KR101048417B1 - Mouse cells knocked down with the expression of micro-RNA and prion genes for the prion gene of mice - Google Patents

Abstract

The present invention is a micro RNA (micro RNA, miRNA) targeting the mouse prion gene, a recombinant expression vector containing the miRNA, transformed with the recombinant expression vector knocked down the expression of the prion gene (knock-down) Relates to mouse cells.

The miRNAs of the present invention and mouse cells transformed thereby can be used to study the function of prion proteins and the pathogenic mechanisms of prion diseases.

Prion, miRNA, knockdown

Description

Micro RNA Targeting Mouse Prion Gene and Prion Gene Knock-down Mouse Cell}

The present invention relates to a mouse cell in which the expression of the micro RNA and the prion gene knocked down the mouse prion gene, more specifically miRNA targeting the mouse prion gene, a recombinant expression vector containing the miRNA, the recombinant expression vector It relates to a mouse cell that is transformed and knocked down the expression of the prion gene.

Prion disease, including mad cow disease, is a disease that causes neurodegenerative diseases in many animals, including humans, and is caused by the conversion of normal prion protein (PrP ^C ), a cell surface glycoprotein, into abnormal pathogenic prion protein (PrP ^Sc ). It has a very long incubation period and is a fatal chronic progressive disease with 100% mortality. To date, no vaccine, antidote or treatment is known.

Although the prion hypothesis has been widely accepted and studies based on studies using Prnp knock-out mice, the function and molecular biological properties of normal prions, the replication methods of pathogenic prions, and pathogenic mechanisms are still clear. Not known. In addition, it is reported that clinical and pathological symptoms differ from one another according to each prion strain, mutant and host species.

The knock-out method is a classical method mainly used for the purpose of suppressing the expression of a specific gene. However, when knock-out techniques were used to inhibit the expression of Prnp , Prnd isotopic expression was induced, in which the gene encodes the Doppler protein during the intergenic splicing process. It is reported that Prnd is controlled by Prnp promoter by intergene splicing. Doppler is a protein similar to prion protein with the N-terminus removed, and unlike prion protein, there is no octamer repeat. Doppler proteins expressed in ZrchII, Ngsk, Rcm0 and Rikn knock-out mice caused degeneration of Purkinje cells and symptoms such as ataxia and dysfunction.

On the other hand, RNA has been recognized as a messenger only in the process of converting the genetic information contained in DNA into protein, but the small RNA known to exist in various forms is involved in the expression and regulation of genes in various forms. It has been found to coordinate the function of cells.

siRNA (small interfering RNA) is a small RNA fragment of 21-25 nucleotides produced by cleavage by Dicer and has been found to specifically bind to mRNA having complementary sequences to inhibit protein expression. siRNA technique is currently considered as the most powerful gene silencing technique. It is largely divided into siRNA, shRNA (short hairpin RNA) and miRNA (micro RNA), and these are collectively called RNAi (RNA interference).

Many researchers have applied RNAi techniques to a variety of studies including animal cell culture and gene function analysis in animal models: 1) research on the function of specific genes, 2) new drug development using cell culture models, 3) Developing a model system related to human disease and researching various genes through research related to the progression of cancer from end to end, 4) development of therapeutic agents for cancer or viral diseases, and 5) siRNA library construction. I use it.

The present invention seeks to establish cells suitable for studying the function of prion proteins and pathogenic mechanisms of prion diseases by knocking down mouse Prnp using miRNA technique, a post-transcriptional gene silencing mechanism.

In one aspect, the present invention provides a miRNA targeting a mouse prion gene.

In another aspect, a recombinant expression vector including the miRNA is provided.

In another aspect, the present invention provides a mouse cell transformed with the recombinant expression vector and knocked down in expression of a prion gene.

Generation of micro RNA (mi RNA / miRNA) consists of two steps of processing. The first microRNA (primary miRNA / pri-miRNA) is made into a pre-mRNA with stem-loop structure of 60-90 nucleotides by RNase type III enzyme called Drosha in the nucleus and then transferred to the cytoplasm. It is cleaved by an enzyme called Dicer and made into mature miRNAs of 21-25 nucleotides. Thus, in the present invention, the miRNA may be a pri-mRNA or pre-mRNA from which the miRNA may be derived.

In the present invention, the miRNA targeting the mouse prion gene means an RNA having a sequence complementary to a part of the mouse prion gene and capable of degrading mRNA or inhibiting translation of the prion gene. Complementarity of 80-90% can inhibit the translation of mRNA, and 100% can degrade mRNA.

MiRNA of the present invention preferably targets 220 to 240 nucleotides (SEQ ID NO: 1) of the mouse prion gene. For this purpose pri-mRNA, pre-mRNA or miRNA comprises a nucleotide sequence having 80%, preferably 90%, particularly preferably 100% homology to the complementary sequence for 220 to 240 nucleotides of the mouse prion gene It is desirable to. In the present invention, the pre-miRNA targeting 220 to 240 nucleotides (SEQ ID NO: 1) of the mouse prion gene is preferably composed of the nucleotide sequence shown in SEQ ID NO: 3.

In addition, the miRNA of the present invention preferably targets 852 to 872 nucleotides of the mouse prion gene. For this purpose pri-mRNA, pre-mRNA or miRNA comprises a base sequence having 80%, preferably 90%, particularly preferably 100% homology to the complementary sequence for 852 to 872 nucleotides of the mouse prion gene It is preferable. In the present invention, the pre-miRNA targeting 852 to 872 nucleotides (SEQ ID NO: 2) of the mouse prion gene is preferably composed of the nucleotide sequence shown in SEQ ID NO: 4.

The miRNA of the present invention can be prepared according to the production method of RNA molecules known in the art. As a method for preparing RNA molecules, chemical synthesis methods and enzymatic methods can be used. For example, the chemical synthesis of RNA molecules can use the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999), and the enzymatic synthesis of RNA molecules is T7, T3. And phage RNA polymerases such as SP6 RNA polymerase are disclosed in the literature (Milligan and Uhlenbeck, Methods Enzymol . 180: 51-62, 1989).

In the present invention, miRNAs can be introduced into mouse cells by themselves or by cloning into a vector, the latter being more preferred. The present invention relates to a recombinant expression vector comprising the miRNA of the present invention. The recombinant expression vector of the present invention preferably comprises both miRNAs targeting 220 to 240 nucleotides of the mouse prion gene and miRNAs targeting 852 to 872 nucleotides.

The recombinant expression vector of the present invention may be constructed by recombinant DNA methods known in the art. The expression vector may be selected from the group consisting of plasmids, lentiviral vectors, retroviral vectors, adenovirus vectors known in the art, used for replication and expression in mammalian cells or other target cell types. As described above, miRNA may be synthesized and inserted into an expression vector.

The present invention relates to a mouse cell knocked down a prion gene prepared by introducing the recombinant expression vector into a mouse cell. The introduction method is not limited thereto, but transfection with calcium phosphate, transfection with DEAE dextran, transfection with microinjection, electroporation and lipofection may be used.

In the present invention, the mouse cells are preferably mouse neuroblastoma (N2a). N2a cells are cells commonly used for prion disease research.

The mouse prion gene knocked down cells of the present invention are suitable for studying the function of the prion protein and the pathogenic mechanism of prion disease at the molecular cell biological level, thereby helping to understand the disease and developing new biomarkers for early diagnosis. It will increase your chances. In addition, miRNAs cloned using a variety of delivery systems currently developed (delivery systen) will be able to accelerate the development of gene therapy using RNAi technology by applying to in vivo experiments.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Design and Synthesis of Pre-miRNAs Targeting Prnp in Mice

For target site selection to down-regulate mouse Prnp , algorithms were used to design pre-miRNA1 targeting the N-terminus and pre-miRNA2 targeting the C-terminus, respectively. Specifically, in order to suppress the expression of the mouse 220 of Prnp from Genebank accession number NM_011170 in mice Prnp 240 nucleotides (TGTGACTATGTGGACTGATGT- SEQ ID NO: 1) from pre-miRNA1 and 852 to target the 872 nucleotide (CCTATTACGACGGGAGAAGAT- SEQ ID NO: 2) Pre-miRNA2 was designed to target. In addition, TGCT (top) at the 5 'end and CAGG at the 3' end were inserted for cloning directly into the expression vector, and the end loop of 19 nucleotides and the inner loop of 2 nucleotides were formed.

Nucleotide sequence of pre-miRNA1 (SEQ ID NO: 3)

5'-TGCTGACATCAGTCCACATAGTCACAGTTTTGGCCACTGACTGACTGTGACTATGGACTGATGT-3 '

Nucleotide sequence of pre-miRNA2 (SEQ ID NO: 4)

5'-TGCTGATCTTCTCCCGTCGTAATAGGGTTTTGGCCACTGACTGACCCTATTACCGGGAGAAGAT-3 '

Example 2: Vector Construction Including Pre-miRNA1 or 2 or Both

2-1: Vector Construction with Single Pre-miRNA

The synthesized pre-miRNA1 and 2 top-strands and bottom-strands were annealed at room temperature for 10 minutes to generate double-stranded oligos. The prepared double-stranded oligo was ligated to pcDNA6.2-GW / EmGFP vector for 30 minutes at room temperature using T4 DNA ligase, and transformed into TOP10 competent E. coil . The cloned pcDNA6.2-GW / EmGFP-miRNA1, 2 and negative miRNA (miRNAneg) were purified and sequenced using Mini-prep to confirm the insertion of pre-miRNA (FIG. 1A).

2-2: Vector Construction with Multiple Pre-miRNAs

Cloned pre-miRNAs 1 and 2 were chained to more efficiently down regulate mouse Prnp through the expression of multiple pre-miRNAs. First, miRNA 1 was digested with Bgl II and Xho I (digestion) as a backbone and miRNA 2 was digested with Bam H1 and Xho I (digestion) as an insert. Digested sections were identified by electrophoresis on 1% and 3% agarose gels, respectively, and purified by gel-extraction. The purified backbone and insert were chained using T4 DNA ligase and transformed into TOP10 competent E. coil . The cloned vector was purified using Mini-prep, and sequencing confirmed the chaining of pre-miRNAs, namely pcDNA6.2-GW / EmGFP-miRNA1-2 (FIG. 1B).

Example 3: Transformation

To transfect cloned pcDNA6.2-GW / EmGFP-miR1, 2, 1-2 and miRNAneg, respectively, to N2a cells, first, a 6-neck cell culture plate was prepared so that N2a cells were 1 × 10 ⁵ cells the day before the experiment. (6 well plate) was subcultured and transfected for 6 hours in a medium containing antibiotics and serum using lipofectamine 2000. After 24 hours, transfection was confirmed under a fluorescence microscope, and drug selection was performed for 2 weeks in a medium containing 20 µg / ml blasticidine S.

Example 4: Transfection Confirmation

Genomic DNA was isolated and purified from each of the transfected cells using the Genomic DNA Purification Kit and diluted to 300 ng / μl. To confirm the insertion of the transfected pre-miRNA, primer sets were designed such that the amplification product size was 352 bp for the pcDNA6.2-GW / EmGFP vector, and PCR was performed using the purified genomic DNA as a template. As a result, 412 bp amplification was observed in N2a cells (N2amiRn) transfected with miRNA1 (N2amiR1), miRNA2 (N2amiR2) and miRNAneg, and 550bp in N2a cells transfected with miRNA1-2 (N2amiR1-2). Amplification products of size could be observed (FIG. 2).

Example 5 Confirmation of Downregulation Efficiency of Prnp by Western Blot

Western blot was performed to evaluate the down regulation efficiency of mouse Prnp in each cell pool where pre-miRNA insertion was confirmed. Each cell was lysed with the addition of RIPA lysis buffer and the protein amount was quantified. Electrophoresis was performed by loading 10 μg of protein into 4-12% gradient SDS-PAGE and blotting the nitrocellulose membrane. A 3F10 anti-prion monoclonal antibody was used as the primary antibody and reacted with a secondary antibody conjugated with HRP. The prion protein reacted with the antibody was confirmed by photosensitive X-ray film using chemiluminescence. After confirming the expression level of the prion protein by 3F10, the primary, secondary and chemiluminescent materials which had already reacted to the membrane were removed by stripping and reacted with the specific cell marker GAPDH. Downregulation efficiency of mouse Prnp was assessed by the amount of prion protein relative to the amount of GAPDH using densitometry. As a result, N2amiRn showed no difference compared to wild type N2a cells. However, N2amiR1 decreased the expression of mouse Prnp to 10.6% and N2amiR2 to 18.7%, and N2amiR1-2 efficiently expressed target mouse Prnp to 77.5%. Knock-down was confirmed (FIG. 3).

Example 6: Discovery of N2amiR1-2 clones with high downregulation efficiency of Prnp

Limited dilution of positive cell clones that efficiently knocked down mouse Prnp expression in the N2amiR1-2 cell pool. First, the N2amiR1-2 cell pool was diluted in a complete medium containing 20 µg / ml blasticidine S at a concentration of 5 cells / ml, and 100 µl was dispensed into 96-well cell culture plates (96well plates). Two weeks later, genomic DNA was isolated from each cell clone and PCR was performed (FIG. 4). Twenty-four cell clones identified with the insertion of miRNA1-2 were evaluated for the degree of mouse Prnp downregulation by the same Western blot method. As a result, it was confirmed that the expression of mouse prion protein was inhibited by 97.7% or more in cell clone 15 (FIG. 5). In addition, the expression of GFP, a reporter gene of the pcDNA6.2-GW / EmGFP vector, was confirmed in the selected cell clone 15 (FIG. 6).

Figure 1a is a schematic diagram of miRNA 1, miRNA2, miRNAneg cloned into the expression vector pcDNA6.2-GW / EmGFP-miR vector.

1B is a schematic of the chained miRNA1-2 cassette.

FIG. 2 shows multiple PCR results for confirming whether miRNAs have been inserted into N2a cells (laneL: 100bp DNA ladder, lane1: vector cloned with bovine Prnp , lane2: vector cloned with miRNAneg, lane3: N2a with miRNA1 inserted) Cell, lane4: N2a cell with miRNA2 inserted, lane5: N2a cell with miRNAneg inserted, lane6: N2a cell with chained miRNA1-2 inserted, lane7: wild type N2a cell, lane8: N2a cell with bovine Prnp inserted , lane9 is a template-free control).

Figure 3 is a result of Western blot to determine the degree of inhibition of mouse Prnp expression by miRNA1, 2 and miRNA1-2 chained, (A) is specific for the monoclonal antibody 3F10 and (above) GAPDH specific for the prion protein Western blot using polyclonal antibodies (below), and (b) is the result of Western blot evaluation using densitometry.

4 is a genomic PCR result for confirming whether miRNA1-2 has been inserted into N2a cells (LaneL: 100bp DNA ladder, lane1: miRNAneg cloned vector, lane2: miRNA1-2 cloned vector, lane3: N2a cell Control, lane4-20: cell clones 1 to 17 obtained via limiting dilution, laneN: control without template).

5 is a result of Western blot to confirm the degree of inhibition of mouse Prnp expression in N2amiRNA1-2 cell clones obtained through restriction dilution, (A) is a monoclonal antibody specific for prion protein 3F10 and (above) GAPDH Western blot using polyclonal antibody (below) and (b) is the result of Western blot evaluation using densitometry.

6 is a micrograph of N2amiRNA1-2 in which mouse Prnp is continuously knocked down, (a) and (b) are wild type N2a cells, (c) and (d) are N2amiRNA1-2 cells, and (a) And (c) are cell photographs confirming the expression of the reporter gene GFP under a fluorescence microscope, and (b) and (d) are cell photographs stained with DAPI.

<110> Seoul National University Industry Foundation <120> Micro RNA Targeting Mouse Prion Gene and Prion Gene Knock-down          Mouse cell <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> mouse prion gene 220-240nt <400> 1 tgtgactatg tggactgatg t 21 <210> 2 <211> 21 <212> DNA <213> mouse prion gene 852-870nt <400> 2 cctattacga cgggagaaga t 21 <210> 3 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> pre-miRNA1 <400> 3 tgctgacatc agtccacata gtcacagttt tggccactga ctgactgtga ctatggactg 60 atgt 64 <210> 4 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> pre-miRNA2 <400> 4 tgctgatctt ctcccgtcgt aatagggttt tggccactga ctgaccctat taccgggaga 60 agat 64  

Claims (16)

MiRNA targeting 220-240 nucleotides of mouse prion gene shown in SEQ ID NO: 1 and 852-870 nucleotides of mouse prion gene shown in SEQ ID NO: 2. delete A pre-miRNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4. delete delete delete delete delete delete delete Recombinant expression vector comprising a pre-miRNA according to claim 3. delete delete Mouse cells transformed with the recombinant expression vector according to claim 11 to knock down the expression of the prion gene. 15. The method of claim 14, Mouse cells are cells characterized in that they are neuroblastoma cells of the mouse. Mouse cells were simultaneously transformed with miRNAs targeting 220 to 240 nucleotides of the mouse prion gene shown in SEQ ID NO: 1 and miRNAs targeting 852 to 870 nucleotides of the mouse prion gene shown in SEQ ID NO: 2 To inhibit prion gene expression.

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