JP3376412B2 - Active ribozyme selection method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently selecting a ribozyme having activity in mammalian cells.

[0002]

2. Description of the Related Art Ribozymes are RNA-based catalysts discovered by Cech et al. In the early 1980s, and have the function of recognizing the base sequence of RNA and cleaving a specific sequence.
Utilizing such functions of ribozymes, it has been attempted in recent years to utilize ribozymes as therapeutic agents for genetic diseases and gene analysis tools.

Although what kind of RNA has a function as a ribozyme has been elucidated to some extent, details are often not clear.
Therefore, in order to effectively use the ribozyme for the above-mentioned uses, it is important to develop a means capable of efficiently selecting an active ribozyme from many RNA molecules which are ribozyme candidates.

Various methods for selecting active ribozymes have been devised in the past, but most of them were selected based on their in vitro activity (Science, Vol. 257, 6).
35-641, 1992). However, since there are many factors that affect the activity and stability of ribozymes in cells,
Ribozymes that show high activity in vitro may show little activity in cells.

As a method for selecting an active ribozyme in vivo, for example, the method of Multirira et al. Is known (Japanese Patent Laid-Open No. 9-98783). Tahira et al.
We have proposed a method for discriminating ribozyme activity using a vector constructed so that a marker protein is expressed by cleavage of RNA. However, this method requires the production of a complicated vector containing multiple promoters, structural genes, etc., and since E. coli is mainly used as a host, the ribozyme selected by this method is actually a human cell. In some cases it may not be active.

[0006]

[Problems to be Solved by the Invention]
This can be solved if a simpler selection system can be constructed using mammalian cells as a host. The present invention has been made under such a technical background, and an object thereof is to provide a novel means for selecting an active ribozyme using a mammalian cell as a host.

[0007]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventor has found that p in Balb3T3 cells is
The inventors have found that active ribozymes and inactive ribozymes can be discriminated by using the presence or absence of expression of 16 genes as an index, and completed the present invention.

That is, the present invention modifies the sequence of a ribozyme as a selection candidate so as to suppress the expression of a tumor suppressor gene, and suppresses the expression of the tumor suppressor gene in a vector expressing the modified ribozyme. The method for selecting an active ribozyme is characterized in that the ribozyme is introduced into a mammalian cell that becomes cancerous, and the ribozyme contained in the cell forming the focus is selected from the cells after the introduction.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The method for selecting an active ribozyme of the present invention is a modification of the sequence of a ribozyme that is a selection candidate so as to suppress the expression of a tumor suppressor gene, and a vector expressing the modified ribozyme, in which expression of the tumor suppressor gene is suppressed The method is characterized in that the ribozyme contained in the cells forming the focus is selected from among the cells after the introduction into the mammalian cell which becomes cancerous. The ribozyme used as a selection candidate may be any type of ribozyme, but a ribozyme that is small and easy to synthesize, such as a hammerhead ribozyme, is preferable.

The suppression of the expression of the tumor suppressor gene can be carried out by making the peripheral sequence of the cleavage site of the ribozyme the same as or complementary to the specific region of the tumor suppressor gene. By having such a sequence in the ribozyme, the mRNA derived from the tumor suppressor gene can be cleaved at a specific site and the expression of that gene can be suppressed. When the tumor suppressor gene is the p16 gene, the cleavage site and its surrounding sequence are ATGGA
GTC CGCTGCA, TTTCG GTC GTACCCC, CGGTC GTA CCCCGAT, TTCA
The expression of this gene can be suppressed by using a sequence corresponding to C GTA GCAGCTC, GGAAC GTC GCCCAGC (underlined is a cleavage site, and each sequence corresponds to R1, R2, R3, R4, and R8 in Examples).

Mammalian cells that become cancerous by suppressing the expression of the tumor suppressor gene are not particularly limited.
Balb3t3 cells can be used. Balb3t3 cells
In a normal state, the tumor suppressor gene p16 is expressed, so that it does not become cancerous, but by suppressing the expression of the p16 gene, it becomes cancerous. Balb3t3 cells are commercially available from Dainippon Pharmaceutical.

[0012] The vector expressing the modified ribozyme may be any vector as long as it contains a nucleotide sequence expressing the modified ribozyme, a suitable promoter and a terminator, but a human tRNA promoter is used as the promoter. preferable. The human tRNA promoter is
It is known that it is advantageous for transcription of short sequences and that its expression level is about 100 times higher than that of an expression system dependent on polymerase II.

If the ribozyme selected as a selection candidate is an active ribozyme, the expression of the tumor suppressor gene is suppressed, so that the cells become cancerous and focus is formed. On the other hand, if the ribozyme is inactive, For example, since the expression of the tumor suppressor gene is not suppressed, the cells do not become cancerous and foci are not formed. Only active ribozymes can be selected from the ribozymes selected as candidates for selection depending on the presence or absence of the formation of such a focus.

[0014]

[Examples] [Example 1] DNAs expressing eight types of hammerhead ribozymes (R1-R8) expected to cleave mRNAs derived from p16 gene were synthesized. The target site of each ribozyme and the base sequence corresponding to that site are shown in FIG. 2 and Table 1, respectively.

[0015]

[Table 1]

Further, a human tRNA promoter having the following sequence was synthesized, and this was inserted into the plasmid pUC1α (Nippon Gene) together with the ribozyme expression DNA to prepare a ribozyme expression plasmid (FIG. 3). Sequence: 5'-ACCGTTGGTT TCCGTAGTGT AGTGGTTATC ACGTTCGC
CT AACACGCGAA AGGTCCCCGG TTCGAAACCG GGCACTACAA AAA
CCAACAA AAAAATTTTT-3 '(SEQ ID NO: 9) As a control, human tRNA without ribozyme expression DNA was inserted.
A plasmid in which only the promoter was inserted was also prepared.

Balb3T3 cells were transfected with each plasmid, and the cells were seeded in a Petri dish containing DMEM medium and cultured at 37 ° C. for 2 weeks to observe the presence or absence of focus formation. The transfection was performed as follows according to the lipofectin method. The day before transfection
Cells were seeded on a 10 cm dish to give 2 × 10 ⁶ cells. twenty four
After a lapse of time, 10 μg of each plasmid and 10 μl of lipofectin (Gibco) were mixed and left at room temperature for 20 minutes. The cell culture medium was changed to a serum-free medium (OPTI-MEM; Gibco), and the above mixed solution was added dropwise. After 12 hours, the medium was exchanged with serum-containing DMEM, and after 2 weeks, the formation of foci was observed.

The relationship between each ribozyme and the number of focus formations is shown in FIG. As shown in FIG. 4, R1, R2, R3, R4, R8
Formation of foci was observed in cells expressing
The cells expressing R3 formed the most foci. Therefore, it was decided to use a plasmid expressing R3 in the following experiments.

Example 2 A plasmid expressing an inactive ribozyme in which G5 at the active site of the hammerhead ribozyme was changed to A was prepared. Balb 3T3 cells were transfected with this plasmid as described above, but no focus formation was observed. [Example 3] Ba expressing active or inactive ribozyme
The expression levels of p16 mRNA and p16 protein in lb3T3 cells were examined by RT-PCR and Western blotting, respectively.

RT-PCR was performed under the following conditions. The primer was 5'-CGGGATCCGC TGCAGACAGA as an upstream primer.
CTGGCCAG-3 '(SEQ ID NO: 10) was used as a downstream primer 5'-GCAAAGCTTG AGGCCGGATT TAGCTCTGCT C-3' (SEQ ID NO: 11). RT-PCR is based on RNA PCR kit (TaKaRa
Company) manual. The amount of amplified cDNA
It was confirmed by 2% agarose electrophoresis. Western blotting was performed as follows.

Proteins were extracted from cells transfected with various plasmids. In each sample, 20 μg of protein was electrophoresed on SDS-polyacrylamide gel electrophoresis at 20 mA for 1 hour. The electrophoresed gel was taken out and electrically transferred onto a PVDF membrane (Funakoshi) at 140 mA for 1 hour and 30 minutes. The p16 protein was detected using a specific p16 antibody (Santa Cruz) according to the manual of the amplified alkaline phosphatase immunoblot kit (Bio-Rad). The results of RT-PCR and Western blotting are shown in FIGS. 5 and 6, respectively. As shown in these figures, the active ribozymes significantly reduced the expression levels of p16 mRNA and p16 protein.

[Example 4] An active ribozyme expression plasmid and an inactive ribozyme expression plasmid were introduced into Balb3T3 cells at a ratio of 1: 1, and the cells were cultured under the same conditions as in Example 1 for 2 weeks and then proliferated. DNA was taken out from the cells and the nucleotide sequence was determined to determine whether it was the active type or the inactive type (FIG. 7). As a control, the cells were cultured in a medium containing G418, and the active form of the DNA extracted from the cells after the culture was determined. The results are shown in Table 2.

[0023]

[Table 2]

As shown in Table 2, all the DNA contained in the cells after the culture expressed active ribozyme. [Example 5] The active site of the hammerhead ribozyme
DNA expressing a ribozyme in which G5 was changed to four types of A, U, C, and G was prepared, and cells obtained by introducing this DNA were cultured in the same manner as in Example 4. DN contained in cells after culturing
When A was examined, all were ribozyme-expressing DNAs whose active site was G.

[0025]

INDUSTRIAL APPLICABILITY Since the method for selecting an active ribozyme of the present invention uses mammalian cells as a host, it is possible to select with high accuracy a ribozyme that is actually active in the human body.

[0026]

[Sequence list]                           SEQUENCE LISTING <110> Director General of the Agency of industrial Science and Technolog y <120> Selection Method for Active Ribozymes <130> 117F0093 <160> 11 <170> PatentIn version 2.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 1 <400> 1 atggagtccg ctgca 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 2 <400> 2 tttcgctcgt acccc 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 3 <400> 3 cggtcgtacc ccgat 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 4 <400> 4 ttcacgtagc agctc 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 5 <400> 5 cacgggtcag gggct 15 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 6 <400> 6 ctggggtcgc ctgcc 15 <210> 7 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 7 <400> 7 tttgtgtacc gctgg 15 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Designated is a target sequence of Ribozyme 8 <400> 8 ggaacgtcgc ccagc 15 <210> 9 <211> 100 <212> DNA <213> Homo sapiens <220> <221> promoter <400> 9 accgttggtt tccgtagtgt agtggttatc acgttcgcct aacacgcgaa aggtccccgg 60 ttcgaaaccg ggcactacaa aaaccaacaa aaaaattttt 100 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Designated is a primer used for RT-PCR <400> 10 cgggatccgc tgcagacaga ctggccag 28 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Designated is a primer used for RT-PCR <400> 11 gcaaagcttg aggccggatt tagctctgct c 31

[0027]

[Sequence list free text]

SEQ ID NO: 1 shows the target sequence of ribozyme 1. SEQ ID NO: 2: Shows the target sequence of ribozyme 2. SEQ ID NO: 3: Shows the target sequence of ribozyme 3. SEQ ID NO: 4: Shows the target sequence of ribozyme 4. SEQ ID NO: 5: shows the target sequence of ribozyme 5. SEQ ID NO: 6: Shows the target sequence of ribozyme 6. SEQ ID NO: 7: shows the target sequence of ribozyme 7. SEQ ID NO: 8: Shows the target sequence of ribozyme 8. SEQ ID NO: 10 shows the primer used for RT-PCR. SEQ ID NO: 11 shows the primer used for RT-PCR.

[Brief description of drawings]

FIG. 1 is a diagram showing an outline of a method for selecting an active ribozyme of the present invention.

FIG. 2 is a diagram showing the target site of the ribozyme used in Example 1.

FIG. 3 is a diagram showing the structure of a plasmid expressing a ribozyme that suppresses the expression of p16 gene.

FIG. 4 is a view showing the focus forming ability of the ribozyme used in Example 1.

FIG. 5 is a graph showing the expression level of p16 mRNa when an active ribozyme and an inactive ribozyme are expressed.

FIG. 6 is a diagram showing the expression level of p16 protein when an active ribozyme and an inactive ribozyme are expressed.

FIG. 7 is a diagram showing an outline of an experiment conducted in Example 4.

Continuation of the front page (56) References JP-A-9-98783 (JP, A) FEBS Letters, 1998, Vol. 436, p. 41-45 Molecular and Celular Biology, 1995, Vol. 15, No. 1, p. 540-551 1997 Research Report on Health Care and Immune Decrease Prevention Research Project for Health Maintenance and Promotion, Human Science Promotion Foundation, July 14, 1998, p. 9-12 Oncogene, 1995, Vol. 11, No. 4, p. 635-645 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12Q 1/68 C12Q 1/02 C12N 15/00-15/90 BIOSIS / WPI (DIALOG) MEDLINE (STN) REGISTRY (STN) CAPLUS (STN)

Claims (3)

(57) [Claims] 1. A ribozyme sequence as a selection candidate is designated as p1.
A vector which is modified so that expression of 6 genes can be suppressed and expresses the modified ribozyme ,
A vector containing the sequence described in 3, 4, or 8 was added to the p16
A method for selecting an active ribozyme, which comprises introducing into a mammalian cell that becomes cancerous due to suppression of gene expression, and selecting a ribozyme contained in cells forming a focus from among the cells after the introduction. 2. The method for selecting an active ribozyme according to claim 1, wherein the vector expressing the modified ribozyme contains a human tRNA promoter. 3. The method for selecting an active ribozyme according to claim 1, wherein the mammalian cell is Balb3T3 cell.