Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/102—Mutagenizing nucleic acids

Definitions

• an inverted repeat DNA containing the base (base sequence) to be introduced into the gene is prepared and introduced into cells using the above-described DNA introduction method. Mutations can be introduced into the target gene.
• the gene therapy described above is applicable to humans as well as non-human vertebrates and invertebrates.
• the gene (SEQ ID NO: 1 in the sequence listing) encoding the Red-shift Green Fluoescent Protein (hereinafter abbreviated as GFP) introduced in Inc.
• GFP Red-shift Green Fluoescent Protein
• the DNA fragment obtained by amplifying the GFP gene with primers U100 hind III and U100 bamh I was digested with Hind III and BamHI to obtain a DNA fragment.
• a bp DNA fragment was prepared.
• the 760 bp HindIII-BamHI fragment in the above plasmid pucGFPO-0 was replaced with this 1.10 bp DNA fragment to construct a plasmid pucGFP0-6.
• pucGFP0-0 has an inverted repeat sequence for the entire length of the gene encoding GFP, and the length of the insert DNA is 1518 bp. In pucGFP0-2, one of the repetitive sequences lacks 38 bases from the stop codon, and the insert DNA is 1479 bp in length. Plasmid pucGFPO-6 has the entire length of the gene encoding GFP and an inverted repeat formed from nucleotides 150 to 250 in the GFP gene, and the insert DNA is 868 bp in length. .
• Fig. 1A illustrates pucGFPO-0
• Fig. IB illustrates pucGFPO-2
• Fig. 1C illustrates pucGFPO-6.
• a 760 bp HindIII-BamHI fragment containing the mGFP gene was isolated from the pDON-mGFP constructed in Example 1, and an episoma 1 mammalian expression vector pCEP4 ( The plasmid was subcloned between HindIII and BamHI sites (Invitrogen) to produce plasmid pcep-mGFP.
• 293 cells (80,000 cells) were seeded on a 48-well plate and cultured in a DEME medium containing 10% FBS. 2 g each of 0-0 i rDNA, 0-2 i rDNA, 0-6 i rDNA (2) Repair of episomal mGFP gene by ir DNA without heat denaturation
• the molar numbers of 6ir DNA are 9.5, 10.1, and 16.6 nmol, respectively. Nevertheless, all three ir DNAs had higher repair rates of mutant bases than the ss oligos. Similar to the case where ir DNA was denatured by heat and transfection was performed, even when transfection was performed without modification, the repair efficiency was high for 0-0 i rDNA containing the entire GFP gene region. The ss oligo was more than 5 times. 0
• Retrovirus particles that introduce the mGFP gene into the chromosome of the cells were produced using Retrovirus packaging kit ampho (Gukafunodi).
• the recombinant retrovirus vector pDON-mGFP described in Example 1 and the packaging vector of the above kit were both simultaneously introduced into 293 cells by the calcium phosphate method. After culturing for 48 hours, the culture supernatant was collected and filtered. Further, the culture supernatant (retrovirus solution) was diluted and added to the medium of 293 cells. The fact that the mGFP gene is contained in the cells cloned from this site (293-10 cells) indicates that the DNA fragment amplified by PCR The sequence was analyzed and confirmed.
• the genomic DNA of the 293-10 cells was extracted, the sequence of the integration site of the retroviral vector and the chromosomal DNA on the cell side adjacent to the site was analyzed, and one copy of the GFP gene was introduced into the cells. I confirmed that I did.
• the cloned cells were used in the following experiments.
• 0 ir DNA was diluted to 75 ⁇ l with Optimen medium and mixed with an equal amount of Optimen medium containing 4 ⁇ g LF2000. After incubating for 20 minutes, the DNA-LF2000 reagent complex was directly applied to the Itoda vesicle, and transfection was carried out with addition of 90 ⁇ l of DEME medium containing 10% FBS. The number of monos of the ss oligo in the medium was 99 2.00 nmol, 0-0 ir DNA was 17.83 nmol, and R0-0 ir DNA was 17.82 nmol. Six hours later, 1.5 ml of a DME medium containing 10% FBS was added. 16 After 18 to 18 hours, change the medium to DME containing 3% FBS.
• the cells were cultured in C for two and a half days. 14 Each was heat denatured at 94 ° C for 5 minutes and quenched with ice water. 2 ⁇ g of ss oligo, 0-0 ir DNA, 0-2 i rDNA, 0-6 i rDNA and 1.5 g pcep — mGFP or pUC 19 (negative control 1) with 37.5 1 Opti
• the medium was diluted with a medium and mixed with an equal amount of Optime ⁇ medium containing 2 / g of LF2000. Negative mix of 37.5 ⁇ l Opti medium with only 3.5 ⁇ g of ⁇ ce ⁇ —mGF Op and Opti medium with 2 g of LF 2000 in equal volume Control 2 was used.
• the number of GFP-positive cells per 10,000 cells which is an indicator of the repair of mutant bases on the mGFP gene, is most often 0-0 ir DNA per 10,000 cells, and the repair effect is about three times that of the ss oligo. there were.
• the U-test of Mann-Whitney in four independent experiments showed that the repair efficiency of 0-0irDNA relative to the repair efficiency of ssoligo showed a significant difference (P ⁇ 0.05).
• Transcription factors are important factors involved in signal transduction to the nucleus, and play a role in regulating gene expression. Many transcription factors are synthesized in the cytoplasm, and always wait in the cytoplasm for their turn. If an activation signal is received, the transcription factor translocates into the nucleus and functions.
• NF_ ⁇ B is present in 293 cells and a DNA sequence capable of binding to it (5′—gattgctttagcttggaaattccggagctg-3 ⁇ SEQ ID NO: 13) has been reported [Eur. J. Biochem, Vol. 268, ⁇ 1828- 1836 (200 1)].
• a DNA sequence capable of binding to it 5′—gattgctttagcttggaaattccggagctg-3 ⁇ SEQ ID NO: 13
• GFP-kBl the base sequence of each primer was Nos. 14, 15, and 16
• an EcoRI-BamHI fragment containing the GFP gene cut out from pucGFP0-0 was used as a ⁇ -type DNA.
• the R reaction was performed to obtain an amplified DNA fragment.
• PCR was performed using GFP-kB2 and Us-EcoRI as a type I of the amplified fragment to obtain an amplified fragment.
• the amplified fragment was subjected to PCR using GFP-kB3 and Us-EcoRI as type I to obtain an amplified fragment.
• the obtained amplified fragment was digested with EcoRI and BamHI, and the fragment was replaced with an EcoRI-BamHI fragment in pucGFR0_0.
• the plasmid constructed in this way was named pucGFPOn f — ⁇ .
• the (m1GFP) gene was subcloned into pCEP4 and transfected into 293 cells to confirm that no fluorescence was seen. Furthermore, using this HindIII-NheI fragment (-36-174) of the mlGFP gene, the HindIII-NheI fragment (—36 — Replaced with 174), and constructed the dap-no-remutation GFP (dmGFP) gene.
• the present invention provides a method for introducing a mutation into a nucleotide sequence on a gene with high efficiency.
• artificial mutation can be introduced into intracellular DNA, or a gene that has been mutated and is not functioning can be repaired.
• the method of the present invention is useful for gene therapy, production of knockout organisms, analysis of gene function, and the like. Sequence listing free text
• SEQ ID N0: 1 Gene encoding red-snifted green fluorescence protein.
• nucleotides 1 to 6 are 2'-0-methylribonucleotides one other nucleotides are deoxyribonucleotides
• SEQ ID NO: 12 PCR primer easy-hindl ll to amplify a portion of gene encoding red-shifted green fluorescence protein
• nucleotides 1 to 6 are 2,-0-methyl ribonucleotides-other nucleotides are
• SEQ ID NO: 14 PCR primer GFP-kBl to amplify a portion of gene encoding red-shifted green fluorescence protein.
• SEQ ID NO: 15 PCR primer GFP-kB2 to amplify a portion of gene encoding red-shifted green fluorescence protein.

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