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Design and Product of Cosmetics Using Intronic RNA Technology

A ribonucleic acid and microRNA technology, applied in the fields of methods and ingredients, skin pigment-related genes and skin aging genes, can solve problems such as obstacles to skin care use

Inactive Publication Date: 2015-09-23
MELLO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These disadvantages hinder the use of type III RNA polymerase-guided RNAi systems for skin care applications

Method used

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  • Design and Product of Cosmetics Using Intronic RNA Technology
  • Design and Product of Cosmetics Using Intronic RNA Technology
  • Design and Product of Cosmetics Using Intronic RNA Technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Construction of a recombinant gene (SpRNAi-RGFP) comprising SpRNAi Synthesis used to generate three different SpRNAi nucleotide sequences, the sequence contains: N1-sense, 5'-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3' (SEQ.ID.NO.12); N1-antisense, 5'-pCGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGGATCC TCTTAC-3' (SEQ.ID.NO.13); N2-sense,5'-pGTAAGAGGAT CCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3'(SEQ.ID.NO.14); N2-antisense,5'-pCGCGTCAGGA GCGCACCATC TTCTTCAAGC GATCGGATCC TCTTAC-3' (SEQ.ID.NO.15); N3-sense,5'-pGTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GTTAACTTGA AGAAGATGGT GCGCTCCTGA-3' (SEQ.ID.NO.16); N3-antisense,5' - pCGCGTCAGGA GCGCACCATC TTCTTCAAGT TAACTTGAAG AAGATGGTGC GCTCCTGCGA TCGGATCCTC TTAC-3' (SEQ. ID. NO. 17); N4-sense, 5'-pCGCGTTACTA ACTGGTACCT CTTCTTTTTT TTTTTGATAT CCTGCAG-3' (SEQ. ID. NO. 18); , 5'-pGTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3' (SEQ. ID. NO. 19). In addition, the two exon fragments are produced by cutting the 208th nucleoti...

Embodiment 2

[0096] Example 2 Construction of a recombinant gene (SpRNAi-RGFP) comprising SpRNAi into the expression vector

[0097] Because the recombinant SpRNAi-RGFP gene has restriction enzymes such as XhoI and XbaI at the five-end and three-end, and can use these cutting positions to insert the recombinant gene into the vector, the SpRNAi-RGFP and the pHcRed-N1 / 1 vector of 3934bp are 1 : 16 (w / w) mix, and lower the temperature from 65°C to 15°C for more than 50 minutes, then add T4 ligase (ligase) and its buffer and mix at 12°C (12 hours). The SpRNAi-RGFP vector can utilize E.coli DH5αLB (50ug / ml kanamycin (kanamycin) (Sigma Chemical, St.Louis, Mo)) to carry out mass replication, and utilize PCR technology and its RGFP specific primer (SEQ.ID. NO.21) and (SEQ.ID.NO.22) were subjected to 30 cycles of reaction at 94°C (1 minute) and 68°C (2 minutes), and their sequences were confirmed by comparison. To insert the recombinant gene into a viral vector, the same procedure can also be pe...

Embodiment 3

[0099] Example three in vivo transfection

[0100] For the transfection vector into cell lines, zebrafish seedlings and mouse skin, the SpRNAi-RGFP expression vector with anti-EGFP, miR-Tyr or miR-Hyal pre-miRNA insert was first combined with FuGene reagent (reagent) (Roche, IN ) after mixing, and follow the instructions of the FuGene reagent (reagent), then directly place or smear the vectors with insert-free RGFP and SpRNAi-RGFP structures on cell lines, zebrafish seedlings and mouse skin , at this time, the anti-HIV gag-p-24 pre-miRNA insert group was used as the negative control group. The appearance of the cells or tissues and their fluorescent staining were observed after 0, 24, 48 and 72 hours after transfection. For in vivo transfection on human skin, the pre-treated SpRNAi-RGFP carrier solution was prepared by mixing 1-1000 μg with anti-EGFP or without anti-EGFP, miR-Tyr or miR- SpRNAi-RGFP carrier of Hyal pre-miRNA insert, 1ml autoclaved ddH 2 0 and 99 ml of 1...

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Abstract

PROBLEM TO BE SOLVED: To provide a method and a composition for producing a non-naturally occurring intron. SOLUTION: Intronic components of the non-naturally occurring intron can be processed so as to provide shRNA (short-chain hairpin RNA) and / or miRNA (microRNA) molecules by skin cells. Therefore, gene silencing effects specific for a gene related to skin pigments in the cells and / or a gene causing aging can be induced. The gene silencing effects thus obtained are not only useful for lightness and whitening of skin color but also effective in suppressing the activity of an unnecessary aging gene in the skin. COPYRIGHT: (C)2009,JPO&INPIT

Description

technical field [0001] The present invention relates to a method of producing cosmetic products for skin maintenance. More specifically, the present invention relates to a method and composition for producing a non-naturally occurring intron whose composition can be broken down in skin cells into small hairpin RNA (small hairpin RNA, shRNA) or microRNA (miRNA) induces gene silencing effects on specific genes in cells, especially for skin pigment-related genes and skin aging genes. The gene silencing effect is not only effective in skin whitening, but also effective in inhibiting aging genes in skin cells. Background technique [0002] Preventing pigmentation build-up (such as sun damage) and skin aging is the main way to have healthy skin. However, much of the skin pigmentation accumulation and aging process is related to the activity of individual genes. For example, tyrosinase is a glycoprotein on the membrane of melanocytes, and tyrosinase is a rate-determining enzyme ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K8/60A61Q19/00A61Q19/02A61P17/16
Inventor 林希龙吴堂熙
Owner MELLO BIOTECH
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