TWI382849B - Method of producing cosmetic product for skin care in ways of gene silencing effect and genetic recombinant nucleic acids encoding gene silencing effector - Google Patents

Description

Method for producing a cosmetic for skin maintenance by gene silencing effect and coding gene Recombinant nucleotide

The present invention is directed to a cosmetic method for producing skin care. More specifically, the present invention relates to a method and composition for producing a non-naturally occurring intron, the component of which is capable of decomposing into small hairpin RNA (shRNA) in skin cells. Or microRNAs (miRNAs) induce gene silencing effects on specific genes of cells, especially for skin pigment-related genes and skin aging genes. The gene silencing effect is not only effective for skin whitening, but also for skin cells to inhibit aging genes.

Preventing pigmentation (such as sunburn) and skin aging is the main method of having healthy skin. However, many skin pigment accumulation and aging processes are associated with individual gene activity. For example, tyrosinase is a glycoprotein on the membrane of melanocytes, which is a rate-determining enzyme for the melanin production process on the skin and hair. In addition, hyaluronan (HA) is a major anti-aging intercellular substance in skin cells, and hyaluronidase (Hyal) causes skin wrinkles by decomposing subcutaneous hyaluronic acid. Therefore, the skin can be young and healthy by inhibiting the activity of these enzyme genes.

Currently, there are no prior art techniques associated with hyaluronidase inhibitors for removing wrinkles. Many prior art techniques for skin whitening use hormonally derived inhibitory peptides, small molecule compounds, and plant extracts to inhibit tyrosinase activity. Sex, wherein the plant extract comprises oligopeptide (Pinel US Pat. No. 7,268,108, and Schonrock US Pat. No. 6,852,699), hydroxytetronic acid derivative (Perricone US Pat. No. 7,019,029), benzene-containing compound (Kim USPat) .No. 6,838,481), hydroquinone (Wortzman USPat. No. 6,998,130 and 7,025,977), analogs of diols and triols (Brown USPat. No. 7,250,157), decanoic acid derivatives (Ancira US Pat. No. 6, 710, 076), Ascomycete-derived enzyme (Mammone US Pat. No. 6, 514, 506) and plant extracts (Nagamine US Pat. No. 7, 192, 617; Lee US Pat. No. 7, 125, 572; Steck US Pat. No. 6,521,267; Pauly USPat. No. 7,105,184; Leverett USPat. No. 6,994,874, 7,060,304, and 7,247,321; Arquette USPat. No. 7,025,957, 7,029,709, and 7,097,866; Chaudhuri USPat. No. 6,649,150 and 6,969,509). Although these substances and methods may be effective in vitro, only a few of them can cause the effects of lightening pigments in clinical trials (Solano et al. (2006), Pigment Cell Res. 19: 550-571). However, all quinone derivatives derived from benzenediol derivatives are cytotoxic. Therefore, the gap between in vivo and in vitro trials suggests the need for more innovative ways to demonstrate the safety and efficacy of these compounds.

Recently, advances in RNAi technology, novel ribonucleic acid reagents have been revealed to have the potential to inhibit specific genes, including the use of short double-stranded ribonucleic acid/small interfering ribonucleic acid (siRNA) (eg dsRNA/siRNA) (Fire et al. (1998) Nature 391: 806-811; Elbashir et al. (2001) Nature 411: 494-498) and deoxyribonucleated ribonucleic acid interference molecules (eg D-RNAi) (Lin Et al. (2001) Biochem. Biophys. Res. Commun. 281:639-644). Therefore, perhaps these ribonucleic acid reagents can be used to develop novel cosmetic designs and products for the skin. In theory, the RNA interference (RNAi) mechanism triggers a post-transcriptional gene silencing (PTGS), in which the transcript gene silences the specific gene at a nanoscale dose, and the ribonucleic acid interference mechanism is confirmed to have a sustained effect. And it is less toxic than traditionally using antisense oligonucleotides or small molecule inhibitor gene knockout methods (Lin et al. (2001) Current Cancer Drug Targets 1 : 241-247). With many papers published (Grant, SR (1999) Cell 96 : 303-306; Elbashir et al. (2001) supra ; Lin et al., (2001) supra ; Lin et al. (2004a) Drug Design Reviews 1 : 247-255 ), small interfering ribonucleic acid (siRNA)-induced gene silencing can last for more than a week, but the effects of deoxyribonucleotide-RNA interfering molecules (D-RNAi) can last for a month. The siRNA/D-RNAi triggers a series of intracellular specific sequence information ribonucleic acid (mRNA) decomposition and its translational inhibition process, however siRNA/D-RNAi may also affect all transcripts with similar sequences, a phenomenon known as co-suppression ( Co-suppression). For example, a nucleotide of a foreign gene or a viral gene is a small ribonucleic acid fragment produced by treatment with RNase III endoribonucleases (Dicer) and RNA-directed RNA polymerases (RdRp). Produce a common inhibition. Based on the established ribonucleic acid interference mechanism, the prior art has focused on the inhibition of tyrosinase activity by synthetic siRNA and double-stranded ribonucleic acid technology (Binetti US Pat. Application Publication No. 20050137151 and Collin-Djangone US Pat. Application) Publication No.20070134188)

Although ribonucleic acid interference technology can inhibit specific gene activity in the skin, its application has not been confirmed to be stable and safe in higher vertebrates, especially in fish, birds, mammals and humans. For example, almost all vertebrate siRNA agents in the form of double-stranded ribonuclease exhibit non-specific ribonuclease-like decomposition by interferon (Stark et al. (1998) Annu. Rev. Biochem. 67 :227-264; Elbashir et al. supra ; US Pat. No. 4, 289, 850 to Robinson; Lau US Pat. No. 6, 159, 714). This cytotoxic response caused by interferon reduces the silencing effect of specific gene genes and decomposes non-specific ribonucleic acids in vertebrate cells. Particularly in mammalian cells, when the size of the siRNA/double-stranded ribonucleic acid is greater than 25 base-pairs, the ribonucleic acid interference effect will decompose the non-specific ribonucleic acid. Transfection of siRNA or shRNA less than 25 base pairs may not overcome the above problems, as Sledz et al. and Lin et al. have demonstrated that high doses of siRNA and shRNA (>250 nM in human T cells) can cause a similarity greater than 25 Severe cytotoxicity by base pair double-stranded ribonucleic acid. This toxicity is caused by the double-stranded ribonucleic acid structure by activation of non-specific ribonuclease-like breakdown and apoptosis caused by interferon-like processes via the PKR and 2-5A system pathways in the cell. It is well known that interferon-induced protein kinase PKR stimulates apoptosis, and activation of 2',5'-oligoadenylate synthase (2-5A system) triggered by interferon will cause widespread The cleavage of a single ribonucleic acid, while both the PKR and 2-5A systems have a double-stranded ribonucleic acid junction. In addition to this problem, the most difficult problem is the transmission of these unstable siRNA/shRNAs into the body, as the activity of RNase is high in vertebrates (Brantl S. (2002) Biochimica et Biophysica Acta 1575 , 15-25).

Since the ribonucleic acid interference is caused by a small ribonucleic acid product (21-25 nucleotide base) derived from a transcription nucleotide of a foreign gene or a viral gene, the Pol-III-mediated siRNA/shRNA expression vector (Pol- III-mediated siRNA/shRNA expression vector) provides a relatively stable ribonucleic acid silencing effect in vivo (Tuschl et al. (2002) Nat Biotechnol. 20 :446-448). Although the prior art (Miyagishi et al. (2002) Nat Biotechnol 20 : 497-500; Lee et al. (2002) Nat Biotechnol 20 : 500-505; Paul et al. (2002) Nat Biotechnol 20 : 505-508) The siRNA approach has successfully maintained a stable gene silencing effect, whereas their strategy has lost the focus of ribonucleic acid silencing on specific cell types or tissues because of the use of a third-type ribonucleic acid polymerase promoter (Pol-III). Promoter). Type III ribonuclease polymerase promoters such as U6 and H1 are activated in almost all cell types such that tissue-specific silence genes are unlikely to occur. Furthermore, because the third type of ribonucleic acid transcription often occurs in a small segment of DNA without a normal transcriptional stop, resulting in a ribonucleic acid greater than 25 base pairs, the longer ribonucleic acid will produce an unexpected similarity. Interferon cytotoxicity (Gunnery et al. (1999) J Mol Biol . 286 : 745 757; Schramm et al. (2002) Genes Dev 16 : 2593-2620). Furthermore, competitive conflicts with the third type ribonucleic acid polymerase promoter and other vector promoters (e.g., LTR and CMV promoters) can also cause problems. In addition, the third type of ribonucleic acid polymerase-guided ribo-interceptor RNAi system produces high concentrations of siRNA/shRNA, which in turn oversaturates the microRNA (miRNA) pathway in the cell, thus producing a wide range. miRNA inhibition and cell death (Grimm et al. (2006) Nature 441 : 537-541). These shortcomings cause the use of a third-type ribonucleic acid polymerase-directed ribonucleic acid interference system to create an obstacle to skin care applications. In short, in order to improve the stability, specificity and safety of the ribonucleic acid interference technology in the delivery system, a novel ribonucleic acid interference mechanism is used to efficiently, stably and safely use genetic engineering methods and components and to inhibit specific genes. Still waiting for its needs.

According to the study of gene transcription (such as the message ribonucleic acid (mRNA)), the exon composition with protein translation function is fully described in the literature, and the intron without protein translation function will be Decomposed (Nott et al. (2003) RNA 9 : 607-617). However, is the intron really a genetic waste without any function? This recent misunderstanding has been corrected by the observation of intronic microRNAs (Lin et al. (2003) Biochem Biophys Res Commun . 310 :754-760; Ying et al. (2004) Gene 342 :25- 28; Ying et al. (2005) Biochem Biophys Res Commun . 326 : 515-520). Intronic microRNAs (intronic miRNAs) are a group of small single-stranded ribonucleic acids derived from introns, in which introns are further broken down into small-clip microRNAs, usually with a miRNA size of 18 Between the nucleotides of 27, and directly degrade the message ribonucleic acid of the specific gene it complements or inhibit the protein translation of the specific gene it complements. From this point of view, the intronic miRNA is similar to the aforementioned siRNA/shRNA, but the difference is that the siRNA/shRNA does not require a second type of ribonucleic acid polymerase to process them. In addition, because introns usually contain many translational stopers for nonsense-mediated dacay (NMD) systems to identify most of the ribonucleic acids are rapidly degraded to avoid accumulation in cells and then produce toxicity (Zhang Et al . (1994) Nature 372 : 809-812; Lewis et al . (2003) Proc. Natl. Acad. Sci. USA 100 : 189-192). It is estimated that approximately 10% to 30% of the spliced introns are retained in the NMD system and transported to the cytoplasm to remain to their decomposition, suggesting that the cells themselves have the original intronic miRNA (Clement et al . (1999) RNA 5 : 206-220).

The present invention provides a gene silencing effect for inducing ribonucleic acid (RNA) splicing and processing correlation in human cells. The method comprises the steps of: step a. constructing a genetic recombinant nucleotide, wherein the genetic recombinant nucleotide comprises at least one meson having an intron of a gene silencing effector (intronic insert) Intron, the gene silencing effector is linked by an exon, and the intron can be separated from the exon to induce RNA-mediated gene silencing. And the exon is linked to be translated into a protein; step b. is to cloning the recombinant nucleotide into the vector; and step c. transfecting the vector into a plurality of human cells, wherein the human cell Generating a plurality of genetically recombinant nucleotides, a precursor RNA, at which time human cells spliceosomes are spliced to the intron and are separated from the precursor ribonucleic acid such that a gene silencing effector is produced Silence has a gene that is complementary and homologous to the gene silencing effector sequence.

The present invention also provides a genetically modified recombinant nucleotide for inducing ribonucleic acid splicing, comprising at least one intronic insert having an intronic insert (also referred to as SpRNAi in the present invention), wherein Introns are ligated by exons and can be isolated by cellular RNA splicing machinery; and a plurality of exons, wherein exons can be joined to form a specific Functional gene.

As shown in Figure 1, the intronic miRNA production process of introns in cells depends on the precursor message ribonucleic acid (pre-mRNA) and intron splicing transcribed by the second type of transcription (polymerase) system. Clustering of (spliceosome), this process occurs in the vicinity of nuclear stains in the nucleus (Lin et al. (2004a) supra ; Ghosh et al. (2000) RNA 6 : 1325-1334). In eukaryotic cells, a gene transcription molecule having a translational protein function (for example, a message ribonucleic acid mRNA) is produced by a second type ribonucleic acid polymerase (Pol-II). The transcription of the gene produces the precursor message ribonucleic acid (pre-mRNA), which has four major components: the 5'-untranslated region (UTR), and the exon (with the ability to translate the protein) Protein-coding exon), a non-coding intron and a 3'-untranslated region, and the recombinant nucleotide of the present invention comprises an intron With exons. . In general, the five-terminal-untranslated region and the three-terminal-untranslated region can be regarded as introns with no translation function; among them, the intron without translation function accounts for most of the sequence of the entire precursor message ribonucleic acid transcription molecule. Wherein each intron has a length in the range of about 30,000 base pairs and needs to be spliced to turn the pre-mRNA into a mature message ribonucleic acid (mRNA). This becomes a mature mRNA process called splicing of ribonucleic acid, where this process is performed by a splicosome. After ribonuclear splicing, some intron ribonuclease fragments are further processed into microRNAs (miRNAs) that individually and efficiently silence specific genes via ribonucleic acid interference gene silencing effects (mechanisms). At the same time, exons are ligated to form mature message ribonucleic acid for protein translation synthesis.

We have demonstrated that the intron of the vertebrate gene is effective in producing mature miRNAs, however this process differs from siRNA and intergenic miRNAs (exonic miRNAs) (Lin et al . (2003) Supra ; Lin et.al. (2005) Gene 356 : 32-38). To confirm the difference, Figure 2 shows the comparison between the original generation process and the ribonucleic acid interference mechanism in siRNA, intergenic miRNA, and intronic miRNA. Wherein, the siRNA is formed by a double-stranded complementary ribonucleic acid which is transcribed by a promoter at the opposite position of the same DNA template and further digested by a third type of ribonucleic acid. The enzyme (RNase III endoribonuclease, Dicer) is processed to a size of 20 to 25 base pairs. Unlike siRNA, the generation process of intergenic miRNAs (eg, lin-4 and let-7) is involved in a transcriptional molecule that does not have a transcriptional function, such as a second-type transcriptional polymerase system or It is a third type of transcriptase polymerase system. However, the intron microRNA is transcribed and released by a second type of transcriptional polymerase system to form a spliced intron. In the nucleus, the precursor microRNA is mediated by a Drosha- like RNase (which acts to generate an intergenic miRNA) or a nonsense-mediated degradation (NMD) system. The miRNA is spliced to form a stem-loop like a hairpin, called a precursor microRNA, and transported to the cytoplasm to act as a mature miRNA. Next, all three ribonucleic acids were finally combined with an RNA-induced silencing complex (RISC). However, the third type endoribonuclease and ribonucleic acid induce a pathway in which the silent complex acts on siRNA and miRNA (Tang, G. (2005) Trends Biochem Sci . 30 : 106-114). Therefore, the effect of miRNA is more specific than that of siRNA, because miRNA is only a single-stranded ribonucleic acid. In other words, siRNA mainly catalyzes the degradation of mRNA, while miRNA can trigger the degradation of specific mRNA or inhibit the synthesis of specific proteins. Because the intronic miRNA pathway is regulated by many intracellular systems that contain the second-type transcriptional polymerase system, the ribonucleic splicing system, and the NMD system, the gene silencing effect of the intronic miRNA is thought to be due to these three ribonucleic acid interferences. Path is one of the most efficient, specific, and safe (Lin et al . (2008) Frontiers in Bioscience 13 : 2216-2230). Meanwhile, the genetic recombinant nucleotide of the present invention is produced by a transcription system, and the transcription system is derived from a second type transcription system (Pol-II), a first type transcription system (Pol-I). And choose one of the viral transcription systems.

The present invention discloses a novel function of an intron for gene regulation, as shown in Figures 3A and 3B, based on the mechanism of splicing and processing of intronic miRNA, a preferred embodiment of the present invention is a second type ribonuclease gene expression system (Pol-II-mediated recombinant gene expression system) can comprise a splicing of intron (designated as SpRNAi), which can inhibit specific gene SpRNAi via complementary sequence. This SpRNAi is derived from a precursor ribonucleic acid (pre-mRNA) formed by a second type ribonucleic acid polymerase system. After splicing, SpRNAi is further processed into mature gene silencers (such as shRNA and miRNA). After the intron is removed, the exon-recombinant transcriptional molecules of the exons are joined to form a mature message ribonucleic acid molecule that is further translated into a protein. The recombinant nucleotide of the present invention further comprises at least one multiple restriction/cloning site for linking to a expression vector of a ribonucleic acid transcription molecule representing a recombinant nucleotide of the gene; and a plurality of The transcription and translation termination sites of the correct ribonucleic acid transcription molecule that forms the recombinant nucleotide of the gene. The intron comprises an intronic insert (ie, a precursor microRNA carrier) having a gene silencing effector, a five-terminal splicing site, a three-terminal splicing site, a branching point region, and a polypyrimidine region.

As shown in Figure 3A, the essential components of SpRNAi contain several identical nucleotide sequences, including a 5'-splice site, a branch point motif (BrP), and a polypyrimidine. A poly-pyrimidine tract and a 3'-splice site. In addition, a shRNA-like precursor microRNA carrier is inserted into SpRNAi , wherein the precursor microRNA carrier is located between the five-terminal splicing and the branching region. This SpRNAi forms a set of lariat structures upon splicing of ribonucleic acids. The U2 and U6 small nuclear ribonucleoproteins (snRNPs) of the splice body are involved in the release and excision of the set of horses to form the precursor microRNA. In addition, the three ends of SpRNAi contain a multi-translation stop region, which is to enable the intronic miRNA splicing and NMD processes to be performed accurately. When the multiple translational stop codon region is expressed in the cytoplasmic message ribonucleic acid, the multi-translating stopr can transmit a message that activates the NMD system pathway to degrade any abnormal ribonucleic acid structure in the cell. However, the shRNA and pre-miRNA inserted into SpRNAi can be individually retained to Dicer for splicing to form mature siRNA and miRNA. Plus, in order to show the cells, we will use Drall SpRNAi restriction sites connect a red fluorescent protein (red fluorescent protein, RGFP) gene (from Heteractis crispa) to form a recombinant gene containing the red fluorescence protein The SpRNAi ( SpRNAi-RGFP ) gene, in which the twenty-eighth nucleotide position of the red fluorescent protein gene is blocked by the restriction enzyme Drall , an AG-GN nucleotide breakpoint is generated, and two breakpoints are generated at the breakpoint. The terminal produces a structure in which three nucleotides protrude, and after the SpRNAi is inserted, the two ends can form a five-terminal splicing site and a three-terminal splicing site. Because the embedding of SpRNAi causes the red fluorescent protein to fail to perform normally, the red fluorescent protein can return to normal after the intron splicing. We can use the red fluorescent protein expression to determine the shRNA/miRNA of the intron. The amount released, at this time, red fluorescent protein can also provide many exonic splicing enhancers (ESE) to help the correctness and efficiency of ribonucleic splicing.

In another embodiment, as shown in Figure 3B, the present invention provides a genetic engineering method using synthetic synthetic ribonucleic splicing and processing elements to generate a non-native gene, such as a man-made five-terminal splicing site, a branching point region, and a polypyrimidine. and a three-terminal region of the splice is formed of SpRNAi an artificial (man-made intron), the SpRNAi this artificial structure comprising at least one RNA of insertion, this structure may be the structure-based or shRNA or miRNA of antisense RNA (antisense RNA). These elements can be chemically synthesized and linked by a DNA synthesizer (from Sigma-Genosys services (Woodlands, TX)) (eg, a spRNAi intron such as a miR-tyr or an intron such as a meson) Mir-302). On the other hand these elements can also be manufactured and linked with restriction enzymes. This SpRNAi line can be directly transfected into cells or co-transfected into cells with different cellular genes to be manipulated by a second type of transcriptional polymerase system. After ribonucleic splicing and message ribonucleic acid maturation, the ribonucleic acid structure inserted in SpRNAi will inhibit the transcription of specific genes via intracellular splices and NMD systems. At this point, the exon can be normally ligated to form a mature message ribonucleic acid to display the function of the gene, such as reporter gene translation or the following marker proteins: red fluorescent protein, green fluorescent protein (green fluorescent protein) , luciferase, lactose gene regulatory group (lac-Z) and derivatives thereof. The presence of reporter/marker proteins is useful for understanding the location of shRNA/miRNA molecules, particularly in identifying gene silencing effects.

In addition, mature message ribonucleic acid may also help to repair damaged or lost genes by traditional gene therapy, and may also help to improve specific gene expression. In other aspects, the invention provides novel components ( spRNAi (artificial intron)) and methods for inducing a cell to generate a gene silencing molecule via an intron ribonuclease splicing and processing mechanism, the silencing molecule via an antisense gene Elimination or RNA interference effects to inhibit specific gene function. Gene silencing molecules derived from the artificial intron SpRNAi include antisense RNA, ribozyme, short temporary RNA (stRNA), double-stranded ribonucleic acid, small interfering ribonucleic acid (small) Interfering RNA, siRNA), tiny non-coding RNA (tncRNA), short hairpin RNA (shRNA), hairpin-like RNA structure, microribose Nucleic acids (microRNAs, miRNAs) and pre/precursor ribonucleic acid structures involved in ribonucleic acid interference. In the present invention, the recombinant nucleotide of the gene is a gene of a cell itself having an intron. Gene silencing reagents derived from ribonucleic acids (recombinant nucleotides) using these introns have an effect on the silencing of specific genes, which are derived from pathogenic transgenes, viral genes. (viral genes), mammalian genes, jumping genes, mutant genes, oncogenes, disease-related small RNA genes, protein-coding genes, and any with or without A gene with a protein translation-related gene and a mixture of the above genes is selected.

The present invention uses the SpRNAi-RGFP expression system of the red fluorescent protein-containing SpRNAi by this second type of transcription polymerase, and we have succeeded in human prostate cancer cells (prostate cancer LNCaP), human cervical cancer. (human cervical cancer HeLa) and rat neuronal stem HCN-A94-2 cell (Lin et al . (2006a) Methods Mol Biol . 342 :295-312) produce mature shRNA with gene silencing effect and miRNAs, as in zebrafish, chicken, and mouse (Lin et al . (2006b) Methods Mol Biol . 342 :321-334). We have tested different precursor microRNAs in zebrafish and many human cell lines against green fluorescent protein and other cellular genes, and learned that the more efficient gene silent microRNA is between the five-terminal splicing and branching regions. between. As shown in Figure 3C, a significant gene silencing effect occurs in the group of trans- GFP pre-miRNAs (line4) transfected with green fluorescent protein (ie, the miR group), however other No effect was detected in the experimental group and the control group; these experimental groups and the control group were sequentially (left to right) 1 and control group (blank vector control, Ctl). 2. A control group for the pioneering microRNA of the HIV virus ( HIV-p24 ). 3. The anti group of the antisense EGFP insert without the hairpin loop structure and the miR* group of the inverted precursor microRNA sequence; the inverted precursor microRNA sequence The lineage is fully complementary to the anti- EGFP pre-miRNA (miR*) against green fluorescent protein. There is no silent phenomenon in non-targeted genes, such as red fluorescent protein and actin, indicating that the RNA interference of the RNA interference of SpRNAi is highly specific. To confirm the ribonucleic acid interference effect of ribonucleic acid splicing in introns, we have tested the expression system of the red fluorescent protein-containing SpRNAi of these different second-type transcriptases, as shown in Figure 3D from left to right. . The red fluorescent protein carrier group without introns 2. The vector expressing RGFP with an intronic anti- EGFP pre-miRNA insert 3. A group of red fluorescent protein carriers having an intron-resistant green fluorescent protein precursor microRNA (five-terminal splicing defect). According to Northern bolting analysis, the mature miRNA (ie, the microRNA of the intron) is only in the red fluorescent protein carrier group with the anti-green fluorescent protein precursor microRNA of the intron. It was formed, thus confirming the need to form an intronic miRNA by an intracellular splicing mechanism.

In addition, we have further determined the structural design of the preferred precursor-pre-miRNA insert to induce optimal gene silencing effects via the RISC complex (Lin et.al. (2005) Gene 356 :32 -38). The RISC complex, a protein-RNA complex, can cause the degradation of specific gene transcriptional molecules or inhibit translation via a ribonucleic acid interference mechanism. For the formation of RISC complexes, double-stranded siRNA plays an important role, the double-stranded siRNAs are functionally distinct, and the RISC complexes tend to respond to one of them. Based on the siRNA model, it is speculated that the ribonucleic acid formed by the miRNA and its complementary ribonucleic acid is also important for the formation of the RISC complex. If this is true, the RISC complex should be both miRNA and its complementary ribonucleic acid. Degraded is correct, however, based on experimental results, the RISC complex has a specific preference for miRNA.

As shown in Figure 4A, two different intronic miRNA-inserted SpRNAi-RGFP expression vectors were named miRNA*-stemloop-miRNA [1] (also This is the circular expression vector indicated above Figure 4A and miRNA-stemloop-miRNA* [2] (also known as the circular expression vector shown below in Figure 4A) (miRNA* represents a miRNA that is complementary to the mature miRNA sequence) ). The two sets of vectors having different microRNAs comprise the same double loop structure, which can silence the nucleotide sequences 280 to 302 of the EGFP gene. After transfection of these vectors (60 ug each) into the zebrafish embryos for 24 hours, microRNAs (miRNAs) with silent effect potential were precipitated via latex beads in the mirVana microribonucleic acid separation column. After sequence alignment, the silencing effect of the microRNA (miRNA) was selected as miRNA-stemloop-miRNA* [2], as shown in Figure 4A. Because mature microRNAs (miRNAs) are only found in zebrafish transfected with miRNA-stemloop-miRNA*[2], it is postulated that RISC complexes tend to interact with miRNA-stemloop-miRNA*[2] rather than miRNA* -stemloop-miRNA [1]. In this experiment, experiments were carried out using zebrafish (Tg (actin-GAL4: UAS-gfp)) expressed by an actin promoter, which has been expressing green fluorescent protein in various cells. As shown in Figure 4B, this zebrafish was transfected with the SpRNAi-RGFP vector and expressed a red fluorescent protein which can be used as an indicator protein. As a result, it was observed that the gene silencing effect of the gastrointestinal part of SpRNAi-RGFP was weak, and it was speculated that the RNase activity of this site was strong. In Figure 4C, Western blotting detected red fluorescent protein expression in miRNA*-stemloop-miRNA[1] (miR* group) and miRNA-stemloop-miRNA*[2] (miR group). However, the gene silencing of the green fluorescent protein was only found in the zebrafish transfected with miRNA-stemloop-miRNA*[2] (miR group), as shown in Fig. 4B. Because the structure of miRNA*-stemloop-miRNA[1] and miRNA-stemloop-miRNA*[2] is very similar, the intronic pre-miRNA of the intron premature-stem-loop May be associated with the formation of mature miRNAs in the RISC complex.

Because the ring structure of the precursor micro-ribonucleic acid (pre-miRNA) is too large, the performance of the SpRNAi-RGFP vector may be poor, so the ring of the trans-ribonucleic acid (tRNA) is used (5'-(A/U) UCCAAGGGGG-3') replaces the larger ring structure, which has been shown to accelerate the transport of microRNAs (miRNAs) from the nucleus to the outside of the nucleus. Recently, the present invention uses a modified pre-mir-302 ring structure (e.g., 5'-GCTAAGCCAGGC-3' SEQ. ID. NO. 1 and 5'-GCCTGGCTTAGC-3' SEQ. ID. NO. 2) It provides the same rapid precursor micro-ribonucleotide (pre-miRNA) transport and does not interfere with the transport of transgenic ribonucleic acid (tRNA). The improved pre-mir-302 ring structure is highly expressed in embryonic stem cells but is less expressed in differentiated cells, and thus the modified pre-mir-302 ring structure does not interfere with its own ribose. Nucleic acid pathway.

Regarding the insertion process of the precursor micro-ribonucleic acid (pre-miRNA), since the restriction enzyme digestion of the recombinant SpRNAi-RGFP is located at the five-terminal and three-terminal, respectively, PvuI and MluI , the intronic insert can be Many other specific gene precursors are replaced by ex-miRNA microRNAs (eg, anti-green fluorescent protein and anti-tyrosin precursor microRNA). By using a precursor microRNA pre-miRNA insert to counter or silence different gene transcription molecules, the intronic miRNA production system of this intron can be applied to induce gene silencing effects. Powerful tool. In order to determine the correct size of the insert, SpRNAi-RGFP (10 ng) can be utilized by PCR (eg, 5'-CTCGAGCATG GTGAGCGGCC TGCTGAA-3' and 5'-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3 ') 25 cycles were replicated at 94 ° C (1 min.), 52 ° C (1 min.), and 70 ° C (1 min.). The final product of the final PCR was further purified by 2% agarose gel chromatography and confirmed by gel extraction kit (Qiagen, CA).

The present invention employs a second type polymerase (Pol-II) SpRNAi-RGFP expression system and utilizes this system to develop novel cosmetics for skin care. In a preferred embodiment, the present invention provides a method of using a non-naturally occurring intron which enables the intron to be treated by shRNA and miRNA by skin cells to induce skin pigment related genes and skin aging. Gene silencing effects on genes. The method comprises several steps: a step provides 1) a gene capable of expressing a target for skin receptors and 2) an intron-producing ribonucleic acid transcription molecule capable of generating a gene silencing effect in a skin receptor (primary Recombinant gene of RNA transcript); step b treatment of the skin receptor with the intron pre-transcription RNA transcript to silence or inhibit the function of the target gene. The skin is capable of expressing the underlying gene in vitro , in vivo , in vitro assays from the body, or ex vivo . At some levels, a precursor microRNA transcriptional molecule (pre-miRNA) that produces a gene silencing effect (like an intronic insert) can target specific genes such as tyrosinase (Tyr), hyaluronidase (Hyal), The receptors for hyaluronic acid such as adhesion molecules CD44, CD168, oncogenes and NF-kappa B have silent or inhibitory effects with other genes involved in pigmentation and skin aging. In other embodiments, a precursor pre-miRNA insert (or a recombinant nucleotide of the invention) can be incorporated into an intron fragment of a cellular gene by genetic engineering. In general, this intron insertion technique involves plasmid-like transgene transfection, homologous recombination, transposon delivery, and deoxyribose DNA ligation, insertion of transgene insertion, jumping gene integration, and retroviral infection.

In another embodiment, the recombinant gene of the invention behaves in association with a precursor message ribonucleic acid (pre-mRNA). The recombinant gene consists of an exon and an intron. Exons can be ligated after ribonuclease splicing to form a functional message ribonucleic acid (mRNA) and then translated into protein, while introns are released from the nucleus and processed into a gene silencing effect with a specific gene silencing function. Gene silencing effector, the gene silencing effector contains antisense RNA, microRNA (miRNA), shRNA, siRNA, double-stranded ribonucleic acid, and precursors of the above ribonucleic acid ( For example, pre-miRNA and piRNA). The intronic gene silencing effector of these introns may comprise a hairpin-like stem-loop structure (approximately equivalent to a small-clamped nucleotide), which is a sequence of this structure. Homologous to 5'-GCTAAGCCAGGC-3' (SEQ.ID.NO.1) or 5'-GCCTGGCTTAGC-3' (SEQ. ID. NO. 2), which promotes splicing of the correct ribonucleic acid molecule and facilitates transport of the molecule from the nucleus to the cytoplasm. Moreover, these intronic inserts contain sequences that are complementary or homologous to a particular target gene. The sequence of the intronic insert of the homologous or complementary intron is between about fifteen base pairs and one thousand five hundred base pairs, preferably eighteen to twenty-seven base pairs. about. These intronic inserts are similar to the complementarity or homology of small nucleotides to the target gene sequence of between about 30% and 100%, preferably between 35% and 49%. An intronic insert is a reverse nucleotide comprising a sequence complementation rate of 30% to 100% for the sequence of the gene to be silenced, and a preferred sequence complementation rate ranges from 90% to 100%.

In addition, the five-terminal end of the non-naturally occurring intron comprises a 5'-splicing site or 5' clip, wherein the five-terminal splicing is a nucleotide sequence homologous to 5'-GTAAGAGK-3 '(SEQ.ID.NO.3) or GU(A/G)AGU sequence (eg, 5'-GTAAGAGGAT-3', 5'-GTAAGAGT-3', 5'-GTAGAGT-3' and 5'- GTAAGT-3'). The 3'-splicing site or 3' clip sequence is homologous to the GWKSCYRCAG (SEQ.ID.NO.4) or CT(A/G)A(C/T)NG sequence (eg, 5' -GATATCCTGCAG-3', 5'-GGCTGCAG-3', 5'-CCACAG-3'). In addition, a branch point sequence is located between the five-terminal and three-terminal splicing, and the branch point region is located in a nucleotide sequence homologous to 5'-TACTWAY-3' (SEQ.ID.NO.5). (eg, 5'-TACTAAC-3' and 5'-TACTTAT-3'), the branching point region contains a branching point, and the branching point is an adenosine (A) capable of forming a set of horses (lariat) structure. In addition to the polypyrimidine A poly-pyrimidine tract is located between the branching point region and a three-terminal splicing region, wherein the polypyrimidine region is a nucleotide sequence having a plurality of thymines (cytosine) and a cytosine (Cytosine), the polypyrimidine The nucleotide sequence of the poly-pyrimidine tract is homologous to 5'-(TY)m(C/-)(T)nS(C/-)-3' (SEQ.ID.NO.6) and 5 '-(TC)nNCTAG(G/-)-3' (SEQ.ID.NO.7). Wherein, "m" and "n" mean a repeating sequence of one or more, preferably the number of sequences of m is between one and three and the number of sequences of n is between seven and twelve. "-" means no nucleotides. All of these introns are linked by a number of linked nucleotides, based on 37 CFR 1.822 (Taiwan "nucleotide and amino acid sequence listing format"), and W is adenine (A) ) or thymine (thymine (T)) / uracil (U), K refers to guanine (G) or thymine (T) / uracil (U), S refers to cytosine ( C) or guanine (G), Y refers to cytosine (C) or thymine (T) / uracil (U), R refers to adenine (A) or guanine (G) and N refers to adenine (A), cytosine (C), guanine (G) or thymine (T) / uracil (U) or others.

In another preferred embodiment of the invention, the recombinant gene nucleotide sequence can be recombinantly incorporated into a performance vector for gene transfection. The expression vector comprises plasmids, cosmids, phagemids, yeast artificial chromosomes, jumping genes, transposons, transgenes (transgenes) ), retrotransposons, retroviral vectors, viral vectors such as lentiviral vectors, lambda vectors, adenoviral vectors (AMV) vectors, adeno-associated viruses Vector (adeno-associated viral (AAV) vectors), modified hepatitis-viral (HBV) vectors, cytomegalovirus (CMV)-associated viral vectors, and plant-related flowers Plant-associated mosaic virus, for example, tabacco mosaic virus (TMV), tomato mosaic virus (ToMV), Cauliflower mosaic virus (CaMV) And poplar mosaic virus (PopMV). In the process of transfection of SpRNAi-RGFP , these vectors which exhibit introns of different gene silencing effectors can be used to achieve the silent effect of a single or multiple target genes. In other embodiments, a plurality of different gene silencing effectors can be generated from the shRNA meson of SpRNAi-RGFP to silence a plurality of genes. The advantage of this approach is to provide a stable and relatively long-term specific gene silencing effect by using transgenic gene transfection or viral infection. Among them, the present invention can generate RNAi-related gene silencing molecules via a cell-nuclear chromosomal splicing and processing mechanism, and the molecule comprises small interfering RNA (siRNA) and microRNA (microRNA). (miRNA)) and small hairpin RNA (shRNA). It is controlled by a specific gene ribonucleic acid promoter in the cell, for example, a second type ribonucleic acid polymerase promoter (Pol-II promoter) and a viral promoter. The viral promoter comprises a cytomegalovirus (CMV), a retrovirus long-terminal region (LTR), a hepatitis B virus (HBV), and an adenovirus (adenovirus). AMV)), adeno-associated virus (AAV) and ribonucleic acid promoters of viruses such as plant-associated mosaic virus. For example, the lentiviral LTR promoter provides more than 5x10 ⁵ copies of the precursor message ribonucleic acid per cell. However, a pair of drug-sensitive repressors are inserted at the front of these viral promoters to control their expression rate. The inhibitor can be inhibited by chemical drugs or antibiotics, including G418, tetracycline, neomycin, ampicillin, kanamycin, and derivatives of the above antibiotics.

In addition to novel skin care applications, the potential applications of the present invention include skin treatments, such as by silencing or inhibiting disease-related genes, epidermal vaccines against viral genes, external treatments for microbial-related pathogens, and skin message conduction pathways. Research and combination of rapid test methods for biochip gene function. The present invention can also be used as a tool for studying gene function on skin cells or as a component and method for improving skin condition, which includes normal, pathological, cancerous, viral infection, microbial infection, physiological disease, genetic mutation. Animal or human skin.

The present invention provides a novel method for producing intronic RNA in cells, preferably to produce mature siRNA, miRNA and shRNA, which can induce ribonucleic acid interference gene silencing effect (RNAi/PTGS - Associated gene silencing effects). Wherein, the cells can act on the intronic insert of the present invention to produce a single or multiple gene silencing effector, for example, the anti-green fluorescent protein precursor microRNA (pre-miRNA) is expressed in the zebra by the foreign body. On the fish, as shown in Figure 4A, two sets of mature microRNAs (mir-EGFP 282/300 and mir-EGFP280-302) were generated, suggesting that a single SpRNAi meson has more than one gene silencing effect. Gene-silencing effectors. Different spliced RNA effectors can produce a sense or antisense configuration. In some instances, the spliced ribonucleic acid can hybridize with some of the underlying gene transcription molecules to form a double-stranded ribonucleic acid to drive the ribonucleic acid interference effect. At the same time, expression of siRNA, miRNA and shRNA on the expression vector of the present invention can alleviate the suspicion that ribonucleic acid is rapidly degraded.

In various embodiments, the invention further provides a method of producing an antisense miRNA of a complementary positive-stranded microribonucleic acid that inhibits a target miRNA in a skin cell. Because microRNAs (miRNAs) carry out ribonucleic acid interference silencing effects, antisense miRNAs have the gene silencing effect of neutralizing positive-sense miRNAs, thus enabling genes that were originally inhibited by microribonucleic acids. Restore function. Unlike the double-stranded siRNA, the antisense miRNA binds to a partial sequence of the sense miRNA, resulting in a mismatched base-paired region that is cleaved and degraded by ribonuclease. The misconnected region may be located in the middle of the stem-arm or in the ring structure of the precursor micro-ribonucleotide (pre-miRNA). Furthermore, on siRNA, this mismatched region has been shown to inhibit the gene silencing effect of siRNA (Holen et al . (2002) Nucleic Acid Res . 30 :1757-1766; Krol et al . (2004) J. Biol.Chem 279: 42230-42239). This phenomenon may be related to intron-mediated enhancement phenomena in plants. According to previous studies of Arabidopsis, intronic inserts play an important role in post-transcriptional modification. The performance is increased or decreased (Rose AB (2002) RNA 8 : 1444-1451; Stoutjesdijk et al . (2002) Plant Physiol . 129 : 1723-1730). The intron promotion phenomenon can be expressed by inhibiting a specific gene expression of a microRNA (miRNA) used to inhibit a specific gene to recover twice or more than ten times.

In order to clearly describe the present invention, the following specific terms are employed herein. However, the invention is not limited to these specific terms. It should be understood that each particular element encompasses all technical equivalents that can be used for similar purposes by similar means.

The term "nucleotide" is used herein to mean a single molecule of deoxyribonucleotide (DNA) or ribonucleotide (RNA) molecules containing pentose, phosphoric acid. Phosphate and nitrogenous heterocyclic base. This base is linked to a five-carbon sugar via a glycosidic bond to form a nucleoside, which is linked to a phosphate by a three-terminal and a five-terminal position of the five-carbon sugar.

The term "oligonucleotide" as used herein means that one molecule contains two More than one DNA or RNA, preferably more than three. Its exact size depends on its optimal functional status. Oligonucleotide lines can be synthesized by chemical synthesis, DNA replication, reverse transcription, and the like.

The term "nucleic acid" as used herein refers to a polymer of nucleotides, either single or double.

The term "Nucleotide Analog" as used herein means that the structure of a purine or pyrimidine nucleotide is different but similar to A, T, C, G or U and can be in a nucleic acid. Replace normal nucleotides.

The term "gene" as used herein means that the sequence of a nucleic acid has the code for RNA or a multi-peptide (protein) which is RNA or DNA.

The term "base pair (bp)" is used herein to mean the pairing of A with T or C with G in a double stranded DNA molecule. In RNA, U is substituted for T to pair with A. In general, base pairs are linked by hydrogen bonding.

The term "precursor messenger RNA (pre-mRNA)" as used herein refers to a gene-promoted RNA transcription in a eukaryotic cell produced by a second-type RNA polyzyme (Pol-II). Primary ribo-nucleotide transcripts, this process is transcription, a precursor message ribonucleic acid sequence contains a 5'-end untranslated region, a 3'-end untranslated region (3'-end untranslated region) ), exons and introns.

The term "intron" is used herein to mean a portion of a gene transcription molecule that has a code for a non-translated region.

The term "exon" is used herein to mean a portion of a gene transcription molecule that has a code for the translation region.

The term "signal ribonucleic acid (mRNA)" as used herein refers to a combination of a precursor message ribonucleic acid exon of an intron cell that has a protein translational code.

The term "complementary deoxyribonucleic acid (cDNA)" is used herein to mean a single strand of DNA complementary to an mRNA sequence which lacks any intron sequences.

The term "sense" as used herein means that the sequence of a nucleic acid molecule is identical to the sequence of mRNA and its composition is homologous to the mRNA. This positive nucleic acid configuration is indicated as "+", "s" or "sense".

The term "antisense" as used herein means that the sequence of a nucleic acid molecule is complementary to the sense of the gene. This antisense nucleic acid configuration is designated "-", "a" or "antisense", such as "aDNA" or "aRNA".

The term "5'-end" as used herein means that a contiguous nucleotide does not have a phosphodiester bond and a third nucleotide at the position of the fifth carbon of the five-carbon sugar. The carbon level to the end of the connection is called the five ends.

The term "3'-end" as used herein means that a contiguous nucleotide does not have a phosphodiester bond and a fifth nucleotide at the position of the carbon of the fifth carbon sugar. The carbon level to the end of the connection is called the three ends.

The term "template" is used herein to mean a nucleic acid molecule which can be negatively produced by a ribonucleic acid polymerase. According to the ribonucleic acid polymerase, the template system can be single-stranded, double-stranded, or partially double-stranded. The synthetic replication nucleic acid is complementary to the template, at least one of the double strands The shares are complementary or partially complementary.

The term "Nucleic Acid Template" is used herein to mean a double strand of DNA, double stranded RNA, DNA-RNA hybrid double stranded or single stranded DNA or single stranded RNA.

The term "conserved" is used herein to mean that a nucleotide sequence is identical to the original sequence.

The term "Complemetary or complementarity or complementation" is used herein to mean a complementary nucleotide, for example, the sequence "AGT" is complementary to the sequence "TCA" or "TCU". Complementation can be between two strands of DNA, between DNA and RNA, between two strands of RNA. Complementary energy is partial or complete. Partial complements have only certain nucleotide base pairings; however, full complementation means that all nucleotide bases are paired. The degree of complementation has an important impact on the efficiency of the two crosses.

The term "homologous or homology" as used herein means that the nucleic acid sequence is similar to the sequence of a gene or mRNA. A nucleic acid sequence may be partially or completely complementary to the sequence of a particular gene or mRNA, for example, homology may also indicate that a certain proportion of similar nucleotides exceeds the total number of nucleotides.

The term "Complemetary base" as used herein refers to the pairing of nucleotides when DNA or RNA forms a double-strand configuration.

The term "Complemetary Nucleotide Sequence" as used herein means that the nucleic acid sequence of a single strand of RNA or DNA is sufficiently complementary to another strand, in which hydrogen bonding forces are used to form a complement.

The term "hybridization" (Hybirdize and Hybridization) is used herein to mean the case where a nucleotide sequence forms a double strand via base complementation. Hybridization, in some cases, provides a DNA polymerase for the initial step of DNA replication after the primer complements a particular nucleic acid sequence.

The term "RNA interference (RNAi)" as used herein refers to a mechanism of transcriptional gene silencing following initiation by eukaryotic cells via small RNA fragments (eg, microRNAs (miRNAs) and siRNAs). These small RNA fragments can be used as gene silencing effectors to interfere with specific gene transcription molecules within the cell to which they are complementary.

The term "microRNA (miRNA)" as used herein means a single-stranded ribonucleic acid (RNA) that is complementary to a particular gene transcription molecule. miRNAs are typically between 17 and 27 nucleotides and are capable of directly degrading the message ribonucleic acid in a cell or inhibiting the translation of a particular gene based on the complementarity between the miRNA and the mRNA. The miRNA of the cell itself can be found in all eukaryotic cells as an important mechanism for preventing viral infection.

The term "pre-miRNA" is used herein to mean a single-stranded ribonucleic acid with a small sandwich structure containing a stem-arm and stem-loop region. The RNaseIII endonuclease interacts to produce a gene silencing effector. This Pre-miRNA can form a complete or partial complement to a specific gene transcription molecule.

The term "small or short hairpin RNA (shRNA)" as used herein means a single strand of a ring structure sequence that has a partial or complete complementarity. Ribonucleic acid. Many microRNAs are derived from shRNA precursors, also known as precursor microRNAs (pre-miRNAs).

The term "Vector" is used herein to mean a recombinant nucleic acid molecule that carries a specific genetic material. Generally, the recombinant nucleic acid molecules are linked into a single loop. This vector is capable of automatic replication in cells. One form of vector is that the episomes can replicate extrachromosomally. Among them, those having the ability to express a gene protein are expression vectors.

The term "Cistron" is used herein to mean a nucleotide sequence having amino acid translation ability and a DNA expression control region at the upper and lower ends of the sequence.

The term "promoter" is used herein to mean a nucleic acid that is recognized by a polymerase to initiate transcription.

The term "antibody" as used herein means a multi-peptide or protein molecule having an interaction with a particular amino acid sequence.

The present invention relates to a method and composition for improving the genetic properties of skin cells by the ribonucleic acid of an intron. This improvement is caused by a gene silencing mechanism of an intron, with an emphasis on this mechanism being induced by a splicing intron (named SpRNAi ). The SpRNAi can carry an intron meson to release the intron of the intron via a mechanism of splicing and processing of ribonucleic acid and inhibit the specific gene in a complementary manner via the RNAi/PTGS silent effect. Generally, as shown in Figures 4 to 7, when the recombinant gene is chemically or lipofected or virally infected into the skin cells, the intronic insert is passed through the second type of ribonucleic acid. The polymerase system is transcribed and released by the splicing of the spliceosome and the mechanism of treatment by the NMD system. After release, the intronic insert of SpRNAi forms a set of lariat RNA and is further processed into gene silencing effectors, including short-temporary RNA. (stRNA)), antisense RNA, siRNA (siRNA), small ribonucleic acid (shRNA), microRNA (miRNA), ribozyme RNA, Piwi-interacting RNA (piRNA) And one of the precursors and derivatives of these ribonucleic acids and the ribonucleic acid mixed with the above ribonucleic acids. These gene silencing effectors will then undergo degradation of their target gene transcriptional molecules or protein translation of the target gene via the RISC complex.

To mimic intracellular pre-mRNA splicing and processing mechanisms, we used intracellular splices and the NMD system to catalyze intron removal and processing of the SpRNAi-RGFP expression system. SpRNAi is released via a cascade of intracellular splices to further form a gene silencing effector. Methods for incorporating splice system identification into SpRNAi are disclosed in Examples 1 and 2, respectively.

Design, construction, and evaluation of a red fluorescent protein ( SRNARNA-RGFP ) expression system that produces a second type of transcriptional polymerase that is a silent effector of introns

The present invention is the use of a second transcription polymerase (Pol-II) of the red fluorescent protein expression vector system (SpRNAi-RGFP expression system) have been demonstrated efficacy, the SpRNAi-RGFP expression system capable of producing gene silencing effectors (gene silencing effector) of SpRNAi, as shown in FIG. 3A and 3B, there are such silent effector miRNA and shRNA. These silent effectors can be released via a cascade of intracellular splices and the NMD system. In other embodiments, however, the ribosomal precursor RNA (pre-rRNA) transcribed by the first-type ribonucleic acid polymerase system can also produce a functionally identical gene silencing effect via the same spirit and principle. Gene silencing effector. In addition, a ribonucleic acid transcription molecule (such as a genetically modified nucleotide precursor ribonucleic acid) that can be used to generate SpRNAi contains a message ribonucleic acid (mRNA), a heteronuclear ribonucleic acid (hnRNA), a ribosome ribonucleic acid (rRNA), a trans-ribose ribose. Nucleic acid tRNA, snoRNA, small nuclear RNA snRNA, precursor microribonucleic acid (pre-miRNA), viral ribonucleic acid (viral RNA) and derivatives and precursors of the above ribonucleic acids.

As shown in Examples 1 to 2 and Figure 3A, SpRNAi was ligated with the Drall restriction enzyme to ligature a red fluorescent protein (RGFP) gene (from Heteractis crispa ) to form a recombination-containing red fluorescent The structure of the SpRNAi ( SpRNAi-RGFP ) gene, the red fluorescent protein can not be expressed normally due to the insertion of SpRNAi , but after the intron is spliced, the red fluorescent protein can return to normal performance, we can use red fluorescent The amount of red fluorescent protein detected at a wavelength of 570 nm is used to determine the amount released by the small ribonucleic acid (shRNA)/microribonucleic acid (miRNA) of the intron. SpRNAi-RGFP construction is based on the structural properties of pre-mRNA, and the major part of SpRNAi (artificial intron) recognition by splices includes a five-terminal splicing site, a branching region, a polypyrimidine region (used to interact with the splice), and Three-end splicing. As shown in FIG. 3B, the SpRNAi (artificial intron) having a silent effector of the present invention comprises a five-terminal splicing site, an intron meson (ie, a precursor microribonucleic acid meson), a branching point region, a polypyrimidine region, and Three-end splicing. In addition, some translation terminators are located near the three-terminal splicing site of SpRNAi .

Generally, the five-terminal splicing is a nucleotide sequence homologous to 5'-GTAAGAGK-3' (SEQ.ID.NO.3) or GU(A/G)AGU sequence (eg, 5 '-GTAAGAGGAT-3', 5'-GTAAGAGT-3', 5'-GTAGAGT-3' and 5'-GTAAGT-3'). The three-terminal splicing is homologous to the GWKSCYRCAG (SEQ.ID.NO.4) or CT(A/G)A(C/T)NG sequence (eg, 5'-GATATCCTGCAG-3', 5'-GGCTGCAG-3 ', 5'-CCACAG-3'). In addition, a branch point sequence is located at the five-terminal and three-terminal splicing, and the branch point region is located in a nucleotide sequence homologous to 5'-TACTWAY-3' (SEQ. ID. NO. 5) (eg, 5' -TACTAAC-3' and 5'-TACTTAT-3'), the branch point region contains a branch point, and the branch point is an adenosine (A) capable of forming a set of lariat structures. In addition, the polypyrimidine region is located between the branching point region and the three-terminal splicing region, wherein the polypyrimidine region is a nucleotide sequence having a plurality of thymines (cytosine) and a nucleus of the polypyrimidine region. The nucleotide sequence is homologous to 5'-(TY)m(C/-)(T)nS(C/-)-3' (SEQ.ID.NO.6) and 5'-(TC)nNCTAG(G/ -)-3' (SEQ. ID. NO. 7). Wherein, "m" and "n" mean a repeating sequence of one or more, preferably the number of sequences of m is between one and three and the number of sequences of n is between seven and twelve. "-" means no nucleotides. All these introns are Some connect nucleotides to connect, based on 37 CFR 1.822, W refers to adenine (A) or thymine (T) / uracil (U), and K refers to guanine ( Guanine (G)) or thymine/uracil, S means cytosine or guanine, Y means cytosine or thymine/uracil, R means adenine or guanine, and N means adenine or cytosine , guanine or thymine/uracil and others. For all splice identification regions, the deoxythymidine is replaced by uridine.

In order to determine the position of the interferon of the SpRNAi in the intron, since the position is determined by the restriction enzyme cleavage, the restriction enzymes are from AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI, Asp718I. , BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II , EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI , NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI and the above restriction enzymes One of the mixed restriction enzymes is selected. The intronic insert is a DNA template comprising a set of serotonin, a short-temporary RNA (stRNA), an antisense RNA, and an siRNA (siRNA). , small ribonucleic acid (shRNA), microribonucleic acid (miRNA), Piwi-interacting RNA (piRNA), ribozyme and precursors of these ribonucleic acids and mixtures of the above ribonucleic acids.

For the convenience of gene delivery to induce specific cell activation, the preferred genetically recombinant SpRNAi of the present invention is incorporated into a expression vector selected from the group consisting of a plasmid, a cosmid, and a phagmid. , a yeast artificial chromosome, a transgene, a transposon, a retrotransposon, a skipping gene, a viral vector, and a vector in which the above vectors are mixed. The vector is introduced into a specific cell or tissue by gene transfection methods, including liposomal transfection, chemical transfection, DNA ligation, and insertion. Transgenic genes, chemical transformation, electroporation, homologous recombination, transposon insertion, jumping gene transfection, Viral infection, reversed viral infection, micro-injection, gene-gun penetration, and a combination of the above methods. In addition, the vector further comprises a promoter of at least one virus or a second type ribonucleic acid polymerase (Pol-II) to express SpRNAi-RGFP and a Kozak consensus translation initiation site to increase in eukaryotic cells. Translation efficiency, a multiple SV40 polyadenylation signal in the downstream region of SpRNAi-RGFP , a pUC origin of replication, at least two restriction enzyme cleavage positions for confluence SpRNAi-RGFP into the vector, an optional SV40 replication initiator in a mammalian cell for expression of the SV40 T antigen, and an SV40 early (early) which exhibits at least one antibiotic resistance gene in prokaryotic cells. Promoter, the location of the above elements incorporated into the SpRNAi-RGFP vector is determined by the restriction enzyme cleavage site. The viral promoter of the above vector comprises a cytomegalovirus (CMV promoter), a retrovirus long-terminal region (LTR) promoter, and a hepatitis B virus promoter (hepatitis B virus). HBV), adenovirus AMV promoter, adeno-associated virus (AAV) promoter, and plant-associated mosaic virus promoter. The restriction enzymes are from AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU111/120I, BsrI/BI/GI, BssHII /SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II, EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI , MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI One of the restriction enzymes of SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI and the above restriction enzymes is selected. Wherein, the expression of the anti-antibiotic gene is used as an indicator for screening a specific cell, and the anti-antibiotic gene line can be resistant to penicillin G (penicillin G), ampicillin, neomycin, and barley. Paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, Combination of rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, G418 and the above antibiotics Antibiotics.

The SpRNAi-RGFP vector has been tested in zebrafish (Tg (actin-GAL4: UAS-gfp)) strain to inhibit green fluorescent protein expression, as shown in Example 3 and Figure 3C, anti-green fluorescent protein The miRNA transfected into the SpRNAi-RGFP group (line4) by liposome showed a significant inhibition of green fluorescent protein (about 80% protein inhibition), but no effect was detected in other experimental groups and control groups. To; these experimental groups and the control group were sequentially (left to right) 1 and control group (blank vector control, Ctl). 2. A control group for the pro-microRNA (pre-miRNA) of the HIV- infected protein ( HIV-p24 ). 3. The anti group of the antisense EGFP insert without the hairpin loop structure and the miR* group of the inverted precursor microRNA sequence; the inverted precursor microRNA sequence The lineage is fully complementary to the anti- EGFP pre-miRNA (miR*) against green fluorescent protein. There is no silent phenomenon in non-targeted genes, such as red fluorescent protein and actin, indicating that the RNA interference of the RNA interference of SpRNAi is highly specific. Figure 3D shows Northern blotting analysis showing mature microRNAs (also known as intron microRNAs) only in anti-green fluorescent protein precursor microRNAs with introns The red fluorescent protein carrier group was formed (the second column in the middle). This phenomenon was not observed in the red fluorescent protein group without introns (the first column on the left), and the red fluorescent protein system could be normally They are joined to form a mature ribonucleic acid for expression.

Assessing the efficiency of intron microRNAs (miRNAs) in cells

Previous experiments have determined that intronic miRNAs of introns can produce gene silencing effects in cells. In order to further evaluate the efficiency of the gene silencing effect of the microRNA in the intron and determine the structure of the preferred microribonucleic acid, zebrafish was selected as the experimental material to further understand the preference of the RISC complex for the microRNA structure. .

In the experiment, it was found that the stem-loop of pre-miRNA determines the combination of RISC complex and miRNA. One of the combinations of RISC complex is different from the complex of siRNA-RISC combination (Lin et.al. (2005) Gene 356 : 32-38). The double strand of siRNA plays an important role in the siRNA-RISC complex, and only one strand of the double stranded siRNA is incorporated into the RISC complex. This phenomenon is due to the dynamic thermal stability of the siRNA double-stranded base pair. Decide. Based on this siRNA pattern, the complementation of the miRNAs with their complementary miRNAs (also known as miRNA*s) is considered to be one of the key steps in the formation of the RISC complex. If the model is correct, there will be no preference for a particular one. However, experiments have found that the ring structure of the intronic pre-miRNA plays an important role in the composition of the RISC complex.

In Examples 1 and 2, the SpRNAi-RGFP vector expressing the anti- EGFP miRNA and the miRNA*-stemloop-miRNA [1] and the miRNA-stemloop-miRNA* [2] (miRNA* representative line and mature microribonucleic acid sequence were used. Complementary microRNAs) Intronic insert expression vectors of these two different microRNAs (miRNAs), which differ in the expression of mesons from DNA synthesis machinery and the insertion of previously prepared SpRNAi- In the RGFP vector. The two sets of vectors contain the same double-loop structure which is capable of silently effecting the nucleotide sequences 280 to 302 of the EGFP gene. Because the intronic insert is linked by the PvuI and MluI restriction enzyme cleavage sites, the meson can be easily manipulated by many other different anti-gene mesons (eg, anti- EGFP , anti-Tyr or anti-Hyal). Replace. By this particular application, the intronic insert expression system of this intron provides a tool for miRNA genetic applications for intracellular development.

To determine which pre-miRNA structure is preferred, the microribonucleic acid with the potential for silent effects in zebrafish is precipitated via latex beads in a mirVana microribonucleic acid separation column (Ambion, Austin, TX). The miR-EGFP (280-302) was shown to exert a silent effect on the nucleotide sequence 280 to 302 of the EGFP gene, as shown in Fig. 4A. Because this potent miRNA is only found to be effective in transfecting zebrafish miRNA-stemloop-miRNA*[2] (that is, the circular expression vector indicated below Figure 4A), it is speculated that the RISC complex is miRNA-stemloop- miRNA*[2] has a structural preference, as shown in Figure 4B, using zebrafish (Tg (actin-GAL4: UAS-gfp)) expressed via the actin-activated promoter to demonstrate The relationship between the visually-characterized gene silencing effect and miRNA expression, this zebrafish will always express green fluorescent protein in various cells. This zebrafish was transfected with the SpRNAi-RGFP vector and expressed a red fluorescent protein which can be used as an indicator protein. After transfecting the SpRNAi-RGFP vector into the zebrafish using FuGene cationic liposomal reagent (Roche, In), it was found that all vectors could completely enter the two-week zebrafish juveniles at 24 hours of transfection, except for the bone and scales.

The indicator protein of the red fluorescent protein was detected in the transfected zebrafish, whereas the silent effect of the green fluorescent protein EGFP was transfected with the miRNA-stemloop-miRNA*[2] pre-miRNA zebrafish It was observed in the group (miR group). As shown in Fig. 4C, Western blot analysis quantitatively determines that the gene silencing effect is lower in the intestinal (GI) system than in other tissues, and this phenomenon may be related to higher RNase activity in the intestine. Since the thermal stability of the five ends of the anti- EGFP pre-miRNA stem-arms is the same, it is speculated that the ring structure of this pre-miRNA is related to the combination of the RISC complex. Therefore, based on the diversity of the circular structure, a set of pre-mir-302 rings (for example, 5'-GCTAAGCCAGGC-3' (SEQ.ID.NO.1) and 5'-GCCTGGCTTAGC-3' (SEQ.ID. NO. 2)) is combined with a RISC complex to exert the utility of the present invention.

Intronic microRNA inhibits tyrosinase and hyaluronidase on mouse skin

Based on the above experiments, a SpRNAi-RGFP vector having a pre-miRNA meson of miR-Tyr or miR- Hyal was used to inhibit a pigment accumulation-related gene such as Tyr or an aging-related gene such as Hyal . The self-designed miR-Tyr and miR-Hyal pre-miRNA lines target a region of high homology between humans or mice. This mouse provides a live laboratory animal for testing the safety and efficacy of the SpRNAi-RGFP vector. In nature, 54 miRNAs of the cell itself have the function of inhibiting tyrosinase (Tyr; 2082 base pairs). These 54 sequences include mir-1, mir-15a, mir-16, mir-31, mir. -101, mir-129, mir-137, mir-143, mir-154, mir-194, mir-195, mir-196b, mir-200b, mir-200c, mir-206, mir-208, mir-214 , mir-221, mir-222, mir-292-3p, mir-299-3p, mir-326, mir-328, mir-381, mir-409-5p, mir-434-5p, mir-450, mir -451, mir-452, mir-464, mir-466, mir-488, mir-490, mir-501, mir-522, mir-552, mir-553, mir-570, mir-571, mir-582 , mir-600, mir-619, mir-624, mir-625, mir-633, mir-634, mir-690, mir-697, mir-704, mir-714, mir-759, mir-761, mir -768-5p, and mir-804. These sequences were analyzed according to the miR database miRBase::Sequence program (http://microrna.sanger.ac.uk), and all anti-Tyr miRNAs were directed to the front end of the tyrosinase (NCBI accession number NM000372). Nucleotides, in addition to nine cells, the miRNAs themselves can be used to inhibit hyaluronidase ( Hyal ; 2518 bp; NCBI accession number NM007312) including mir-197, mir-349, mir-434-5p, mir-549, Mir-605, mir-618, mir-647, mir-680, mir-702 and mir-763. Among these miRNAs, mir-434-5p is the only miRNA inhibitory sequence that is unique against both Tyr and Hyal genes, and is the sequence that does not interfere with other genes. However, because almost all miRNAs affect more than 50 specific target genes, these miRNAs are not specific, making skin care safe.

To test the feasibility of miRNA for skin whitening, the pre-mir-434-5p of the cell itself was expressed on the skin of mice by the SpRNAi-RGFP expression vector of the present invention. As shown in Figure 5, mottled albino skin was formed in melanin-dissected mice by continuous subcutaneous transfection with a pre-mir-434-5p expression vector (50 μg) for four days (200 μg total) (This is the microribonucleic acid group), in which the tyrosinase Tyr-membrane protein catalyzes the rate determining step in the process of forming melanin. Two weeks after the first transfection, it was observed that the melanin of the skin and hair was significantly reduced. Conversely, the blank group and the small interfering ribonucleic acid group transfected with the third type polymerase (Pol-III) did not have this effect at the same dose. The sample was taken from the hair follicle and analyzed by Northern blot. Pre-mir- The 434-5p expression vector reduced the Tyr performance by 76.1% ± 5.3% after two days of transfection, whereas other control groups such as the small interfering ribonucleic acid group (such as the GAPDH group) did not have this silent effect. Grimm et al. reported in 2006 that high concentrations of siRNA/shRNA produced by a third type of polymerase (Pol-III) would over-saturate miRNA pathways in cells and cause extensive dysregulation of miRNAs. Thus, the miRNA of the second type ribonucleic acid polymerase system (Pol-II) provides a more efficient, more compatible, and safer method of skin care. However, because pre-mir-434-5p also inhibits the other five cellular genes including TRPS1, PITX1, LCOP, LYPLA2, and Hyal.

In order to improve the specificity and safety of miRNA, the redesigned mir-434-5p sequence can form a 3-25 nucleotide sequence of Tyr or a 459-482 nucleotide region of Hyal, wherein the sequence The pre-miRNA insert sequence used to silence the Tyr gene is 5'-GTCCGATCGT CGCCCTACTC TATTGCCTAA GCCGCTAAGC CAGGCGGCTT AGGCAATAGA GTAGGGCCGA CGCGTCAT-3' (SEQ.ID.NO.8), which forms a hairpin-like ribonucleic acid gene. Silent effector, and processed to miR-Tyr miRNA sequence contains or homologous to 5'-GCCCTACTCT ATTGCCTAAG CC-3' (SEQ.ID.NO.9), in other examples, Hyal inhibited pre-miRNA Is 5'-GTCCGATCGT CAGCTAGACA GTCAGGGTTT GAAGCTAAGC CAGGCTTCAA ACCCTGACTG TCTAGCTCGA CGCGTCAT-3' (SEQ.ID.NO.10), which forms a different hairpin-like ribonucleic acid and is processed into a miR-Hyal miRNA sequence containing or Homologous to 5'-AGCTAGACAG TCAGGGTTTG AA-3' (SEQ. ID. NO. 11). Although the pre-miR-Tyr and pre-miR-Hyal constructs are based on the same mir-434-5p and mir-302 ring structures, the mature miR-Tyr And the miR-Hyal line is completely different from each other. As shown in Fig. 6, the miR-Tyr group and the miR-Hyal group are transfected on the skin of mice to inhibit tyrosinase by more than 90%, respectively, and hyaluronidase is inhibited. More than 67%, without any non-specific silent suppression, the expression of mature miR-Tyr and miR-Hyal miRNA was quantified by Northern bot analysis, while tyrosinase and hyaluronan The acidase system was quantified by Western blot analysis.

Silence specificity and safety of miR-Tyr and miR-Hyal genes on human skin

The miR-Tyr and miR-Hyal RNA interference effectors of the intron of the present invention, which are known for the gene silencing effect, for further testing of the SpRNAi-RGFP vector line having miR-Tyr and miR-Hyal in human skin cells and tissues Specificity and security. To efficiently transfect the vector into human epidermal cells, SpRNAi-RGFP vector solution (1 μg/ml) (100 μg of SpRNAi-RGFP vector with 99 ml of DNase-free glycerin and 1 ml of sterile deionized water) Mixed into) transfected into cells. Among them, DNase-free glycerin can be used to help miR-Tyr form cysts to increase the efficiency of transfection into deep skin and cell membrane penetration. This is the main ingredient used to whiten the skin. In other embodiments, it can also be a finished cosmetic by adding other ingredients to enhance odor, color, effectiveness and stability. As shown in Fig. 7A, the Asian male arm skin was treated with 2 ml of this main component, and after three days, compared with the glycerol control group of the SpRNAi-RGFP vector without any silent effector, it was found to be more fair.

The cultured skin cell line is approved by the donor and taken from the donor, and all treatments are processed with the consent. The vector was transfected into these skin cells as shown in Examples III and 6. Figure 7B shows that the reduced tyrosinase and the melanin produced by the Western blot analysis were statistically significant (p > 0.001). The decrease in the amount of tyrosinase is directly proportional to the dose of the melanin receptor and the transfected miR-Tyr. This phenomenon indicates that the dose increase of miR-Tyr is positively correlated with the tyrosinase and its melanin receptor. This reduction was not observed in other experimental groups such as the glycerin group of the SpRNAi-RGFP vector without any miRNA insert and the miR-gfp group of the SpRNAi-RGFP vector expressing the anti- EGEP pre-miRNA insert. When transfected with the miR-Tyr expression vector (1 μg/ml), the maximum silent effect of tyrosinase was about 55% to 60% and the melanin silent effect was about 30% to 45%, while the actin system was not affected by miR-Tyr. The interference is silent, thus suggesting that the invention has a safety specificity phenomenon. Figure 7C further shows a significant reduction in skin melanin, as indicated by the bright-field (BF) of skin cell culture, whereas in normal skin cells without miR-Tyr, the melanin system precipitates near the nucleus. The phenomenon of melanin precipitation in cells transfected with miR-Tyr is very rare, indicating that miR-Tyr has a whitening effect in cells. According to this melanin reduction phenomenon, the skin cells transfected with miR-Tyr are also accompanied by a decrease in tyrosinase, which can be analyzed by immunocytochemical staining analysis (ICC), as shown in Fig. 7C. Therefore, based on FIG. 7A to, for example, 7C, the SpRNAi-RGFP vector of the miR-Tyr miRNA can successfully inhibit tyrosinase and melanin in cells.

After the gene silencing effect of miR-Tyr was established in human skin cells, ribonucleic acid was analyzed using biochips (GeneChip U133A & B arrays, Affymetrix, Santa Clara, CA) to evaluate more than 32,668 human genes with miR-Tyr Comparing the cells stained with cells without miR-Tyr transfection, the results showed that the gene silencing of specificity was much more silent than that of genes without specificity. The entire RNA of each test cell strain was purified by RNeasy spin column (Qiagen, Valencia, CA). As shown in Figure 8A, biochip analysis of no miR-Tyr (i.e., miR-) and cell lines with miR-Tyr (i.e., miR+) now shows that only two genes are altered by more than 1.5 fold (i.e., gene expression). There are more than 50% changes), the two genes are the tyrosinase gene and the TRP1 gene interacting with it (as shown in Figure 8B), suggesting that the gene silencing effect induced by miR-Tyr is for tyrosinase The department is very specific. In addition, no other cytotoxicity or PKR/2-5A pathway was affected, suggesting that the miR-Tyr gene silencing effect is quite safe for dermal applications (Figure 8B). Further, Northern blotting analysis was also used to compare the degree of expression of these genes recognized by the biochip (as shown in Fig. 8C) to confirm the results of the experiments shown in Figs. 8A and 8B. Further comparing the miR-Tyr transfection group (miR+) (Fig. 8A right) with the untransfected group (miR-), the correlation coefficient (correlation coefficiency) of the 32558 genes was 99.8%, while the mir-434-5p was transferred. The correlation coefficient between the stained group and the untransfected group (miR-) was 77.6%, which means that only 65 genes in the miR-Tyr transfection group were affected by miR-Tyr, but 7317 genes were affected. The effect of mir-434-5p transfection changed. Because miRNAs have silent functions against a wide range of genes based on their mismatched stem-arms structure, the present invention demonstrates that redesigning this stem-arms structure is essential for this miRNA safety.

Therefore, the present invention uses an intronic hairpin-like miRNA/shRNA expression vector to provide an effective method for intracellular skin care, particularly for pigmentation and aging prevention. At the same treatment, Pol-II-transcribed intronic miRNA does not cause any detectable cytotoxicity, as does non-specific mRNA degradation induced by Pol-III-directed siRNA (Sledz et.al.supra ; Lin (2004b) Supra ). This emphasizes that the intronic miRNA is an efficient and specific method without the cytotoxicity of the double-stranded siRNA. Because the intronic miRNA-mediated gene silencing pathway works synergistically with numerous intracellular regulatory systems, gene silencing effectors work together by systems including the Pol-II transcription system, the RNA splicing system (intron excision mechanism), and the NMD system. The intronic miRNA silencing effect is considered to be more efficient, specific, and safe in all current known RNAi pathways, based on all the advantages, using redesigned intronic miRNAs to provide a long-term, effective, specific and safe skin care Gene regulation method can avoid non-specific gene silent cytotoxicity such as traditional siRNA

The present invention has been described by the above-described related embodiments, but the above embodiments are merely examples for implementing the present invention. It must be noted that the disclosed embodiments do not limit the scope of the invention. Conversely, the spirit and scope of the scope of the patent application Modifications and equal arrangements are intended to be included within the scope of the invention.

The following experimental design is illustrative, but does not limit the scope of the invention. Reasonable changes can be made to those skilled in the art without departing from the spirit and scope of the invention.

Example 1 Construction of a recombinant gene comprising SpRNAi ( SspRNAi-RGFP ) for the production of three different SpRNAi nucleotide sequences comprising: 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); N4-antisense, 5'-pGTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3' (SEQ. ID. NQ. 19). In addition, the two exon fragments were generated by the 208th nucleotide of the red fluorescent RGFP gene (SEQ.ID.NO.20) by DraII restriction enzyme digestion, and the cleaved five-terminal system was further amplified by T4 DNA polymerase. Form a blunt end. The cut void can be filled by an artificial SpRNAi intron. This RGFP is linked to a red fluorescent protein gene which is formed by the addition of an additional aspartate to the amino acid group 69 of HcRed1 chromoproteins (derived from Heteractis crispa ). (BD Biosciences, CA) developed less denaturation and nearly twice the intensity of red fluorescent 570 nm wavelength radiation. The SpRNAi-RGFP expression vector was modified with the pHcRed1-N1/1 vector (Clontech BD Biosciences, Palo Alto, CA; catalog #6365-1), and the pHcRed1-N1/1 vector already contained the red fluorescent protein gene of HcRed1, of which HcRed1 protein It is purified from sea anemone Heteractis crispa . Using pHcRed1-N1 / 1 vector with XhoI and XbaI restriction enzyme was isolated and purified using 2% agarose gel electrophoresis RGFP gene of the full length 769 bp fragment of 3934bp of free RGFP vector.

N1-sense and N1-antisense, N2-sense and N2-antisense, N3-sense and N3-antisense, and N4-sense and N4-antisense individually heat each complementary sequence to 94 ° C (2 minutes) after 70 ° C (10 Minutes) and 1x PCR buffer (50 mM Tris-HCl, pH 9.2 at 25 ° C, 16 mM (NH ₄ ) ₂ SO ₄ ). Then, by gradually cooling N1-N4, N2-N4, N3-N4 (1:1), N1, N2, N3 to N4 are connected, and the temperature is applied from 50 ° C to 10 ° C for more than 1 hour, after which the enzyme is coupled with T4 ligase and The corresponding buffer (Roche, IN) was mixed with it for 12 hours (12 ° C), so that the intron could be inserted into the appropriate position of the exon. After the RGFP exon fragment was added to the reaction (1:1:1), the T4 ligase and the corresponding buffer were adjusted to carry out the ligation reaction for another 12 hours (12 ° C). In order to access the correct size of the recombinant gene RGFP into the vector, 10 ng of the ligated nucleotide sequence utilizes PCR technology and a pair of RGFP- specific primers 5'-CTCGAGCATG GTGAGCGGCC TGCTGAA-3' (SEQ.ID.NO) .21) and 5'-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3' (SEQ.ID.NO.22) in PCR conditions at 94 ° C (1 minute), 52 ° C (1 minute), 68 ° C (2 minutes) and 30 times The same reaction. The product of the PCR and the nucleotide sequence of about 900 bp were isolated by a 2% agarose gel and purified by a Gel Extraction kit (Qiagen, CA). The SpRNAi- functional RGFP gene of this 900 bp nucleotide was further determined by sequence alignment.

Because the recombinant gene uses the XhoI and XbaI restriction enzyme sites to insert the recombinant gene into the vector at the five-terminal and three-terminal ends, the vector must have skin compatibility and characteristics in the organism, and the expression carrier contains the substance. Plasmids, cosmids, phagemids, yeast artificial chromosomes, jumping genes, transposons, retrotransposons, retroviral vectors. In addition, since the inserted recombinant gene is ligated to the five-terminal and three-terminal positions by the PvuI and MluI restriction enzyme cleavage sites, it is also possible to use the same restriction enzyme cleavage site to introduce other different recombinant genes into the vector. Preferably, the inserted recombinant gene is a gene silencing effector similar to a small clip, and the gene silencing effector can be directed to a green fluorescent protein, a luciferin gene, a lactose control group (lac-Z), a virus. Sequences of genes, bacterial genes, plant genes, animals, and human genes are silenced. These silent effector mesons have a complementarity or homology rate of about 30% to 100% with their target gene sequence, and between 35% and 49% for hairpin-shRNA mesons, for sense-stRNA For antisense-siRNA mesons, the preferred sequence complementarity ranges from 90% to 100%.

Example 2 constructing a recombinant gene containing SpRNAi ( SspRNAi-RGFP ) into an expression vector

Because the recombinant SpRNAi-RGFP gene has restriction enzymes such as XhoI and XbaI at the five-terminal and three-terminal ends, and can use the cleavage to insert the recombinant gene into the vector, the SpRNAi-RGFP and the 3934 bp pHcRed-N1/1 vector are 1 : 16 (w/w) was mixed and cooled from 65 ° C to 15 ° C for more than 50 minutes, after which T4 ligase and its buffer were added and mixed at 12 ° C (12 hours). The SpRNAi-RGFP vector can be replicated in large quantities using E. coli DH5 α LB (50 ug/ml kanamycin (Sigma Chemical, St. Louis, Mo.)) and utilizes PCR technology and its RGFP- specific primer (SEQ.ID.NO.21). And (SEQ. ID. NO. 22) at 94 ° C (1 minute), 68 ° C (2 minutes) and 30 cycles of reaction and alignment confirmed the sequence. In order to access the recombinant gene into a viral vector, the same procedure can also be carried out using the XhoI/XbaI- linearized pLNCX2 retroviral vector (BD). Since SpRNAi is ligated to the five-terminal and three-terminal ends by the PvuI and MluI restriction enzyme cleavage, these cleavage sites can be used to remove or link anti- EGFP shRNA or miR-Tyr or miR-Hyal sequences.

Synthetic nucleotide sequences can be used to generate many different SpRNAi artificial introns containing more different miR-Tyr or miR-Hyal mesons, which are similar to the small-clamp precursor microRNA, as follows: miR-Tyr -sense,5'-GTCCGATCGT CGCCCTACTC TATTGCCTAA GCCGCTAAGC CAGGCGGCTT AGGCAATAGA GTAGGGCCGA CGCGTCAT-3'(SEQ.ID.NO.8);miR-Tyr-antisense,5'-ATGACGCGTC GGCCCTACTC TATTGCCTAA GCCGCCTGGC TTAGCGGCTT AGGCAATAGA GTAGGGCGAC GATCGGAC-3' (SEQ. ID.NO.23); and miR-Hyal-sense, 5'-GTCCGATCGT CAGCTAGACA GTCAGGGTTT GAAGCTAAGC CAGGCTTCAA ACCCTGACTG TCTAGCTCGA CGCGTCAT-3'(SEQ.ID.NO.10); miR-Hyal-antisense, 5'-ATGACGCGTC GAGCTAGACA GTCAGGGTTT GAAGCCTGGC TTAGGTTCAA ACCCTGACTG TCTAGCTGAC GATCGGAC-3' (SEQ. ID. NO. 24). These mesons can complement the miR-Tyr-antisense and the miR-Hyal-sense to miR-Hyal-antisense by hybridization, such as miR-Tyr-sense. These miR-Tyr and miR-Hyal expression vectors can be efficiently replicated using E. coli DH5 α LB (50 ug/ml kanamycin (for pHcRed1-N1/1 vector) or 100 ug/ml kanamycin (for pLNCX2 vector)). The SpRNAi-RGFP vector was purified and isolated from the Mini-prep or Maxi-prep plasmid extraction kit (Qiagen, CA). As for the pLNCX2 vector, the packaging cell lines PT67 (BD) can be used to generate an infected but not replicable virus. The infected PT67 cell line is in 1xDMEM medium (10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 ug/ml streptomycin). Sulfate and 50 ug/ml neomycin (Sigma Chemical, MO) at 37 ° C, 5% CO ₂ or less), the virus dose is about 10 ⁶ cfu / ml.

Example 3 in vivo transfection

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 is first mixed with FuGene reagent (Roche, IN). And following the instructions of FuGene reagent, the vector with the structure of insert-free RGFP and SpRNAi-RGFP was directly placed or smeared on the cell line, zebrafish seedling and mouse skin at this time. The pre-miRNA insert group of HIV gag-p-24 was used as the negative control group. The cell or tissue appearance and its fluorescent staining were observed at 0, 24, 48 and 72 hours after transfection. For in vivo transfection on human skin, the previously processed SpRNAi-RGFP vector solution is prepared by mixing 1-1000 μg with anti- EGFP or without anti- EGFP , miR-Tyr or miR-Hyal pre-miRNA insert SpRNAi-RGFP vector, 1 ml autoclaved ddH ₂ O and 99 ml of 100% DNase-free glycerin (or glycerol). Then, the mixed solution can be directly applied and massaged on human skin to produce its effect.

Embodiment 4 Northern Blot Analysis

RNA (20 μg total RNA or 2 ug poly[A ⁺ ] RNA) was separated by electrophoresis on 1% formaldehyde-agarose gels to adsorb RNA onto the nylon membrane (Schleicher & Schuell, Keene, NH). The synthetic probe design is complementary to a 75 bp junction sequence between the 5-terminal exon of RGFP and the anti- EGFP pre-miRNA insert or complementary to miR-Tyr (SEQ.ID.NO.9) or miR-Hyal (SEQ. ID. NO. 10), wherein the miR-Hyal line was calibrated by the Prime-It II kit (Stratagene, La Jolla, CA) and using [ ³² P]-dATP (>3000 by random primer extension technique) The sequence was calibrated by Ci/mM, Amersham International, Arlington Heights, IL) and purified 10 bp-cutoff Micro Bio-Spin chromatography columns (Bio-Rad, Hercules, CA). Reduced by mixing 50% deionized formamide (pH 7.0, 5x Denhardt's solution, 0.5% SDS, 4 x SSPE and 250 mg/mL denatured salmon sperm DNA fragments (18 hr, 42 ° C)) Non-specific signal. After that, the impurities were washed off with 2 x SSC, 0.1% SDS (15 min, 25 ° C) and 0.2 x SSC, 0.1% SDS (45 min, 37 ° C), and (automatic) radiography was performed ( Autoradiography).

Example 5 SDS-PAGE and Western Blot Analysis

For immunoblotting of specific proteins, the isolated cell strain from which the culture solution was removed was subjected to ice PBS wipe, and the protease inhibitors, Leupeptin, TLCK, TAME, and PMSF were supplemented with CelLytic-Mlysis/extraction reagent (Sigma, MO) according to the instructions. After the cells were mixed with a shaker for 15 minutes at room temperature, the cells were scraped into a test tube and the cell residue was precipitated by centrifugation at 12000 x g for 5 minutes, and the cell extract containing the protein was collected and stored at -70 ° C until use. Proteins were quantified using an SOFmax software package on an E-max microplate reader (Molecular Devices, Sunnyvale, CA). Each 30 μg of cell extract was added to SDS-PAGE sample buffer (with or without reduction of 50 mM DTT) and the sample was boiled for 3 minutes. Before injecting the sample into 8% polyacylamide gels, 2~3 μl of protein marker (Bio-) Rad) is injected into it. SDS-polyacrylamide gel electrophoresis is performed according to standard procedures (Molecular Cloning, 3rd ED). After protein chromatography, the protein was adsorbed onto a nitrocellulose membrane by electroblotting and incubated with Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 1 to 2 hours at room temperature. And use the antibody primary antibody to directly target specific proteins (EGFP (1: 5,000; JL-8, BD), RGFP (1: 10,000; BD), Tyr (1: 2,000; Santa Crutz), or Hyal (1: 2,000; Santa) Crutz)) Calibration, acting overnight at 4 °C. Then washed three times with TBS-T buffer, and then treated with nitrocellulose membrane (goat anti-mouse IgG conjugate with Alexa Fluor 680 reactive dye (1:2,000; Molecular Probes)) at room temperature. After one hour of action, the cells were washed three times with TBS-T buffer and imaged and images were recorded with Li-Cor Odyssey Infrared Imager and Odyssey Softeare v. 10 (Li-Cor).

Example 6 Intronic RNA-mediated Gene Silencing in Introns of Zebrafish

This strain of Tg ( actin- GAL4: UAS-gfp) zebrafish juvenile was raised in a container with 10 ml of 0.2x serum-free RPMI 1640 medium for transfection, and 60 μl of FuGene liposomal transfection reagent (Roche) was first transfected. Biochemicals, Indianapolis, IN) was mixed with 1 ml of 1x serum-free RPMI 1640 medium. The SpRNAi-RGFP vector having no anti- EGFP pre-miRNA insert was mixed as described in Examples 1 and 2, and finally mixed with the above mixed culture solution for transfection, and the mixture was placed. After 30 minutes on ice, they were placed directly into the container of zebrafish juveniles. All three doses were separated by 12 hours (all 60 μg), and samples were collected and observed at 60 hours after transfection.

Example 7 mir-434-5p and miR-Tyr-mediated Gene Silencing of in vivo mouse skin introns (Intronic mir-434-5p and miR-Tyr-mediated Gene Silencing)

Albino skin mottled melanin knockout mice (W-9 black strain) were serially injected subcutaneously with SpRNAi-RGFP expression vector with mir-434-5p pre-miRNA insert (meson) for four days (a total dose of about 200 μg) or The liposome-encapsulated SpRNAi-RGFP expression vector with miR-Tyr pre-miRNA was injected twice daily for six days (a total dose of about 240 μg). To generate a SpRNAi-RGFP expression vector with a native mir-434-5p pre-miRNA meson, use as described in Example 2, except that the synthetic mir-434-5p pre-miRNA is not used as an intron meson (intronic insert) (5'-GTCCGATCGT CUCGACUCUG GGUUUGAACC AAAGCUCGAC UCAUGGUUUG AACCAUUACU UAAUUCGUGG UUUGAACCAU CACUCGACUC CUGGUUCGAA CCAUCCGACG CGTCAT-3' (SEQ.ID.NO.25)). To more efficiently transfect the vector into a particular tissue, the carrier was mixed with FuGene liposomal transfection reagent (Roche, IN) as described in Examples 3 and 6.

Example 8: Intronic miR-Tyr-mediated gene silencing effect in human skin

In order to efficiently transfect the vector into the human skin layer, the 1 μg/ml SpRNAi-RGFP vector solution can be prepared by mixing 100 μg of the carrier in 1 ml of autoclaved ddH ₂ O with 99 ml of 100% DNase-free glycerin or glycerol. DNase-free glycerin is used to form miR-Tyr into a sac-like shape to facilitate transport of the vector deep into the cell. Therefore, in this way, the inventor's left arm was directly coated with 2 ml of the above mixture with miR-Tyr (right side of the arm) and 2 ml of no miR-Tyr mixture on the left side of the arm. Two days later, it was found that The arm treated with the miR-Tyr mixture is more white, which further confirms its efficacy on human skin.

Example 9 Immunocytochemical (ICC) Staining Assay

A chemical immunostaining test group (Imgenex San Diego, CA) and instructions for manufacturing use. The sample sections were first immersed in PBS three times and soaked in Zeller's solution (10 mM Tris, 100 mM MgCl ₂ , 5% fetal calf serum, 1% BSA and 0.5% Tween-20, pH 7.4) for 30 minutes, then the sample was taken. The sections were immersed in a primary antibody (diluted with Zeller's solution) overnight and stored in a 4 °C freezer. The next day, the sections were washed with TBST and soaked in the secondary antibody for two hours, at this time biotinylated goat anti-rabbit or horse The anti-mouse antibody is a secondary antibody (Chemicon, Temecula, CA). The samples were washed once with TBST and then stained with streptavidin-HRP as the tertiary antibody and colored with a DAB substrate. The results were observed with a 100x microscope and at 100x and 400x (TE2000 inverted microscopic quantitation system).

Example 10 Microarray Analysis

In order to prepare a well-targeted probe for hybridization with a gene on a biochip, the extracted whole ribonucleic acid (2 μg) was converted into a double-stranded cDNA using a Superscript Choice system kit (Gibco/BRL, Gaithersburg, MD) and modified oligh (dT) ₂₄ -T7 promoter primer such as 5'-GGCCAGTGAA TTGTAATACG ACTCACTATA GGGAGGCGG-(dT) ₂₄ -3', the subsequent steps are as described by the user. After the double-stranded cDNAs were extracted with phenol/chloroform, they were precipitated with ethanol and re-dissolved to 0.5 μg/μl in diethyl pyrocarbonate (DEPC)-treated ddH ₂ O. Phase-Lock Gel (5'Prime→3' Prime, Inc., Boulder, CO) can be used to increase the extraction rate. In vitro transcription can be obtained with T7 ribonucleic acid polymerase with 1 μg of cDNA, 7.5 mM unlabeled ATP and GTP, 5 mM unlabeled UTP and CTP and 2 mM biotin-labeled CTP and UTP (biotin-11-CTP, biotin-16-UTP , Enzo Diagnostics) co-reacted to generate cRNA at 37 ° C for 4 hours, then cRNA was purified using RNeasy spin columns (Qiagen, CA), and then confirmed by 1% agarose gel, followed by heating to 94 ° C for 35 minutes at 40 mM Tris-acetate, pH 8.0, 100 mM KOAc/30 mM MgOAc allowed the cRNA to be randomly broken down to a size of approximately 50 nucleotides.

A four-nucleotide biochip (GeneChip U133A&B arrays, Affymetrix, Santa Clara, CA) was used, which contained a total of 32,668 genes for hybridization, and the hybridization reaction was carried out in 200 μl of AFFY buffer (Affymetrix) at 40 ° C for 16 hours. Complete with continuous agitation. After completion of the hybridization reaction, the biochip was swabbed with 200 μl of 6x SSPE-T buffer (1 x 0.25 M sodium chloride/15 mM sodium phosphate, pH 7.6/1 mM EDTA/0.005% Triton) and then with 200 μl. Wash 6x SSPE-T buffer at 50 ° C for one hour, then rinse twice with 0.5X SSPE-T and 0.5x SSPE-T at 50 ° C for 15 minutes, then use 2 μg / ml streptavidin-phycoerythrin (Molecular Probes) After staining with 1 mg/ml acetylated BSA (Sigma) in 6x SSPE-T (pH 7.6), the biochip was placed in a confocal scanner (Molecular Dynamics) for interpretation and Affymetrix Microarray Suite version 4.0 software for analysis. After normalizing the sample by the average difference between the perfectly matched probe and the mismatched probe, a signal with a signal difference greater than twice is collected.

Example 11 Statistical analysis

Biochip results were presented as mean ± SE. Statistical analysis of these sample data was calculated by one-way ANOVA. When there were statistically significant differences, Dunnett's post-hoc test was used to identify sample groups that differed from the standard. In order to compare between the two groups, a two-tailed sudent t test was used. For comparison over two groups, ANOVA was aligned by post-hoc multiple range test, and the probability value p < 0.05 was considered to be statistically significant, all p values were determined by two-tailed test.

Figure 1 is a diagram showing the mechanism of intronic mircoRNA (miRNA) production in the intracellular intron of the present invention.

Figure 2 shows the siRNA, exonic (intergenic) microRNA and intronic microRNA pathway mechanisms.

3A to 3D are diagrams showing the structural components of the SpRNAi-RGFP recombinant gene (Fig. 3A) and its principle (Fig. 3B) to produce an artificial microRNA of an intronic miRNA mimicking an intron. . Intracellular assays The SpRNAi-RGFP expression vector inhibits green fluorescent protein directly in zebrafish and shows more than 85% knockout effect on the green fluorescent protein gene, as confirmed by Western blot analysis. Intronic miRNAs and their splicing precursors with inactive introns of green fluorescent protein can be visualized by 1% formaldhyde agarose gel electrophoresis and after Northern blot analysis.

Figures 4A through 4C show the intronic RNA inserts of the different introns of the SpRNAi-RGFP construct of the present invention for efficient microRNA formation and efficient green production in zebrafish. Fluorescent protein silencing effect confirms that the RISC complex is not between 5'-miRNA*-stemloop-miRNA-3'[ 1 ] and 5'-miRNA-stemloop-miRNA*-3'[ 2 ]hairpin RNA Symmetrical preference (Figure 4A). In vivo gene silencing was only found in transfected precursor microRNA structures [ 2 ], rather than in transfected structures [ 1 ], confirming their preference. Because the color of green fluorescent protein and red fluorescent protein overlap, the red color is larger than the dark orange color as shown in the figure, so the amount of green fluorescent protein is much lower than that of red fluorescent protein in the precursor microRNA structure [ 2 ] The transfection group, at this time the indicator of the vector red fluorescent protein is the average performance (Fig. 4B). Analysis of the amount of green fluorescent protein by Western blot confirmed that there was a significant gene silencing effect in the transfected precursor microRNA structural group [ 2 ] (Fig. 4C). No gene silencing effect occurred in other experimental groups such as Liposome only, empty vector group without any meson (Vctr), and siRNA (siR) group.

Fig. 5 is a diagram showing the gene silencing effect of the present invention which utilizes intracellular tyrostinase gene production in mice to remove pigment by mir-434.

Figure 6 is an artificial miR-Tyr meson of the present invention expressed on mouse skin A specific tyrosinase gene silencing effect is produced.

Figures 7A through 7C illustrate the silencing effect of the tyrosinase gene of more than 50% on the human arm skin (Figure 7A) and the primary skin culture cells (Figures 7B and C) using the miR-Tyr meson.

8A to 8C are gene chip analysis (Affymetrix human GeneChip U133A & B, CA) of a precursor ribonuclease (pre-miRNA) transfected skin cell line of artificial tyrosinase of the present invention, showing that the present invention is specific A gene silencing effector such as mir-434, which is higher than a general miRNA.

<110> Meiluo Biotechnology Co., Ltd.

<120> Using intronic microRNA technology for novel cosmetic designs and products

<130> US 61/000797

<150> US 61/000797

<151> 2007-10-30

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Claims (54)

A method of producing a cosmetic for skin maintenance by a gene silencing effect, comprising the steps of: a. constructing a recombinant nucleotide, wherein the recombinant nucleotide comprises at least one intron The intron has an intronic insert, the intron of which encodes a gene silencing effector, and induces a ribonucleic acid-mediated gene silencing effect (RNA-mediated gene silencing). The sequence of the gene silencing effector is redesigned from the sequence of mir-434 microRNA (miRNA), wherein the sequence of the mir-434 microribonucleic acid precursor (pre-miRNA) comprises SEQ. ID.NO.25; and b. cloning the recombinant nucleotide into a vector; wherein the intron passes through an intron excision mechanism in the human cell Separating at least one precursor RNA from the recombinant nucleotide of the gene such that the gene silencing effector is produced to silence the tyrosinase and/or hyaluronidase genes . A method for producing a finished product for skin maintenance as described in claim 1, wherein the intron is linked by an exon and is separable from the exon to induce ribonucleic acid Mediating the silencing effect of the gene; the exon can be translated into a protein. A method of producing a cosmetic for skin maintenance by a gene silencing effect, as described in claim 1, wherein the human cell comprises human skin cells. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 2, further comprising a step of synthesizing the recombinant nucleotide of the gene, wherein the recombinant nucleotide comprises the intron With the exon. A method for producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 1, wherein the genetic recombinant nucleotide is chemically synthesized or linked via a DNA synthesizer. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 1, wherein the genetic recombinant method is linked by a genetic engineering method, and the genetic engineering method is selected from the group consisting of In homologous gene combination, DNA ligation, transgene insertion, transposon delivery, jumping gene integration, and A group consisting of retroviral infections. A method of producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 1, wherein at least one of the introns comprises a hairpin-like RNA structure. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the patent application, wherein the recombinant nucleotide is cleaved by a genetically engineered restriction enzyme and deoxygenated by the intron Ribonucleic acid joins to produce a non-native gene. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the invention, wherein the genetic recombinant nucleotide is a cellular gene having the intron. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 9, wherein the gene of the cell itself is selected from the group consisting of a viral gene, a mammalian gene, a jumping gene, and a protein encoding (protein) -coding) A group of genes and non-protein-coding genes. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 9, wherein the intron is genetically engineered into a gene of the cell itself, and the genetic engineering method Selected from homologous gene combination, DNA ligation, transgene insertion, transposon delivery, jumping gene integration And a group consisting of retroviral infections. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 1, wherein the intron is a nucleotide comprising the gene silencing effector Introns, a branch point motif, a poly-pyrimidine tract, a 5'-splice site (5'clip), and a three-terminal splicing site (3'-splice site, 3'clip). The production as described in item 1 of the patent application is used by the gene silencing effect A skin-maintaining cosmetic method, wherein the intronic insert is a nucleotide having a gene silencing effector, and the gene silencing effector is selected from the group consisting of a set of chromosomal ribose Lariat-form spliced RNA, short temporary sense RNA (stRNA), antisense RNA (aRNA), short-hairpin RNA (shRNA), microribonucleic acid (short-hair spliced RNA) MicroRNA, miRNA), ribozyme RNA, and a group of precursors and derivatives of the above ribonucleic acids. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the patent application, wherein the intronic insert is a reverse nucleotide comprising a sequence for a gene to be silenced 90% to 100% sequence complementarity. A method for producing a cosmetic for skin maintenance by a gene silencing effect, as described in claim 1, wherein the intronic insert is a similar small clip nucleotide comprising a gene for silencing The sequence has a sequence complementation rate of 30% to 100%. A method for producing a cosmetic for skin maintenance by a gene silencing effect, as described in claim 1, wherein the intronic insert is a hairpin-like nucleic acid. ) comprising a stem-loop structure comprising SEQ.ID.NO.1 or SEQ.ID.NO.2. The production as described in item 1 of the patent application is used by the gene silencing effect A skin-maintaining cosmetic method, wherein the intronic insert is a hairpin-like precursor microRNA (pre-miRNA), and the similar small clip precursor microRNA sequence comprises the SEQ .ID.NO.8 or SEQ.ID.NO.10. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the invention, wherein the gene silencing effector is a microRNA (miRNA), the microribonucleic acid sequence Include SEQ.ID.NO.9 or SEQ.ID.NO.11. A method for producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 1, wherein the intronic insert is incorporation into the intron by At least one restriction site is used for the merging, and the restriction sites are selected from the group consisting of AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II, EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, A group consisting of PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI , and XmaI . The production as described in item 12 of the patent application is used by the gene silencing effect A skin-maintaining cosmetic method, wherein the branch point motif comprises a branch point, and the branch point is an adenosine (A) and is located in a SEQ. ID. NO. 5 nucleotide sequence of the sequence. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 12, wherein the branching point region comprises a branching point, and the branching point is an adenosine (A) and Located in a nucleotide sequence comprising 5'-TACTAAC-3'. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 12, wherein the polypyrimidine region is a nucleotide sequence having a plurality of thymine and Cytosine The nucleotide sequence of the polypyrimidine region comprises SEQ.ID.NO.6 and SEQ.ID.NO.7. A method of producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 12, wherein the five-terminal splicing is a nucleotide sequence and comprises SEQ.ID.NO.3. A method of producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 12, wherein the five-terminal splicing is a nucleotide sequence and comprises 5'-GTAAG-3'. A method of producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 12, wherein the three-terminal splicing is a nucleotide sequence and comprises SEQ.ID.NO.4. a method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 12, wherein the three-terminal splicing is a nucleotide sequence And it contains 5'-CTGCAG-3'. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 1, wherein the carrier is a performance carrier selected from the group consisting of a plasmid and a cosmid. ), a group consisting of phagmid, yeast artificial chromosome, transgene, transposon, retrotransposon, jumping gene, and viral vector. A method of producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 1, wherein the vector comprises at least one virus or a second type ribonucleic acid polymerase promoter, Kozak translation start (Kozak The consensus translation initiation site), polyadenylation signals, and a plurality of restriction enzyme cleavage sites. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 28, wherein the viral promoter comprises a cytomegalovirus (CMV promoter), and a retroviral long terminal sequence is initiated. (retrovirus long-terminal region (LTR) promoter), hepatitis B virus (HBV), adenovirus AMV promoter, adeno-associated virus (AAV) Promoter) and plant-associated mosaic virus promoter. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 28, wherein the restriction enzyme cleavage restriction enzyme is selected from the group consisting of AatII, AccI, AflII/III, AgeI, ApaI. /LI, AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI /II, EagI, Ecl136II, EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI , NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI And the group of XmaI . A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the invention, wherein the vector further comprises a pUC origin of replication, and the prokaryotic cell has at least one The SV40 early promoter of the antibiotic resistance gene and an optional SV40 replication initiator in the mammalian cell. A method for producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 31, wherein the antibiotic-resistant gene line is resistant to penicillin G, penicillin, new Neomycin, paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin , tetracycline, rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, A group consisting of tylosin and G418. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to claim 1, wherein the vector is transfected into the human cell by a gene transfection method, and the gene transfection method is selected from the group consisting of Liposomal transfection, chemical transfection, chemical transformation, electroporation, homologous recombination, transposon delivery Transposon insertion), jumping gene transfection, viral infection, micro-injection, and gene-gun penetration. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the invention, wherein the genetically modified nucleotide precursor nucleic acid is produced by a transcription system selected from the group consisting of A group consisting of a second type of transcription system (Pol-II), a first type of transcription system (Pol-I), and a viral transcription system. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the invention, wherein the genetically modified nucleotide precursor nucleic acid comprises a message ribonucleic acid (mRNA), a heteronuclear ribonucleic acid (hnRNA) ), ribosomal ribonucleic acid (rRNA), transduction ribonucleic acid tRNA, snoRNA, small nuclear RNA (snRNA), precursor micro-ribonucleic acid (pre-miRNA), viral ribonucleic acid (viral RNA) and derivatives of the above ribonucleic acid Things and precursors. A method for producing a cosmetic for skin maintenance by a gene silencing effect as described in claim 1, wherein the intron excision mechanism is selected from the group consisting of a ribonucleic splicing system and nonsense-mediated decay (nonsense-mediated decay) (NMD)) A group of systems. A method for producing a cosmetic for skin maintenance by a gene silencing effect according to the first aspect of the patent application, wherein the gene silencing effect is by posttranscriptional gene silencing, ribonucleic acid interference (RNA) Interference) or NMD system action. A genetic recombinant nucleotide encoding a gene silencing effector, comprising: at least one intronic insert having an intronic insert, wherein the intron of the intron encodes a gene silencing effector Expressing the gene of tyrosinase and/or hyaluronidase; the sequence of the gene silencing effector is redesigned from the sequence of mir-434 microRNA (miRNA) , wherein the sequence of the mir-434 microribonucleic acid precursor (pre-miRNA) comprises SEQ.ID.NO.25. The recombinant nucleotide of the gene silencing effector according to claim 38, further comprising a plurality of exons, wherein the intron is linked by the exons and can be ribose in the cell The cells are separated from the exons by treatment with a cellular RNA splicing machinery; the exons can be joined to form a gene with a specific function. Such as the genetic recombination nucleotide described in claim 38, further package Containing: at least one multiple restriction/cloning site for linking to a expression vector of a ribonucleic acid transcription molecule representing a recombinant nucleotide of the gene; and a plurality of corrective nucleotides for forming the gene Transcription and translation termination sites of ribonucleic acid transcriptional molecules. The genetic recombinant nucleotide according to claim 38, wherein the intron further comprises: a five-terminal splicing site and a three-terminal splicing site; a branching dot region; and a polypyrimidine region. The genetically modified nucleotide according to claim 38, wherein the intronic insert has a gene silencing effector nucleotide, and the gene silencing effector is selected from the set of horses. Lariat-form spliced RNA, short temporary sense RNA (stRNA), antisense RNA (aRNA), short-hairpin RNA (shRNA), micro A group consisting of microRNAs (miRNAs), ribozyme RNAs, and precursors and derivatives of the above ribonucleic acids. The recombinant nucleotide of the gene according to claim 38, wherein the intronic insert is a reverse nucleotide comprising a silent base Because of the sequence 90% to 100% sequence complementarity. The genetically modified nucleotide of claim 38, wherein the intronic insert is a similar small-clustered nucleotide comprising 30% to 100% of the sequence complementary to the sequence of the gene to be silenced. rate. The genetically modified nucleotide of claim 38, wherein the intronic insert is a similar hairpin-like nucleic acid comprising a circular structure (stem- Loop structure), the similar small nucleotide sequence comprises SEQ.ID.NO.1 or SEQ.ID.NO.2. The recombinant nucleotide of the gene according to claim 38, wherein the intronic insert is a hairpin-like precursor microRNA (pre-miRNA), which is similar. The small clip pioneer microRNA sequence comprises SEQ.ID.NO.8 or SEQ.ID.NO.10. The gene recombinant nucleotide according to claim 41, wherein the gene silencing effector is a microribonucleic acid (miRNA), and the microribonucleic acid sequence comprises SEQ.ID.NO.9 or Is SEQ.ID.NO.11. The recombinant nucleotide of claim 41, wherein the branching point region comprises a branching point, and the branching point is an adenosine (A) and is located in a SEQ. ID. NO. 5 nucleotide sequence of the sequence. The recombinant nucleotide of claim 41, wherein the branching point region comprises a branching point, and the branching point is a nucleoside and the nucleotide sequence comprises 5'-TACTAAC-3 '. The recombinant nucleotide of claim 41, wherein the polypyrimidine region is a nucleotide sequence having a plurality of thymines and Cytosine, and the nucleotide of the polypyrimidine region The sequence comprises SEQ.ID.NO.6 and SEQ.ID.NO.7. The recombinant nucleotide of the gene according to claim 41, wherein the five-terminal splicing is a nucleotide sequence and comprises SEQ.ID.NO.3. The recombinant nucleotide of the gene according to claim 41, wherein the five-terminal splicing is a nucleotide sequence and comprises 5'-GTAAG-3'. The recombinant nucleotide of the gene according to claim 41, wherein the three-terminal splicing is a nucleotide sequence and comprises SEQ.ID.NO.4. The recombinant nucleotide of the gene according to claim 41, wherein the three-terminal splicing is a nucleotide sequence and comprises 5'-CTGCAG-3'.