applied to the transgenic animals, the amount of GFP detected was measured to determine the degree of transcription of hsp16A induced by the shock.

As a result, GFP, which is rarely expressed in the control group, was strongly expressed in the hypoxic condition or the experimental group subjected to the heat shock (see FIG. 2 ), which indicates that the transcriptional regulation of the hsp16A gene, previously known as the heat shock protein, is not only induced by heat shock but also by hypoxia. It was the first time that it was controlled by impact.

Existing studies on hsp16A have been conducted on its transcriptional regulation centered on the response to heat shock, and it has been reported that there is a heat shock responsive element (Stringham, EG, et. al., Molecular Biology of the Cell , 3: 221-233, 1992; Kay, RJ, et al., Molecular and cellular Biology, 6: 3134-3143, 1986). However, it was confirmed from the results of the present invention that the hsp16A gene is induced not only by heat shock but also by hypoxic shock, and to identify a hypoxia repsonse regulatory region distinguished from the heat shock reactive element, hsp16A-GFP Promoter analogs were prepared to perform promoter analysis.

The hsp16A-GFP promoter analogs prepared for promoter analysis in the present invention were prepared as follows with a GFP fusion gene construct comprising a full-length promoter or promoter deletion at various sites.

JS301; Includes all 340 bp full length promoters.

JS304; Using the XbaI restriction enzyme site, remove the middle region of the promoter, 150 bp, and include a 190 bp promoter that fused half of the far heat shock response element and half of the near heat shock response element.

JS306; A 260 bp long promoter with 80 bp removed from the front end of the promoter, containing both heat shock response elements.

JS307; 85 bp long promoter with 255 bp removed from the front of the promoter, containing only half of the near heat shock response element.

JS308; A 240 bp long promoter with 100 bp removed from the front end of the promoter, with only half of the far-end heat shock response elements and all of the near-end heat shock response elements.

JS312; Removes the front 120 bp of the promoter and removes the far heat shock response element with a 220 bp promoter and includes only the near heat shock response element.

JS314; JS313, which includes only the basic promoter TATA as a 75 bp long promoter by removing the front 265 bp of the promoter; And a 325 bp long promoter with 15 bp removed including the heat shock response element on the near side of the promoter.

As a result of investigating how the transcription of GFP responds to hypoxic conditions in the various promoter analogs prepared as described above, the heat shock-responsive element for the transcription of the hsp16A gene to be controlled by heat shock is the proximal element or the terminal region. while the hypoxia-responsive element, which is present in the distal element, is most important for transcription control by hypoxic conditions, is between -260 and -241 nucleotides at the hsp16A gene promoter site set forth in SEQ ID NO: 1 . Present (see Table 1 and Table 2 ). The -241 to -241 nucleotide region has a nucleotide sequence set forth in SEQ ID NO: 2 . From this, it can be seen that the mechanism by which the transcription of the hsp16A gene is induced by hypoxic shock is due to the hypoxic-reactive promoter site existing independently of the transcriptional regulation by heat shock.

The present invention also provides a method of selectively inducing RNA interference to a gene of interest using the hypoxic-reactive promoter of the hsp16A gene.

The inventors of the present invention confirmed that transcription of hsp16A is regulated in response to hypoxic conditions in addition to heat shock. As a method of selectively inducing RNA interference to a gene of interest, an experiment in which RNAi can be easily performed in vivo using the hsp16A promoter The method was developed.

Existing in vivo RNAi produced a transgenic organism that introduced two expression vectors simultaneously by connecting desired genes to both hsp16.2 or hsp16.41 promoters in both directions (sense and antisense), and then heat shocked these individuals. The method has been used to make double stranded RNA in vivo by addition. However, the method has a problem in that long-term treatment can be difficult as well as secondary results due to heat shock by applying a strong stimulus called heat shock to the entire subject. On the other hand, if the transcription induction mechanism by the hypoxic impact of the present invention is used, it is possible to produce a strong RNA interference effect while solving these problems.

In order to confirm that the hypoxic-reactive promoter of hsp16A of the present invention can be used for the development of in vivo RNAi test methods, the intermediate chain gene of the nematode retinal-associated complex-1 at the back of the full-length promoter of hsp16A (medium chain gene) apm-1 was produced in the gene construct connected in the sense or antisense direction. After injecting DNA extracted from the sense and antisense constructs of the apm-1 gene into nematodes to prepare transgenic animals, the degree of inhibition of apm-1 gene function was observed in the transgenic animals under hypoxic conditions.

As a result, the function of the apm-1 gene was inhibited and lethality phenotype was observed in embryonic and larval stages, and the frequency was similar or slightly higher than that of the heat shock experiment (see Table 3 ). (See FIG. 6 ).

From the above results, the in vivo RNAi method using the hypoxic-reactive promoter of the present invention selectively interferes with in vivo RNA to a gene of interest without causing a defect in the entire subject as compared to the in vivo RNAi method using a heat shock. It can be used to analyze the function of genes by inducing interference. The method including the nematode (Caenorhabditis elegans) plants, fruit flies (Drosophila), Fano tree dressage Brewer can be applied to organisms, such as sage (Tripanozoma brucei), planarians (planarian) or a mammalian cell line.

The in vivo RNAi method using the hypoxic-reactive promoter of the present invention can be embodied in two ways as follows.

The first method comprises the steps of: 1) preparing an expression vector in which a target gene in the sense direction is connected to the hsp16A hypoxic-reactive promoter and an expression vector in which the target gene in the antisense direction is connected to the hsp16A hypoxic-reactive promoter; 2) preparing a transformed organism by simultaneously injecting an expression vector having a target gene in the sense direction to the hsp16A hypoxic-reactive promoter and an expression vector having a target gene in the antisense direction linked to the hsp16A hypoxic-reactive promoter; And 3) inducing expression by applying hypoxic shock to the transgenic organism (see FIG. 7A).

In this step, the hypoxic shock may be applied by immersing the appropriate amount of water in the medium for culturing the transgenic organisms to submerge the subjects or submerging the subjects and submersing them in buffer or water without shaking on the tube. As a result of the hypoxic shock, transcription is promoted from the hsp16A hypoxic-reactive promoter, resulting in RNA of the sense or antisense strand of the desired gene from each expression vector, and they form a double strand with each other to interfere with RNA. .

The second method comprises the steps of: 1) preparing an expression detector in which the gene of interest is simultaneously linked to the hsp16A hypoxic-reactive promoter in the sense and antisense directions; 2) preparing a transformed organism by micro-injecting the expression vector into the individual; And 3) inducing expression by applying hypoxic shock to the transgenic organism (see b of FIG. 7 ).

In the expression vector of the second method, the sense strand and the antisense strand are simultaneously transcribed in one RNA molecule so that they form a double strand, thereby interfering with RNA, since the sense and antisense strands cannot be physically dropped compared to the first method. RNA interference with the gene of interest is more effective.

On the other hand, the hypoxic-reactive promoter of the present invention can be used for mass production of useful proteins since it promotes transcription of the gene of interest under hypoxic conditions.

The method for producing a large amount of useful protein under hypoxic conditions using the promoter comprises the steps of 1) preparing an expression vector linked to a gene of interest in the hypoxic-reactive promoter of claim 1; 2) preparing a transformed organism by micro-injecting the expression vector into the organism; And 3) subjecting the transgenic organism to hypoxic shock to induce the expression of the target gene linked to the hypoxic-reactive promoter.

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 Discovery of Genes Inducing Transcription Under Hypoxic Conditions

The present inventors found a promoter in which the expression is rapidly increased in hypoxia conditions in the process of finding genes whose transcription expression is changed in nematodes exposed to alcohol (ethanol, 7%) by microarray analysis. First, a microarray assay for selecting genes whose transcription expression is changed in alcohol-exposed nematodes was performed as follows.

<1-1> Microarray Assay

Nematodes were cultured on 80 sheets of 100 mm plates, harvested with S basalbuffer, and then divided in half and ethanol with buffer itself (200 mL, control) and final 7% (v / v). The added S buffer solution (200 mL, experimental group) was mixed and liquid cultured at 20 ° C. and 250 rpm in a 1 L flask. The experimental group exposed to ethanol was used for 15 different experiments in different time periods: 2 times for 15 minutes, 2 times for 30 minutes, 2 times for 1 hour, 2 times for 2 hours, 2 times for 4 hours or 6 hours. 5 times exposure to ethanol. At this time, the inlet of the flask was sealed with parafilm. After harvesting the nematodes by centrifuging each of the culture broths, trizol reagents (GIBCO BRL, 15596-018) were added to a final volume of 40 ml. The cells were repeatedly frozen and thawed at least 7 times. Crushed. After centrifugation again, only the supernatant containing RNA was taken, and isopropanol was added thereto in the same amount as the supernatant and centrifuged to precipitate RNA. The precipitate was washed with ethanol and dissolved in 4 ml of 10 mM Tris-Cl buffer (pH 7.4) to obtain total RNA of the experimental and control nematodes.

In order to purely separate mRNA from the total RNA, oligo (dT) -Cellulose Type 7 (Amersham Pharmacia Biotech, 27-5543-02) resin was used. The total RNA was mixed with the resin to bind the mRNA to the resin. And washed with 1 * NETS (100 mM NaCl, 10 mM Tris-Cl [pH 7.4], 10 mM EDTA, 0.2% SDS) using a 10 ml Poly-Prep Column (biorad, 731-1550) and 10 mM Eluted with Tris-Cl (pH 7.4 heated to 70 ° C). Isopropanol and NaOAC were added thereto, mixed well, and then centrifuged. The precipitate was dissolved in 10 mM Tris-Cl (pH 7.4) to obtain mRNAs of the experimental and control groups.

In order to perform microarray analysis with mRNA isolated from the experimental group and the control group, each mRNA was labeled with a different fluorescence, and then the microarray was photographed with about 20,000 gene fragments expected to have nematodes. Hybridization (Stanford university, Stuart K. Kim). At this time, the experimental group mRNA was reverse-transcribed to be labeled with Cye3-dUTP, the control mRNA was labeled with Cye5-dUTP, and the image was made by converting Cye3 into green and Cye5 into red during the scanning process to make the two images overlap. Genes with high expression in the experimental group show strong green spots on the image.

As a result, the present inventors are one of the genes whose transcription is increased in the fastest time when exposed to ethanol, but not expressed in the control group, but in the experimental group to which ethanol is added, the open reading frame (ORF) is markedly increased. ) And was named T27E4.2 ( FIG. 1 ). In Figure 1 , the green spots indicate that transcription was increased, and the gray spots indicate no data. The gene began to increase its transcription within 15 minutes and the extent of the increase continued after 6 hours of exposure. As a result of sequencing analysis, the gene has a nucleotide sequence set forth in SEQ ID NO: 1 , and the same base as hsp16A, the gene of which the gene has been previously reported through homology with other genes in a database. It was confirmed that the sequence has. The hsp16A gene is one of heat shock proteins induced by heat shock, and much research has already been made on the transcriptional regulation mechanism of heat shock.

<1-2> Transcription Induction by Hypoxic Conditions

The present inventors found that the gene is promoted not only by alcohol but also by hypoxic conditions in the process of investigating the sensitivity of hsp16A to alcohol, which is increased by alcohol exposure from nematodes identified in Example <1-1>. Was identified. Specifically, in order to investigate the response of the gene to alcohol, nematodes were treated with or without adding alcohol to the minimal medium of the liquid. At this time, if the culture in a shaking state of about 250 rpm is a normal oxygen supply, but if cultured without shaking the oxygen supply is not made normally is a low oxygen state. In the hypoxic state as described above, the hsp16A gene of the present invention was confirmed that transcription induction occurs at a very high speed regardless of the presence of alcohol.

Example 2 Reactivity Test for Hypoxia and Thermal Shock of hsp16A

The inventors of the present invention confirmed that transcription of the heat shock protein gene, hsp16A, was promoted in a hypoxic state, and performed the following experiment to compare the transcription induction of the thermal shock by measuring the reactivity to hypoxic conditions.

<2-1> Construction of hsp16A-GFP Fusion Plasmid and Transgenic Animals

First, plasmid JS301 was prepared by fusion of green fluorescent protein (GFP) at the back of the entire coding region, including the entire regulatory element (340 bp) of hsp16A. After culturing E. coli with the plasmid JS301, DNA was extracted purely using DNA Midi prep kit (Qiagen), followed by rol-6 marker DNA ( Mol-6 marker DNA, Molecular and Cellular Biology, 10: 2081-2089). , 1990; Transgenic Research, 4: 323-340, 1995). The mixture was microinjected into the germline of hermaphrodite which reached the young adult stage using a microinjection system including Karl Zeiss' Axiovert microscope and a transgenic animal carrying a labeled DNA. animal). The transgenic animal is easily distinguished from the wild type because it contains a rol-6 label and shows a phenotype of a roller moving in a circle instead of going back and forth on an anatomical microscope. Thus, the amount of GFP detected from the transgenic animals subjected to hypoxic conditions or heat shock will indicate the extent of transcription of hsp16A induced by the shock.

<2-2> Reactivity test for low oxygen and heat shock

In order to give hypoxic shock to the transgenic animals, nematodes fully grown in plates with M9 buffer were harvested and placed in 1.5 ml eppendorf tubes, which were then incubated without shaking in a 20 ° C. incubator. On the other hand, in order to apply a heat shock, a plate in which nematodes were fully grown was placed in a 30 ° C. incubator and cultured. While culturing these for 3 hours, the level of GFP expression was measured at each time period. The expression level of GFP was determined to be 1300 x 1030 pixel at 630-fold magnification by placing the impacted and non-impacted transgenic nematodes on slide glass with a digital camera (Axiocam) on a Zeiss fluorescence microscope (Axioplan). The resolution was taken at a constant exposure of 3000, and the difference between the intensity of GFP and the site of nematodes was observed.

As a result, as shown in FIG . 2 , GFP, which is hardly expressed in the control group, can be seen to be strongly expressed in the experimental group to which hypoxic conditions or heat shock were applied. These results indicate that the transcription of the hsp16A gene, known as heat shock protein, is regulated not only by heat shock but also by hypoxic shock.

Example 3 Promoter Analysis of hsp16A

Previously, the promoters of the hsp16A gene were analyzed based on the heat shock induced. As confirmed in Example 2, the hsp16A gene is regulated not only by heat shock but also by hypoxic conditions, so that the necessary promoter is required. Need to reestablish. Therefore, the inventors prepared hsp16A-GFP promoter analogs including deletions of full-length promoter sites or various sites as follows to investigate the degree of transcription induction for their hypoxic conditions.

<3-1> Construction of promoter analogs of hsp16A

The construction of the poromotor analogs of the hsp16A gene preferentially targets the T27E4.2-2 (+490 site recognition) and HindIII restriction enzyme recognition sites as set forth in SEQ ID NO: 3 , which contains the nematode genomic DNA as a template and includes the BamHI restriction enzyme recognition site. Polymerase Chain Reaction (PCR) using a T27E4.2-3 (-340 site recognition) labeled primer pair as set forth in SEQ ID NO: 4 , comprising nucleotides from -340 to +490 830 bp long DNA fragments were amplified. Where +490 denotes the last A of TAA, the last stop codon of the protein coding region, when A of ATG, which is a translation start site, is +1, GFP fusion plasmids were prepared from the entire region including the promoter region and the coding region of the gene. This is because the GFP fusion protein produced from the fusion plasmid is the same as the actual expression of endogenous hsp16A because nematodes do not contain a coding region in the case of nematodes, and thus the GFP fusion protein produced is restricted in its expression site. To show the aspect.

The DNA fragment was inserted into the HindIII / BamHI restriction site of pPD95.77 (A. Fire, Carnegie Institute, Baltimore, USA) vector to prepare a GFP fusion construct JS301 comprising a full-length promoter. Self-ligation of the DNA fragment obtained by cleaving JS301 with XbaI using a restriction enzyme site (XbaI) formed in the fusion construct JS301 to produce a gene construct smaller than JS301, JS304 Was prepared. In addition, JS306 in which the amplified DNA fragment was inserted into the PstI / BamHI site of the vector pPD95.77, the DNA fragment was partially digested with XbaI, followed by electrophoresis, and then a relatively short fragment in an agarose gel. And long fragments were eluted from the gel and inserted into the XbaI / BamHI site of vector pPD95.77, respectively JS307 (575 bp; coding region of 490 bp and promoter region of 85 bp) and JS308 (730 bp; 490 bp) Coding region and a promoter region of 240 bp). In addition, new primers were designed and PCR were carried out in the same manner as above, and the amplified DNA fragment was cloned into the pPD95.77 vector to construct gene constructs. T27E4.2-6 described in SEQ ID NO: 6 and SEQ ID NO: 3 JS312, SEQ ID NO: 8 and SEQ ID NO: 710 bp long DNA fragments amplified by PCR using (-220 site recognition) /T27E4.2-2 primer pair inserted into the HindIII / BamHI restriction site of pPD95.77 vector A 565 bp long DNA fragment amplified by PCR using T27E4.2-8 (-75 site recognition) /T27E4.2-2 primer pair described as 3 is inserted into the SalI / BamHI restriction site of the pPD95.77 vector. A 250 bp long DNA fragment amplified by PCR using a pair of T313E4.2-3 / T27E4.2-7 (-90 site recognition) primers set forth in JS313, SEQ ID NO: 4 and SEQ ID NO: 7 was identified as pPD95.77 vector. JS314 was inserted into the HindIII / SalI restriction enzyme site of ( FIG. 3 ).

In the present invention, the cloning vector pPD95.77 used for the fusion (translational fusion) of GFP to hsp16A is one of the Fire vector 95 series having frame = 2, and multiple cloning is arranged in the order of HindIII, SphI, PstI, SalI, XbaI, and BamHI. After the multi-cloning site, the GFP gene is included, and the DNA of interest is inserted into the ORF (open reading frame) to express the GFP-fused protein in nematodes.

<3-2> Reactivity of Low Oxygen and Heat Shock

In order to confirm how transcription of GFP reacts to hypoxic conditions in the various promoter analogs prepared as described above, hypoxic conditions were added in the same manner as in Example 2 to investigate the reactivity of each fusion gene construct. Table 1 and Table 2 are shown.

Results of promoter analysis to identify hypoxia-responsive regions Construct HN AI MI PI P BM H JS301 +++ +++ +++ +++ +++ +++ +++ JS304 + ++ + ++ - - - JS306 +++ +++ +++ +++ +++ +++ +++ JS307 + ++ - ++ - - - JS308 ++ +++ +++ +++ ++ + + JS312 + +++ +++ +++ + - - JS313 - - - - - - - JS314 ++ +++ +++ +++ ++ + + HN; head neuron, AI; antirior intestine (intestinal of the anterior region), MI; mid intestine (intestinal tract), PI; posterior intestine (intestinal of the posterior region), P; pharynx (pharyngeal), BM; body wall muscle, H; hypodermis

In Table 1 , +++ indicates the degree to which JS301 is expressed at the sites of HN, AI, MI, PI, P, BM, H, + indicates the degree of weakest expression, and ++ is expressed at moderate intensity. do. -Indicates that JS301 was not expressed at all. Therefore, JS313 with only the basic promoter was not expressed in any of HN, AI, MI, PI, P, BM, H under hypoxic conditions, and JS301 and JS306 were not expressed in HN, AI, MI, PI, P, BM, H It was strongly expressed at all sites of.

Comparison of Hypoxic Shock Response and Heat Shock Response Construct Hypoxic shock Thermal shock JS301 +++ +++ JS304 - - JS306 +++ +++ JS307 - - JS308 + +++ JS312 + +++ JS313 - - JS314 +++ +++

As shown in Table 1 and Table 2 , the heat shock-responsive element for the hsp16A gene to be regulated by heat shock is sufficient only by the proximal element or the distal element, whereas the transcription by hypoxic conditions is sufficient. It can be seen that the most important hypoxia-responsive element is between -260 and -240 nucleotides in order to be regulated. The hypoxic-reactive element acts independently of the heat shock-reactive element and consists of the nucleotide sequence set forth in SEQ ID NO: 2 .

<3-3> Reactivity of Hypoxic Conditions in hsp16 Gene Family

In addition to hsp16A, nematodes also contain other hsp16 genes. These hsp16 genes also examined whether their transcription was regulated in response to hypoxic conditions. Among the hsp16 genes, hsp16.2 and hsp16.41, which have the most similar amino acid sequence to hsp16A and amino acid sequence, were examined to determine whether these two genes were induced by hypoxic conditions. Low oxygen shock was applied in the same manner as in Example <2-2>.

As a result, these hsp16 genes hardly induced expression for hypoxic conditions ( FIG. 4 ). In Figure 4 , A and B are the expression patterns of JS306 of hsp16A, C and D are the expression patterns of hsp16.2, E and F are hsp16.41. On the left panel, transcription is induced by thermal shock, and on the right panel, transcription is induced by hypoxic conditions. As shown in FIG. 4 , all three genes were promoted by heat shock, but only hsp16A gene was promoted by hypoxic conditions. Through these results, it was confirmed that the hsp16A promoter found by the present inventors is the only promoter of the hsp16 gene family that responds to hypoxic conditions.

Example 4 Design of an RNAi Experiment in Vivo Using the hsp16A Promoter

In Example 3, the present inventors confirmed that the promoter of hsp16A is regulated in response to hypoxic conditions in addition to heat shock, while the low oxygen-reactive promoter of hsp16A solves the problems raised in the RNAi method using heat shock. In order to confirm that it can be used in the development of in vivo RNAi experimental method to perform the following experiment using the apm-1 gene.

<4-1> Construction of apm-1 Gene Construct Including hsp16A Promoter

The apm-1 gene is one of the medium chain genes of the nematode clathrin-associated complex-1. First, in order to prepare a gene construct for RNAi of apm-1, PGEM of a 340 bp long DNA fragment obtained by PCR using a primer pair of SEQ ID NO: 4 and SEQ ID NO: 4 and T27E4.2-3 / T27E4.2-4 (+3 site recognition) of SEQ ID NO. Plasmid JS302 was prepared by inserting into a -T easy vector (Promega). Plasmid JS305 was prepared by subcloning the 340 bp long DNA fragment obtained by treatment of the plasmid JS302 with restriction enzyme HindIII / EcoRI into a pBluscript KS + vector treated with the same restriction enzyme. In order to produce therefrom the apm-1 sense constructs (sense construct) of the gene, first, the apm-1 cDNA (Shim et al ., Molecular Biology of the Cell, 2000, 11, 2743-2756) as a template and SEQ ID NO: PCR with primer pairs of site recognition including TTA, the termination codon of CE16 APM-1 cDNA having the nucleotide sequences set forth in SEQ ID NO : 9 and SEQ ID NO: 10 / CE17 (site recognition including ATG, the start codon of APM-1 cDNA) The 1240 bp long DNA fragment was treated with restriction enzyme NdeI / BamHI and subcloned into plasmid JS305 treated with the same restriction enzyme to construct a sense construct MC12. In addition, the anti-sense construct of the apm-1 gene contained a DNA fragment of approximately 360 bp in length containing the promoter region of the hsp16A gene obtained by treating plasmid JS305 with the restriction enzyme XhoI / BamHI. Antisense construct MC13 was constructed by subcloning into the inserted plasmid pJL241 (apm-1 cDNA inserted at the cloning site of NheI / BamHI of pRSET-B vector [invitrogen]).

<4-2> Preparation of Transgenic Animals and Reactivity Test for Hypoxia and Heat Shock

According to the method of <2-1> of Example 2, DNA was purely extracted from the sense and antisense constructs MC12 and MC13 of the apm-1 gene, and then transformed into nematodes using the same. In order to observe the in vivo RNAi effect of the amp-1 gene by the hypoxic-reactive promoter of hsp16A in the transformed nematode, it was subjected to hypoxic conditions or heat shock in the same manner as in <2-2> of Example 2, followed by fluorescence microscopy. The expression level of GFP was measured and the results are shown in Table 3 and FIG. 6 .

In vivo RNAi Effects Using Heat Shock or Hypoxic Conditions Elapsed time after impact (hours) Thermal shock (% lethality) Hypoxic shock (% lethality) S AS S + AS S AS S + AS 0-8 17.6 23.9 40 0 12.2 46.3 8-16 5.2 16 29.4 8.2 19.2 42.3 16-24 5.2 19.4 34.9 0.6 21.5 53 24-36 9.6 22.9 30.1 6.6 30 30.2

In Table 3 , S is a transgenic nematode with a sense construct, AS is a transgenic nematode with an antisense construct, and S + AS is a transgenic nematode with a sense construct + antisense construct. Indicates. In addition,% fatality refers to the percentage of the total eggs laid that did not become adult and died during the growth process. As shown in Table 3 , the offspring born by heat shock killed at least 5.2% and up to 17.6% by time due to the side effect of heat shock even when the sense construct was introduced. On the other hand, progeny born by hypoxic conditions have less minor side effects when the sense construct is introduced (from 0% to 8.2%), but the lethality by RNAi is up to 53% when the sense and antisense constructs are introduced at the same time. Reached to. Thus, in vivo RNAi under hypoxic conditions showed better interference effects than in vivo RNAi due to heat shock and had less side effects.

Figure 6 shows the phenotype of the apm-1 gene observed in the in vivo RNAi experiments using the hypoxic-reactive promoter of hsp16A, A and D is a normal morphology, B, C, E, F and G is apm The phenotype exhibited by in vivo hypoxic RNAi is shown. In FIG. 6 , A and D are photographs of a 2-fold stage pear and a L1 larval stage nematode under a 630 × magnification microscope under normal growth, and B, C, E, F, G is a photograph of a nematode killed by in vivo RNAi of the apm-1 gene under a microscope of 630 × magnification. As shown in Figure 6 , the nematode by in vivo RNAi of the apm-1 gene induced problems in the development of the larvae (Larval development) as well as in embryonic development (Embryogenesis) to induce abnormal development of the nematodes.

As discussed above, the hypoxic-reactive promoter of the hsp16A of the present invention has a function of genes by selectively inducing RNA interference in a desired gene without causing a secondary defect in the entire subject as compared to a heat shock promoter. Not only can it be usefully used for the analysis, but it can also be easily used for mass production of useful proteins using hypoxic conditions.

<110> LEE, Junho SHIM, Jaegal KWON, Jae Young <120> HYPOXIA-RESPONSIVE PROMOTER AND METHOD FOR SELECTIVELY INDUCING RNA INTERFERENCE TO TARGET GENE BY USING THE SAME <130> FPD / 200103/0070 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 438 <212> DNA <213> Caenorhabditis elegans T27E4.2 cDNA (hsp16A) <400> 1 atgtcacttt accactattt ccgtccagct caacgttctg tttttgtga tctcatgaga 60 gatatggctc agatggaacg tcaatttactat ccagaagatt tgaaaattaa tttggatgga catacattat caattcaagg agaacaagaa 240 ttaaagactg aacatggata ttcaaagaaa tcattttctc gtgttattct tctacccgaa 300 gatgttgatg ttggtgcagt tgcttcaaat ctttcagaag atggaaaact ttcaattgaa 360 gcaccaaaga aggaagcgat tcaaggaaga tctattccaa ttcagcaagc gcccgttgaa 420 caaaaaactt ctgaataa 438 <210> 2 <211> 2 0 <212> DNA <213> -260 ~ -241 nucleotides of hsp16A promoter <400> 2 cagccgttta gaatgttcta 20 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer T27E4. 2-2 <400> 3 cggatccttc agaagttttt tgttc 25 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer T27E4.2-3 <400> 4 caagcttgaa gtttagagaa tgaacag 27 <210 > 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer T27E4.2-4 <400> 5 cataatgtgt agtttgaaga tttcac 26 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer T27E4.2-6 <400> 6 ataagcttca aaatacaccc catag 25 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer T27E4.2- 7 <400> 7 agtcgacgag ctgcttcttg caaa 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer T27E4.2-8 <400> 8 agtcgacgga aatgagtata aatac 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer CE16 <400> 9 gggatcctta ggtcattctc atttg 25 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer CE17 <400> 10 gcatatgtcg atttccggtc tc 22

Claims (7)

A hypoxia-responsive promoter having a nucleotide sequence set forth in SEQ ID NO: 2 . 1) preparing an expression vector linked to a gene of interest in the hypoxic-reactive promoter of claim 1; 2) preparing a transformed organism by micro-injecting the expression vector into an organism other than a human; And 3) subjecting the transgenic organism to hypoxic shock to induce the expression of the gene of interest linked to a hypoxic-reactive promoter, A method of expressing a target gene under hypoxic conditions using the promoter of claim 1. A method of selectively inducing RNA interference in a target gene present in the organism, comprising expressing a target gene introduced into an organism other than a human by the method of claim 2. The method of claim 3, wherein 1) preparing an expression vector in which the target gene in the sense direction is connected to the hypoxic-reactive promoter of claim 1 and an expression vector in which the target gene in the antisense direction is connected to the hypoxic-reactive promoter of claim 1; 2) A transgenic organism is micro-injected into an organism other than a human by injecting the expression vector in which the target gene in the sense direction is connected to the hypoxic-reactive promoter of claim 1 and the expression vector in which the target gene in the antisense direction is connected to the hypoxic-reactive promoter of claim 1. Manufacturing step; And 3) subjecting the transgenic organism to hypoxic shock to induce the expression of the target gene linked to the hypoxic-reactive promoter. The method of claim 3, wherein 1) preparing an expression vector in which the target genes in the sense and antisense directions are simultaneously connected to the rear of the hypoxic-reactive promoter of claim 1; 2) preparing a transformed organism by micro-injecting the expression vector into an organism other than a human; And 3) subjecting the transgenic organism to hypoxic shock to induce the expression of the target gene linked to the hypoxic-reactive promoter. The hypoxic shock of claim 3, wherein the hypoxic shock is applied to the buffer or water in a state in which the medium for culturing the transformed organism is poured into water to submerge the organism or shaken on a tube after harvesting the organisms. Carried out by locking. The method according to any one of claims 3 to 5, characterized in that the organism is a nematode ( Caenorhabditis elegans ).