TW201009073A - Generation of human embryonic stem-like cells using intronic RNA - Google Patents

Abstract

This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.

Description

201009073 VI. Description of the Invention: [Technical Field] The present invention relates to a method for cultivating, generating and screening human embryonic stem cells. The method comprises a microRNA reagent capable of transducing introns. . In particular, the invention relates to a method and compound for producing non-naturally occurring introns. The compound of this intron can generate small hairpin-like precursor microRNA (miRNA) in human cells via reaction. Therefore, the silent effects of certain genes involved in differentiation and development are induced in the cells, and the cells are then transdifferentiated into differentiated pluripotent stem cells like embryonic stem cells. More specifically, the hairpin-like pre-miRNA comprises mir-302a, mir-302b, mir-302c, mii-302d or a derivative or precursor homologous or similar to the ribonucleic acid described above. . [Prior Art] Recent studies on human stem cells have shown their potential for transplantation therapy. However, the sources that can be used to replicate human stem cells are limited and it is difficult to control their purity and quality. In 1998, James Thomson et al. (US Patent Nos. 5,843,780, US 6,200,806, US 7,029,913 and US 7,220,584) isolated the first human embryonic stem cell from human embryos (Thoms〇n α/., (1998). ) 282: 1145-1147). Two typical cell lines, HI and H9, are derived from the above isolated stem cells. Two years later, Gearhart et al. (such as US 6,090,622, US 6,245,566 and US 6,331,406) also developed the isolation of the original human 201009073 embryonic stem cells (hES) from post-blastocyst after human embryos. -like primordial germ cells). Because these methods of isolating embryonic stem cells must destroy the original embryos, many moral, humanistic and religious concerns have escalated, arguing about the legitimacy of these embryonic stem cells for clinical treatment. In recent years, people have gradually paid attention to the safety concerns of using these embryonic stem cells. For example, long-term culture of embryonic stem cells requires some unknown factors released by mammalian feeders to maintain stem cell pluripotency. Therefore, these embryonic stem cells need to be cultured in mouse or human fibroblasts. In the layer of mammalian cells. Prior art of Reubinoff et al. US 6,875,607 attempts to accomplish this method, however, mammalian cells of fibroblasts have completely different antigenic properties and may contaminate embryonic stem cells and cause immunological rejection of the patient. In addition, although some culture conditions have been developed that do not require mammalian cells, none of them maintain stable, undifferentiated embryonic stem cells for a period of time. This is because the purity of the isolated human embryonic stem cells ® is usually not high. No embryonic stem cells today can achieve 100% high purity in a culture environment. Even under optimal culture conditions, about 3 to 5% or more of embryonic stem cells tend to differentiate into other cell types and lose their stem cell characteristics. Teratoma is a common phenomenon after embryonic stem cell differentiation. A fetal tumor is a tumor derived from a human germ cell line, which often contains a variety of cancerous cell types similar to endoderm, mesoderm, and ectoderm. Therefore, how to avoid breast cancer cell contamination and increase stem cell purity are two major issues in stem cell research today. 5 201009073 Induced Pluripotent Stem (iPS) cell line was published by Takahashi and Yamanaka in 2006 (ce" 126: 663-676). By using a transgenic gene to transfer four transcription factors (〇必/4, the heart into a mouse fibroblast, successfully in vitro, such as vitro), transgenic somatic fibroblast cells into embryonic stem cells. In addition, 'Y et al. developed a new iPS cell from human fibroblasts by similar transgenic genes ((9cM, 5bjc2, iVimog, and LIN28)) (YU Zhan, (2007) - ce 318: 1917- 1920). The application of iPS can not only solve the problem of morality and purity, but also provide a tailor-made treatment for patients with somatic cell nudear transfer (SCNT) (Meissner ei α/., (2006) iVifliwre 439: 212-215) The so-called jpg cell-based somatic cell nuclear transfer therapy technique has been shown to treat sickle-type anemia in a mouse model (Hanna ei a., (2007) 5 Wewce 318: 1920-1923). However, the application of ips is still flawed. These defects are in the way of cell production; one is the potential danger of using retroviruses; the other is the use of transcription factors with oncogene properties. The retrovirus transfection method is The only way to simultaneously transfect four full-length transcripts into somatic cells. However, the possibility of random insertion of retroviral vectors into cellular genomes may affect non-target genes and cause Expected results, especially when one of the transfected genes is at risk for oncogenes. It is quite difficult to accurately transfer the four full-length transcription factors to the correct position. Wire, ips technology requires precise The transcription factor 201009073 regulates each other and stimulates a variety of message transmission pathways and exhibits many developmental factors. Although the detailed mechanism remains unclear, 〇ct4-Sox2-c-Myc-Klf4 Fluor Oct4-Sox2-Nanog The basic effect of -LIN28 produces a developmental factor message that blocks the early differentiation of cells. Although there is only one embryonic stem cell marker, OcM, other genes used to generate ips cells are usually associated with developmental pathways. These genes are usually expressed in different embryos. The cell stage or location directs the cell to a specific differentiation path. By disposing these genes, the normal pathway of cell differentiation is disrupted by ©, and these cells are transdifferentiated to a state similar to embryonic stem cells. It is not natural to achieve the goal. In natural fertilization, maternal materials are mainly Regulates the maintenance and replication of stem cells. This is also the same differentiation pluripotency of embryonic stem cells before the cell cycle of 128. Maternal substances are produced during the period of oogenesis and coexist in mature eggs for Early embryonic development. In mouse eggs, ribonucleic acid accounts for a large proportion of maternal material, © about 450/〇 in the whole genetic material (Stitzel ei ar/·, (2007) 316: 4〇7_4〇8 ). In the maternal-zygotic transition, the maternal material is rapidly degraded, and the transcription process of the zygote begins to generate embryonic development like the two-cell period (0, Farrell, (2〇〇4) · R35-45). It can be understood that many maternal substances are inhibitors of the binding gene product, which can be transduced into pluripotent cellular states in the early stage (4). Thus the secret of 'stem cell maintenance and replication should be in the maternal material' rather than the developmental message that emerges after the differentiation of pluripotent cells. 7 201009073 In short, in order to generate and maintain human embryonic stem (hES) 4ike cells as if they had a maternal material, a novel way is needed to transfer the isolated maternal material to human stem cells or somatic cells. It can maintain stem cell characteristics or transdifferentiate somatic cells into hES cells. Therefore, there is still a need for an efficient, simple, and safe method of transferring genes to deliver maternal material, especially maternal ribozyme nuclei. SUMMARY OF THE INVENTION The present invention is a method for culturing, generating and screening human embryonic stem cells, which expresses mir_302a, mir_302b, by using intron expression of hairpin-like microRNAs. Mii:-302c, mir-302d and homologues or compositions of precursor microRNAs in which the ribonucleic acids are recombined with each other. Microribonucleic acids are typically about 18 to 27 nucleotides in length and are capable of directly degrading their target message ribonucleic acid (mRNA) or inhibiting the translation of the target protein. Direct degradation or inhibition of protein translation is due to the degree of complementarity of microRNAs (miRNAs) with the target (complementarity^mij^i^ family (mir_302s) is highly conserved in all mammals, this family consists of four A highly homologous microRNA (>90% homology) consisting of mir-302a, mir-302b, mir-302c, and mir-302d. The mir-302 family is represented by a gene group. The method exists in a long RNAtranscript and is mir-302b-mir-302c-mir-302a-mir-302d-mir-367 linked from the 5th to the 3rd end 201009073 (Suh β //.,(2004) £)ev.〇/· 27〇: 488-498). Although mir-367 is also present in the mir-302 family of genes, mir_367 actually performs less than the mir-302 family. The mir-302 family has been found to be extremely high in mouse egg cells and human embryonic stem cells (Tang Kou, (2007) Genes Dev. 21: 644-648; Suh et al., (2004) Dev. Biol. 270 : 488-498). The egg cells of mice lack Dicer, a highly consistent ribonuclease for microRNA production, and egg cells restricted to the division phase of meiosis I. This phenomenon indicates that microRNA plays an important role in the process of egg cell formation (Murchison ei α/., (2007) Gewas Dev. 21: 682_693). Therefore, the mir-302 family is very likely to be the main maternal material that maintains stem cell function and re-replication. Compared with previous iPS cell technology, the expression of four cell transcription factor genes is different. Each member of the mir-302 family can regulate more than 445 cells ® genes. In addition, according to the database of miRBase::Sequencesrhttp://micr〇ma ggRger.ac.uk/), the common target genes of each member of the mir-302 family are almost identical. Many of the target genes of mir_3〇2 are essentially developmental messages involved in the differentiation of cells involved in embryogenesis. Therefore, the function of mir_3〇2 is likely to block or suppress the massive generation of developmental messages rather than interfering with specific message paths. For example, 'insulin-like growth factors (IGF)) are potential developmental messages for neuronal stem cells and their precursor cell lines.

Ras/Ra仏mitogen-activated protein kinase MAPK pathway or via 9 201009073 phosphatidylinosital 3-kinase (PI3K)/Akt message pathway. More than μ of the IGF receptor (IGFR) and Ras/PI3K message pathways were found to be the target genes of mir_3〇2. This phenomenon also shows a tight regulation in mammalian egg cells and embryonic stem cells to prevent neuronal differentiation. The mir_3〇2 family complementarily binds to homologous sequences of the transcriptional molecules of the underlying gene' and thereby inhibits their protein translation via RRNA interference. A similar mir_3〇2 family inhibitory effect was found in many different cellular tissues. According to the above evidence, the mir_3〇2 family is likely to play an important role in the maintenance and self-replication of embryonic stem cells. By inhibiting the genes involved in cell development and differentiation, the mir-302 family may be used to transdifferentiate somatic cells into visceral stem cells and maintain the differentiation pluripotency of the cells and the ability of self-replicating embryonic stem cells.

In order to test the embryonic stem cell production and maintenance function of mir-302, the present invention uses a Pol-II-based intronic miRNA expression system to express in different human somatic cells. The mir-302 family, which is used to mimic the native intronic miRNA biogenesis pathway, is shown in Figure 1. The intron microRNA production process is related to the relationship between the precursor message transcription factor and intron splicing/resection of the intracellular second transcription system, usually occurring in the nucleus near the chromosomal chromatin (Lin β). β/. (2〇〇4) Drug Design Reviews 1: 247-255; Ghosh et al. (2000) RKA 6: 1325-1334). In eukaryotic cells, a gene transcription factor such as a message ribonucleic acid having a protein translation ability is produced by a type II RNA polymerase (type-II RNA 10 201009073 polymerases). Usually the gene transcription process generates the precursor message 餹 餹 re-_A), which contains four main parts: the 5'-untranslated region (UTR), containing the protein translation codon exon (protein- Illustratoron), introns with no protein translation ability (in1r〇n) and three-terminal non-translated regions (3'-UTR). Broadly speaking, the five-terminal and three-terminal untranslated regions can be considered as specific introns. The intron accounts for a portion of the primordial message that has no transcribed sequence in the ribonucleic acid. Each intron has a base from 30 bases to thousands of test sites, and must be cut off before the message ribonucleic acid matures. The process by which the precursor message RNA excises the intron is called RNA splicing and is usually spliced via intracellular spliceosome. After ribonucleic splicing, a small number of intron-derived ribonucleic acid fragments are further processed to form miRNA-like. derivative molecules, which can effectively and individually suppress their miRNA-like. Target gene. The above silent inhibition mechanism is via an RNAi-like mechanism, in which the exon of the pre-mRNA of the precursor message is joined together to form a mature message ribonucleic acid. For protein synthesis. We have demonstrated that effective microRNAs can be produced by introns in vertebrate genes, a mechanism different from that of small interfering ribonucleic acid (siRNA) or intergenic miRNA (Lin β d (2003). Biochem Biophys Res Commun. 310: 754-760; Lin et al. (2005) 356: 32-38). To confirm this difference, Figure 2 shows the intracellular biosynthesis process and RNAi machinery in small interfering ribonucleic acid (siRNA), exon micro 11 201009073 ribonucleic acid (intergenic miRNA) and intron microRNA (intr〇 Comparison between nic miRNAs). It is generally speculated that the siRNA is formed by two fully complementary ribonucleic acids, which are transcribed from a promoter (Pmo) at the opposite position on the same DNA template, in a hybridized form. It is then cut into two-nucleotide nuclear nucleic acids of about 20 to 25 by the endonuclease (RNasein endoribonucleases, Dicer). Unlike siRNA models, intergenic miRNAs (such as lin-4 and let-7) contain a long non-coding precursor RNA transcript (pri-miRNA). This molecule is directly from the second. A type (ρ〇ΐ-π) or a third type (Pol-III) ribonucleic acid promoter is transcribed, whereas an intron pri-miRNA is only transcribed from a second type promoter and will be ribose Nucleic acid splicing is spliced. In the nucleus, pri-miRNAs are further composed of Doxa CDms/w-like RNases (for the formation of intergenic miRNAs) or splice and exosomal components (for the formation of intronic miRNAs) to form small clips and ridges. A hairpin-like stem-loop precursor (also known as an intron micronucleic acid intronic miRNA), which is then transported to the cytoplasm for processing by the miRNA-associated dextran . Subsequently, the above three forms of ribonucleic acid are finally integrated into an RNA-induced silencing complex (RISQ), in which the double-stranded siRNA or single-stranded miRNA is contained in the complex. The induction of the silent complex is used to generate a different pathway between the siRNA and the miRNA pathway (Tang, G. (2005) and bc/zem 5W. 30: 106-114). Therefore, the miRNA effect 12 201009073 is used as a general tmiRNA is more specific and less unpaired than siRNA, because m has only a single strand with A. In other words, siRNA mainly catalyzes the brain __, while miRNA can induce silk fibroin _ degradation or inhibit protein biosynthesis Because the miRNA pathway of introns is controlled by intracellular multiplex regulation mechanisms, which include a second type of transcriptional machinery, RNA splicing, and exosomal depletion (exosome digesti〇r^ nonsense-mediated degradation (; nonsense_mediated decay (; NMD) processing), therefore the gene silencing effect of intron micronuclear ribonucleic acid is considered to be the most efficient, specific, and safest path among all three RNAi pathways (Lin α/ (2〇〇8) plus 5/osc Fine ce 13: 2216-2230) The present invention discloses a new function of introns for gene regulation and its relative application. As shown in Figure 3A and Figure 3B, intronic RNA splicing (intronic RNA splicing) And a processing mechanism, a preferred embodiment of the invention is a second type of recombinant transcriptional transcriptional Θ system comprising at least one spliced intron, said intron being referred to in the present invention The function of a gene with a suppressor gene or a gene that is highly complementary to a sequence is formed by co-transcription of a ribonucleic acid transcribed with a second-type transcription system, and the intron is ligated after RNA splicing. Released. Thus 'spliced and further processed into mature gene silencers (such as small hairpin RNA (shRNA)) and microribonucleic acid (miRNA), which can stimulate RNAi-related Gene Silence 13 201009073 Effect. After the intron is spliced and removed, 'recombinant gene exon transcription molecules are joined to form a mature message ribonucleic acid molecule' For the translation to synthesize a marker or functional protein. As shown in Figure 3A, specifically comprising a plurality of highly conserved nucleotide fragments comprising a five-terminal splicing site (5'-splice site), a branch point motif (BrP), a poly-pyrimidine tract, and a three-terminal splicing site (3, _splicesite). In addition, a class of small ribonucleic acid (s_A-like) precursor microRNA sequences are inserted into SpRNAi. This pioneer micronuclear nucleic acid sequence is inserted between the five-terminal splicing site and the branching point region. This part of the process and the heart and intron in the nuclear splicing and processing (RNA splicing and processing), can form a set of horses (1 pull) structure. In addition, the three ends contain a multi-translation stop sub-region (multiple

Translational stop codon region (T codon)) ' to improve the accuracy of intronic RNA splicing and nonsense-mediated degradation processes. When this multi-translation stop appears in the cytoplasmic message ribonucleic acid, this multi-translating stopr can transmit a message that activates the nonsense-mediated degradation pathway for the degradation of any abnormal ribonucleic acid structure in the cell. However, the shRNA and the precursor microRNA (pre-miRNA), which have a high degree of secondary structure, can be individually retained to the sputum splicing to form mature siRNA and miRNA. In addition, in order to express in the cell, we use technology to insert (4) a restriction enzyme cleavage site into a red fluorescent protein (gravity/household) gene (mute protein from mca anemone). And the formation of a genetically recombined red fluorescent protein containing 14 201009073 toilet / / and the Qing gene, in which the red fluorescent protein gene of the two hundred and eight nucleotide position of the restriction enzyme plus " After that, the breakpoint of -AG-GN nuclear bitter acid will be generated, and three nuclear-protrusion structures will be produced at both ends of the breakpoint. φ Fine energy can be embedded by the upper-end cleavage and the three-end splicing . Because the secret tour of the silk red (four) normal performance 'but after the intron splicing, the red fluorescent protein can resume normal performance' so we can use the red fluorescent protein in the transfected cells The amount released by the s_/miRNA of the sorghum. The scaly red fluorescent protein gene also provides many exon splicing enhancers (ESE) to improve the correctness and efficiency of ribonucleic acid splicing (RNA _cing). 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. The region and the three-terminal © splicing to form an artificial (artificial intron), the artificial _/ contains at least one embedded ribonucleic acid structure, which may be an antisense RNA or S_A or miRNA structure. In addition, these components can be chemically synthesized and linked by a DNA synthesizer. On the other hand, the connection of these elements can also be connected by a restriction enzyme. This intron can be directly transfected into a cell or recombined with an integrated cell gene to be synthesized by a second type of transcriptional polymerase system to synthesize a gene transcriptional molecule (e.g., pre-mRNA). After RNA splicing and message ribonucleic acid 15 201009073 mature, the human microribose structure will act through the intracellular splice and nonsense-mediated degradation mechanisms, and the structures can pass through Complementary and intensify the cranes that donate gene molecules. The squamous, New Zealand-inducible exons can be joined to form a mature message nucleus nucleic acid, which exhibits the function of the gene, such as reporter translation or the following proteins: red glory protein, green Fluorescent protein (green flu〇rescem ρ_ίη), glover _ferase), lactose gene regulatory group (lac_z) and derivatives thereof. The presence of the reported/marked proteins allows for the presence of mosquito shRNA/miRNA translocations, especially in identifying gene silencing/RNAi effects. In addition, the mature message ribonucleic acid formed by the connection of exons may also contribute to the specific gene expression of the silkworm, silky shed or lost. In other aspects, the present invention provides a novel compound (to induce a cell to produce a gene silencing molecule via an intrcmic RNA splicing mechanism, which inhibits specific gene function via an antisense gene-mediated knockout or nuclear-nuclear interference effect (RNAi) The gene silencing molecule derived from the artificial intron 包含 contains the antisense ribonucleic acid, ribozyme, ribozyme, short ribonucleic acid (stRNA), double-strand ribozyme Nucleic acid, small interfering nucleoside nucleic acid (smau 丨 两 两 ,, siRNA), tiny codon-free nuclear nucleic acid (such as 丫 non_c〇ding RNA, tncRNA), short hairpin RNA (shRNA) , hairpin_like RNA structure, micronucleus listening nucleic acid (microRNA, miRNA) and related to RNA interference (RNAi) before / pioneer 16 201009073 nuclear nucleic acid structure. The gene silencing reagent derived from the ribonucleic acid (recombinant nucleotide) of these introns contributes to the silent effect of specific genes 'the specific genes from the pathogenic transgenic genes 7 to the transgenes), viral genes ( Viml genes), mutant genes, oncogenes, disease-reiated small RNA genes, and any genes with or without protein translation and genes mixed with them One. © By the expression of the red fluorescent protein contained in the second type of transcriptional polymerase, we have successfully used human prostate cancer cells (prostate cancer LNCaP), human cervical cancer (HeLa). And rat neuronal stem cells (HCN-A94-2 cells) (Lin et al (2006a) Methods Mol BzW. 342: 295-312) produce mature shRNAs and miRNAs with gene silencing effects. The same effect was observed in zebrafish, chicken, and mouse (Lin β α/. ❹ (2〇〇6b) Mei/wds M?/342: 321-334). We have tested different precursor microRNAs in zebrafish and many different human cell lines for the expression of green fluorescent protein and other cellular gene transcriptional molecules, and learned that the more efficient gene is silent microRNA The five ends of the sequence segment between the five-end splicing and the branch point region. As shown in Figure 3C, a significant gene silencing effect occurred in the transfection of the green fluorescent protein (antkEGFP pre-miRNA) group (lane4 miR group), but in other experimental groups and control groups or This effect was not detected in the control group; these experimental groups 17 201009073 and the control group were sequentially (from left to right): 1. The control group; 2. The precursor microRNA for the HIV virus (month 7F-p24) Control group; 3, anti group against anti-intentional green fluorescent protein and no splicing structure (antisense GFP insert without the hairpin loop structure); and 5, reverse miR* group of precursor microRNA sequence' The miR* group of the transgenic microRNA sequence is fully complementary to the anti-green fluorescent protein precursor microRNA (anti-five pre-miRNA, miR*). Regarding other non-target genes, such as red fluorescent protein and actin (β-actin), there is no silent phenomenon, indicating that the micronucleic acid-mediated nuclear nucleic acid interference is highly specific. To demonstrate the effect of ribonucleic RNA splicing on intron-induced ribonucleic acid interference (intronic-like enthalpy), we have tested three different performance systems, as shown in Figure 3D (from left to right): 1. No inclusion The red fluorescent protein carrier group of your sub-genus does not have any precursor microRNA; 2. The red fluorescent protein carrier of intronic anti-EGFP pre-miRNA with intron Group expressing (expression expression ❹ system expressing RGFP with an intronic anti-EGFP pre-miRNA insert); 3. Red fluorescent protein carrier group with intron-resistant green fluorescent protein precursor microRNA (five-terminal splicing defect) . Analysis by Northern Bolting showed that 'mature microRNAs (ie, microRNAs in introns) are only red fluorescent proteins with intron-resistant green fluorescent protein precursor microRNAs. The vector group was formed (lane2), and the GF cadaveric expression system was identical to the lane4 expression system of Figure 3C, as 18 201009073. This confirmed the need to rely on the intracellular splicing mechanism (cellu ar splicing) to form the intronic miRNA 〇* Based on the above observations, we further determined the preferred precursor microRNA (pre-miRNA) structure for the most significant gene silencing effect induced by RNA-indueed silencing c〇mpiex (RISC) (Lin β .β/. (2005) Ge from 356: 32-38). The RISC complex, a protein-RNA complex, can cause degradation of specific gene-transfer molecules or inhibition of translation via a ribonucleic acid interference mechanism. For RISC complex

In the formation of the complex, the double-stranded siRNA plays an important role. The double-stranded siRNA is functionally different, and the RISC complex is only inclined to react to one of the strands. This kind of _ diligence per share of Wu Linji is determined by the age of the contribution. Based on the siRNA model, it is speculated that the double-stranded ribonucleic acid formed by m with A and its complement is also important for the formation of RISC complex. If this is true, there should be no functional tendency in the pre-m surface A. L〇叩) β, displayed in the structure. However, we found that in the zebrafish, the stem4oop structure of the intronicpre-miRNA has an effect on the stock selection of the orphan C complex and the formation of miRNA. As shown in Fig. 4A, two expression vectors of intronic nuRNA with different introns are named miRNA stemloop-tn^Ai^] and miRNA stemi (8) surface (the meridian* represents the ability and maturation). miRNAs with complementary miRNA sequences). The precursor micronucleus-acids of the two groups have the same double-stranded structure (stem-arm 19 201009073 structure). The bent structure can perform a silent effect on the nucleotide 280 to 302 nucleotide sequences of the five GF_P genes. Specifically, the miRNA is identical to the full-length intact mature microribonucleic acid sequence, while the miRNA* is complementary to the sequence of the mature microRNA. Transfection of these vectors (6〇ug * each) via liposome into the two-week old zebrafish embryos 24 hours later (Lin β α/. (2005) 356: 32-38) via mirVana microribose The latex beads in the nucleic acid separation column precipitate microRNAs (miRNAs) with the potential for silent effects. After sequence alignment, the more silent effect of microRNA mi (miR-EGFP (280-302)) was picked and identified as miRNA_steml--miRNA*[2], as shown in the shaded gray in Figure 4B. Because mature microRNAs (miRNAs) are only found in zebrafish transfected with miRNA-stemloop-miRNA* [2], it is concluded that RISC complexes tend to interact with miRNA_stemloop-miRNA* [2] rather than miRNA*- The pioneer microRNA of stemloop-miRNA[1]. In this experiment, the experiment was carried out using a zebrafish (Tg (actin·ΟΑΙν4: υΑ8$φ)) expressed by an actin promoter. This zebrafish has been expressing green fluorescent protein in various scorpion cells. As shown in Fig. 4C, after the zebrafish is transfected with the vector, the green fluorescent protein gene is silenced and the red fluorescent protein which can be the indicator protein is expressed. The observation revealed that the gene silencing effect of the gastrointestinal part was weaker than that of other tissues, and it was speculated that the RNase activity of this part was strong. In Figure 4D, Western blotting can detect the red fluorescent protein expression of miRNA*-stemloop-miRNA[1] (group 1) and miRNA_stemlo〇p-miRNA* [2] (group 2), 20 201009073 The gene silencing of green fluorescent protein is only found in zebrafish transfected with miRNA-stemloop-miRNA*[2] (2 groups), and should also be echoed by Figure 40 due to miRNA*-stemloop-miRNA[l] and The thermodynamic stability of the five-terminal stem-arm structure of miRNA-stemloop-miRNA is 2, so we infer the intron micronuclear nucleic acid (intr〇nic. stem-loop) may be The RISC complex forms a stock selection of mature microRNAs. The cut position of the stem-arm is determined by the selection of mature microRNAs (Lee β α/. ρ〇03) ΛΓ(10)/re 425: 415-419), it is the stem_1〇〇p of the precursor microRNA of the intron that may determine the identification of the special cleavage. Because the structure of the above-mentioned precursor micronuclear domain nucleic acid (stem_i00p) is too large, the performance of the vector of _/-7?6^ may be poor, so the use of transduction ribonucleic acid (tRNA) is bent (l〇op ) (5 '-(A/U)UCCAAGGGGG-3,) (S£Q.ID.N0. 29) to replace the original larger bending structure 'this smaller bend (tRNAmet loop) ❿ proved to be able Accelerate the transport of microRNAs (miRNAs) from the nucleus to the extranuclear, and the transport pathway can be via the intranuclear and extracellular transport mechanisms such as Ran-GTP and Exportin-5 (Line < 3/ (2005) Gene 356: 32-38). Recently, the present invention uses a pair of modified pre-mir-302 bent l〇op structures (such as N0.1) and 5'-GCCTGGCTTAGC-3, (SEQ.ID.N0.2), which provide the same fast The pioneer micronuclear nucleic acid transports and does not interfere with the transport of the transduced nuclear nucleic acid. The improved pioneer microRNA l00p was designed by mimicking the short stem-loop structure of mir_3〇2s, which can be expressed in embryonic stem cells algebra 21 201009073 but is less expressed in differentiated cells. Therefore, the improved pioneering microribonucleic acid loop does not interfere with the transport path of its own microRNA. Regarding the insertion of the precursor microRNA, because the restriction enzyme in the insertion of the intron of the \(4) jGM3 recombinant gene is located at the five-terminal and three-terminal insertions of the / and Mw/' introns (intr) 〇nic insert) can be replaced by the excision of many other specific genes, the precursor microRNA (pre_miRNA) (for example, anti-green fluorescent protein and mir-302 pioneer microRNA). By changing the precursor microRNA insertion to counter or silence different ❿ gene transcriptional molecules' Therefore, the microRNA-generating system of this intron can be applied to the priming gene ζ·« Wiro And a powerful tool for m Wvo silent effects. In order to confirm the correct insert size, a porcine expression vector (10 ng) with pre-miRNA can be used by PCR technology and using a primer (for example, 5'-CTCGAGCATG GTGAGCGGCC TGCTGAA-3' (SEQ. ID.N0.23) and 5'-TCTAGAAGTT GGCCTT-CTCG GGCAGGT-3, (SEQ.ID.N0.24)) at 94 ° C (1 min.), 52 ° C (1 〇 min.), and 70 °C (1 min.) replicates 25 cycles (cycies) and replicates in large quantities (inserts). The final product of the final PCR was further chromatographed via 2% agarose gel and purified using a gel extraction kit (Qiagen, CA) and sequence confirmed. The present invention employs an experimentally demonstrated second type polymerase (p〇l_H) expression system and utilizes this system to develop a family of mir-302 genes for expression in human or mouse cells. In a preferred embodiment, the present invention provides a method of using a non-naturally occurring intron and a compound thereof, which can be treated by a human or mouse cell into a ribo-like ribonucleic acid molecule for induction of cell development 22 201009073 Or the gene silencing effect of differentiation-related genes. The above method comprises the steps of: a) providing 1) a cell which exhibits a plurality of genes, mir_3〇2s for a gene involved in 'development or differentiation; and 2) an expression-competent composition, the compound Containing a recombinant group

Therefore, the gene can produce a primary RNA transcript of an intron having a gene silencing effect in the above-mentioned cells, and the molecule can generate a mir-302-like ribonucleic acid molecule from the intron ( a mir-302-like RNA molecule for the step of knocking down or inhibiting a target gene in a cell via a milk splicing and related mechanism; the step of treating the cell with the above compound, the treatment being under certain conditions, The function of the target gene in the conditional cell is inhibited. Thus, the cells are transformed into embryonic stem cells, which express embryonic stem cell markers (such as the five hearts and the S syndrome). The cell can express the target gene in vitro (additional, in vivo (沅vz.w), in vitro in vivo or in vivo (exWw). In a broad sense, the intron is in the gene. A non-coding sequence comprising a full-length intron and a five-terminal non-translated region and a three-terminal non-translated region. In some aspects, the mir_3〇2 ribonucleic acid of the present invention is designed. The molecule contains the first 17 nucleotides of mir-302a, mir-302b, mir-302c or mir-302d (eg 5'-UAAGUGCUUC CAUGUUU-3, (SEQ.ID.N0.3)). All mir- The 302 family of microRNAs possess the same 17 nucleotides from the five-terminal number. In other embodiments, the mir_3〇2 ribonucleic acid molecule can also design 23 201009073 intron regions in cellular genes. Internally, and in common with it. In general, the technique of 'intron insertion includes plasmo-like transgene transfection, homologous recombination, transposition (transposon delivery), DNA ligatio n), insertion of transgene insertion, jumpiiig gene integration, and retroviral infection or a hybrid method of the above method. In another embodiment, the recombinant gene of the present invention A precursor message ribonucleic acid (pre-mRNA) is expressed. The recombinant gene consists of an exon and an intron. The exon can be ligated after ribonuclease cleavage to form a dynamite b. The ribozyme nucleic acid (mRNA) is then translated into a protein for the recognition of the release of the ribonucleic acid of the intron, at which time the intron is released from the nucleus and is processed to have a gene silencing effect with a specific gene silencing function. Gene silencing effector 'The gene silencing effector can be antisense RNA, microRNA (miRNA), short ribonucleic acid/small ribonucleic acid (shRNA), siKNA, double-stranded ribonucleic acid The precursor of the above ribonucleic acid (for example, pre-miRNA and Piwi-interacting RNA (piRNA)). The ribonucleic acid molecules of these introns may contain a type of hairpin bend. A hairpin-like stem-loop structure (approximately equivalent to a small ribonucleic acid), this structure comprising a sequence homologous to 5,_GCTAAG-CCAGGC-3' (SEQ.ID.N0.1) Or 5,-GCCTGGCTTAGC-3, 201009073 (SEQ.ID.NO.2), which promotes the correct splicing of the ribonucleic acid molecule from the intron and promotes transport of the molecule from the nucleus to the cytoplasm. Moreover, the bent structure of the nucleic acid molecules of these introns (stem_arms) contains sequences complementary or homologous to a particular target gene. The sequence of the homologous or complementary ribonucleic acid molecule of the intron ranges from about 15 base pairs to about 1500 base pairs, preferably from about 18 to 27 base pairs. The complement or homology of these intron ribonucleic acid molecules to the target gene sequence is between about 30% and 100%, preferably between 35% and 49%. For the intronic RNA of the intron of the small clip, the complement or homology rate is between about 30% and 100%; for the linear intr〇nic RNA of the linear intron 90 % to 1 〇〇

In addition, the five-terminal end of the non-naturally occurring (artificial) intron comprises a 5'-splice site or 5' clip, wherein the five-terminal splicing is a nucleotide sequence and is homologous to 5 , -GTAAGAGK-3, (SEQ.ID.N0.4) or GU(A/G)AG U sequence (eg, 5'-GTAAGAGGAT-3, (SEQ.ID.NO. 30), 5,-GTA AGAGT-3', 5'-GTAGAGT-3' and 5'-GTAAGT-3'). At this point, the 3'-splice site or 3, clip sequence is homologous to the GWKSCYRCAG (SEQ.ID.N0.5) or CT(A/G)A(C/T) NG sequence (eg , 5,-GATATCC TGCAG-3' (SEQ. ID. N0.31), 5'-GGCTGCAG_3, 5, -CCACAG-3'). In addition, the branch point region sequence is located between the five-terminal and three-terminal splicing regions, and the branch point region is located in a nucleotide sequence homologous to 5 '-TACTWAY-3, (SEQ. ID. N0.6) (eg, 5'-TACTAAC-3' and 5,-TACTTAT-3,), the branch point zone package 25 201009073 3 is a fulcrum, and the branch point is an adenosine (A) capable of forming a set of horses ( Lariat) The structure of the intron ribonucleic acid, which is produced by the synergistic action of serotonin synthase (2'-5'-oligoadenylate synthetases) and splices. In addition, the polypyrimidine region is located between the branch point region and the three-terminal splicing region, wherein the polypyrimidine region is a nucleotide sequence having a high density of thymine and Cytosine, and a polypyrimidine region The nucleotide sequence is homologous to 5'-(TY)m(C/-)(T)nS(C/-)-3' (SEQ.ID.N0.7) and 5,-(TC)nNCTAG (G /-)-3' (SEQ.ID.N0.8). Wherein "m" and "n" mean a multiple repeating sequence of greater than or equal to one. 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. Some linked nucleotides can be used to join the above intron fragments. Based on 37 CFR 1.822 (Taiwan ', the format of nucleotide acid and amino acid sequence list is also the same), W refers to adenine (adenine or thymine (T)) / uracil (U) )), K means guanine (G) or thymine (T) / uracil (U), S means cytosine (〇 or guanine (G), Y means cytosine (〇) Or thymine (T) / uracil (U), R refers to adenine (A) or black mites (G) and N refers to adenine (A), cytosine (〇, guanine (G) Or thymine (T) / uracil (U) or others. In another embodiment of the invention, the recombinant nucleotide can be recombinantly incorporated into a performance vector for gene transfection. The expression vector is selected from the group consisting of DNA transfection Genes (DNA transgenes), plastids, jumping genes, transposons, retrotransposons, retroviral vectors, lentiviral vectors, glands Viral vector 26 201009073 (adenoviral (AMV) vectors), adeno-assodated viral (AAV) vectors, modified hepatitis-viral (HBV) vectors, Cytomegalovirus-associated viral vectors (Cyt〇megai〇vims (CMV)-associated viral vectors). These are the gene silencing effectors that express different introns during the transfection process and the GFP expression system. The vector 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 self-organize the intronic hairpin RNA insert of the intron. Derived to silence many genes. The advantage of this method 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 is via the cell nucleus chromosomal splicing and processing mechanism. An RNAi-related.gene silencing effector, which contains small interfering RNA (siRNA), microRNA (miRNA), and sputum Small hairpin RNA (shRNA). Intracellular specific

Controlled by a gene ribonucleic acid promoter selected from the group consisting of a second type ribonucleic acid polymerase promoter (Pol-II promoter), a third type nuclear nucleic acid polymerase promoter (Pol-IIIpromoter), and a virus. Promoter. The viral promoter may be a Pol-II-like RNA promoter selected from the group consisting of a cytomegalovirus (CMV) and a retrovirus long-terminal region (retrovirus long-terminal). Region ¢11113⁄4,: hepatitis B virus (HBV), adenovirus (adenovirus 27 201009073 (AMV)), and adeno-associated virus (AAV). For example, lentiviral LTR The promoter can provide more than 5x1 〇 5 units of the precursor message ribonucleic acid per cell. In addition, a pair of drug-sensitive repressors are inserted at the front of the lentiviral promoter to control the gene silencing. The manner in which the effector expresses the rate is one of the possible ways. The inhibitor can be inhibited by a chemical or antibiotic selected from the group consisting of G418, tetracycline, neomycin, ampicillin, kanamycin, and derivatives of the above antibiotics. . The present invention provides a novel method for producing intron ribonucleic acid (intr〇nic RNA) in a cell for the production of mature siRNA, miRKA and shRNA, which can induce gene silencing effects of ribonucleic acid interference gene silencing effects) siRNA, miRNA and s_A In a cell, a single or multiple gene silencing effector can be produced by the expression of a cellular mechanism and the processing of the meson of the intron of the first shRNA of the present invention. For example, as previously described, the anti-green fluorescent protein precursor microRNA (pre-miRNA) is heterologously expressed on zebrafish, as shown in Figure 3A, resulting in two sets of mature micronucleated nucleic acids of different lengths, such as (mir-EGFP 282/300 disk mir-EGFP280-302). This clearly shows that a single implant can produce more than one gene-silencing effectors. A sense or antisense DNA configuration can produce the same or a different gene silencing effector for complementation with the target gene molecule. In some instances, the gene silencing effector of the intron of 28 201009073 can hybridize with some of the underlying gene transcriptional molecules (eg, mRNA) to form a double-stranded ribonucleic acid that drives the ribonucleic acid interference (RNAi) effect. The gene silencing effector system is continuously produced by the expression vector, and thus the present invention can alleviate the concern caused by rapid degradation of small ribonucleic acid. Furthermore, the present invention can be used to produce stem cells. The potential application of the present invention includes human embryonic stem cells without mammalian cells. The strain maintains and avoids the cell line differentiation of the above cell line, in vitro replication and culture of the differentiated stem cell strain, purification of the homogenous stem cell population, and transplantation using the above stem cells. The present invention may also be used for studying stem cell function and mechanism. Alternatively, the stem cell properties can be altered for specific use. In various embodiments, embryonic stem cells such as the invention can be selected from normal somatic cells, cancerous somatic cells, mammals (eg, humans, monkeys, rats, and mice). The mature stem cells are derived. 实施 Embodiments In order to clearly describe the present invention The following specific terms are used herein. However, the present invention is not intended to be specific to these specific terms. It should be rotated to include all technical equivalents, which can be similarly achieved by similar means. 0 "Nudeotide" - The word here means - a single molecule of deoxyribotmcleotide XDNA or a rib〇nude〇tide (RNA) molecule 'these molecules contain five carbons _em〇se), sulphate (10) 〇Sphate 29 201009073 and nitrogenous heterocyclic base. This test is a nucleoside linked to a five-carbon sugar via a glycosidic bond, which is a micronucleotide linked to the phosphate at the three-terminal and five-terminal positions of the five-carbon sugar. The term "oligonucleotide" as used herein means that a molecule contains more than two DNA or RNA, preferably more than three, and usually more than ten. Its exact size depends on its optimal functional status. The nucleotide system can be formed by chemical synthesis, DNA replication, reverse transcription, and a mixture of the above. The term "nucleic acid" is used herein to mean 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, but Substitution of normal nucleotides in nucleotides. The term "Nucleic Acid Composition" is used herein to mean a nucleic acid compound relating to a polyphosphate (p〇lynucle〇tide) such as DNA or RNA, which is present in single or double stranded form. The term "gene" as used herein means that the sequence of a nucleic acid has a codon of rnA or a multi-peptide (protein) which may be 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, the base pair is connected by hydrogen bonding. The term "precursor messenger RNA (pre-mRNA)" as used herein refers to a gene produced by eukaryotic cells by the second type of anti-dimerization 30 201009073 enzyme (Pol-II). The primary ribonucleotide transcripts, which are transcriptions, a precursor message, the nuclear nucleic acid sequence contains a 5'-end untranslated region, and a three-terminal non-translated region (3' -end untranslated region), exon and intron 〇 "intron" as used herein means a fragment of a non-translated region of a gene transcription molecule such as in- Frame intron, five-terminal non-translated region 0 and three-terminal non-translated region. The term "exon" is used herein to mean a portion of a gene transcription molecule that has a portion of a protein translation region. The term "message ribonucleic acid (mRNA)" as used herein refers to a precursor RNA plasmon exon which consists of an intron after cleavage by a nuclear splicing mechanism, and which has a protein translational code for the mRNA. The term "complementary deoxyribonucleic acid (cDNA)" is used herein to mean a single strand of DNA complementary to a reference sequence which does not have any intron sequences. The term "sense" as used herein means that the sequence of a nucleic acid molecule is identical to that of a homologous mRNA. This positive nucleic acid configuration is designated as ', +,,,, 's' or ''sense'. The term "antisense" as used herein means that the sequence of a nucleic acid molecule is complementary to the mRNA. This anti-nucleic acid configuration is designated as, _,,,, 'a' or ''antisense', such as ''a DNA'' or ''aRNA''. The term "5'-end" is used herein to mean that a contiguous nucleotide is not in the position of carbon at the carbon of the five carbons, No. 31, 201009073, and is not a phosphodiester bond (ph〇sph〇diester b〇nd). The end point of the carbon number of the next nucleotide is called the five ends. There is at least one phosphate displayed at the endpoint. The term "3 -end" is used herein to mean that a continuous nucleotide is not in the position of the carbon of the five carbon sugars, and that the phosphodiester bond (10) 〇Sph〇diester b〇nd) and the next nucleotide The end of the fifth carbon position connection is called the three ends. The endpoint is typically a hydroxyl group.

The term "template" as used herein refers to a nucleic acid molecule that can be replicated by a ribonucleic acid polymerase. The template system can be single-stranded, double-stranded, or partially double-stranded depending on the ribonucleic acid polymerase. The synthetic replicating nucleic acid is complementary to a template wherein at least one of the double strands is complementary or partially complementary. Both RNA and DNA are synthesized from the five-terminal to the three-terminal direction. The two strands of nucleic acid are usually aligned together to form a double strand. The term "Nucleic Acid Template". herein means a double-stranded 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 a precursor sequence. The term "Complemetary or complementarity 〇r complementation" is used herein to mean a method of base pairing between polynucleotides, 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. Mutual complement can be partial or complete. Partial complements have only certain nucleotide base pairings; however, complete complementation means that all nucleotide bases are paired. The degree of complementarity has an important impact on the efficiency of the hybridization of the two 32 201009073 shares. Complementation is important for complex t (amplificati〇n) reactions. It is also important to detect binding of nucleic acids. The complementarity rate is the ratio of the number of mismatched bases to the total number of all the bases in a single strand of nucleic acid, so the 50% complementarity is half of the base unpaired and half of the base pairing. Even double strands with different base numbers can be paired. In this case, the complement occurs in the longer base of the partial base pair for the shorter one. The term "homologous or homology" as used herein means that a polynucleotide sequence is similar to the sequence of a gene or mRNA. For example, a nucleic acid sequence may be partially or completely homologous to the sequence of a particular gene or mRNA. Homology may also indicate the ratio of the number of similar nucleotides to the total number of nucleotides. The term "Complemetary base" is used herein to mean a paired nucleotide when DNA or RNA forms a double-strand configuration. The term "Complemetary Nucleotide Sequence" as used herein means that the nucleotide 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 "Hybirdize and Hybridization" is used herein to mean the case where the nucleotide sequence is double-stranded by base complementation. Hybridization sometimes provides a DNA polymerase for the initial step of DNA replication after the primer complements a particular nucleic acid sequence (e. g., a template). There is a non-random and specific interaction between the two complementary polynucleotides. The term "RNA interference (RNAi)" as used herein means "post-transcription" driven by small RNA fragments (eg, 'microRNA33 201009073 and small interfering ribonucleic acid (siRNA)) in eukaryotic cells. Gene silencing mechanism." These small RNA fragments can be used as gene silencing effectors to interfere with the expression (transcription or translation) of specific genes in their complementary cells. The term "microcrRNA (miRNA)" is used herein to mean a single-stranded ribonucleic acid (RNA miRNA typically between about 17 and 27 nucleotides in length) that is complementary to a particular gene transcriptional molecule. It can directly degrade the message ribonucleic acid in the cell or inhibit the translation of the specific gene through the complementation between 11111^1111 and 1111^. The miRNA of the cell itself can be found in all eukaryotic cells, and it is an important mechanism for preventing virulence virus infection. And regulate the performance of genes for growth and development of animals and plants. The term "pre-miRNA" refers to a single-stranded ribose acid with a small sandwich structure containing a bent structure region (stem) -arm and stem-loop region) are used to interact with R^aselll endonuclease to generate a gene siiencing effector (such as miRNA). This miRNAs can inhibit genes complementary to miRNAs. Pre- The bent structure region of the miRNA can form a complete or partial hybrid with the double-stranded specific gene transcription molecule, and the bent structure region forms a circular or a hairpin-like bending structure with one end of the double strand. nuclear The term "small interfering RNA (siRNA)" as used herein, refers to a short double-stranded ribonucleic acid having a length of about 18 to 25 base pairing nucleotides that is capable of degrading a gene transcription molecule that is fully complementary thereto. The term "small or short hairpin RNA (shRNA)" 34 201009073 herein means a single-stranded ribonucleic acid having a partially or fully complementary sequence of bent structures, the sequence of the bent structure being unpaired Nucleotide-forming hairpin structures. Many microRNAs (miRNAs) 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 (e.g., rDNA) that is capable of movement and expression in a different genetic environment. Generally, the recombinant nucleic acid molecules are joined in a loop form. This vector is capable of being automatically replicated in cells, as the fragments in it are also replicated. A preferred form of vector is that the episome can replicate extrachromosomally. A preferred vector automatically replicates and expresses the genetic material carried. A vector in which a gene expressing a specific multi-peptide is expressed is an expression vector, and specifically, the vector can also recombine cDNA by forming a mRNA via a reverse transcriptase. The term "Cistron" is used herein to mean a nucleotide sequence having an amino acid-translated codon and having a DNA expression control region at the top and bottom of each sequence. The term "promoter (4)" is used herein to mean a transcriptional reaction that can be detected by a polymerase-identified nucleic acid. The promoter of the present invention may also be a polymerase junction, an enhancer and other sequences capable of initiating a polymerase reaction. The term "antibody" as used herein means a multi-peptide or protein molecule having a consensus amino acid sequence joined to a receptor. While the embodiments of the present invention have been described in the drawings, it is to be understood that the disclosed embodiments are not intended to On the contrary, modifications and equivalents of the spirit and scope of the invention are included in the scope of the invention. The present invention relates to a novel compound and method for altering the genetic properties of eukaryotic cells by intron-derived ribonucleic acids. This genetic change is produced by a gene silencing mechanism of an intron, with a focus on transducing an intron containing at least one RNA splicillg splicing (named <57^40) The recombinant gene enters the target cell or organic tissue. The yA can carry the intron ribonucleic acid meson such as Jiajia: ^八心坤' releases the ribonucleic acid of the intron via the mechanism of splicing and processing of the cellular nucleus a meson, and a gene transcriptional molecule that complements the intron of the ribonucleic acid of the intron by a silencing effect of the RNAi/post-transcriptional gene @ silencing (PTGS). In general, such as Figure 4 and Figure 5 show that when the recombinant gene is transfected by chemical means or liposome (lip0Sme) or virally infected into eukaryotic cells, the intron ribonuclease RNA insert is passed through the second The ribonuclease polymerase system transcribes and is released by the action of ribonucleic acid and processing such as spliceosome, exosomes and nonsense-mediated degradation systems. During the splicing period of the nucleic acid, the intronic RNA of the intron forms a set of lariat RNA and is further processed into gene silendng effectors, such as short-lived nucleoside nucleic acid (short). -temporary RNA (stRNA», antisense RNA, small interfering ribonucleic acid (siRNA), small ribonucleic acid (shRNA), microRNA (miRNA), Piwi interaction ribonucleic acid (piRNA) and these ribose The nucleic acid precursor and the derivative and the ribonucleic acid mixed ribonucleic acid are selected. Thereafter, the gene silencing effector will pass 36 201009073 via the RISC complex and the RNAi-induced initiator of transcriptional silencing (RITS). In order to mimic the translation of their target gene transcriptional or inhibitory proteins, in order to mimic the intracellular precursor message ribonucleic acid (pre-mRNA) splicing and processing mechanisms, we use intracellular splices, exosomes and NMD systems to catalyze The intron removes and processes the expression system. It is identified by a series of intracellular splice compositions in the snRNP (4) Regions (eg, snRNPs Ul, U2 ❹ and U4/U6U5 tri-snRNP) are combined to be released to further form gene silencing effectors. The integration of the artificial snRNP recognition region and the incorporation of the recombinant expression system will be It is disclosed in the first and second embodiments. Design, construct, and evaluate the regulation of red fluorescent protein that can induce the intron ribonucleic acid (D2) transcriptional polymerase. The use of 糸 〇 〇 驱 驱 细胞 细胞 细胞 细胞 细胞 细胞 细胞 细胞 细胞 细胞 内核 内核 内核 内核 内核 内核 内核 内核 内核 内核The mechanism has been demonstrated in vivo, and the present invention utilizes a recombinant gene vector system using a second type of transcription polymerase (Pol-II) (referred to as intronic gene silencing effectors). As shown in Figures 3A and 3B, the expression system has an artificial splicing intron, such as miRNA and hairpin-like shRNA, which will utilize genetic engineering methods and several The synthetic DNA ligation method incorporates the red fluorescent protein gene 37 201009073 (i?GFP), as disclosed in Example 1 and Example 2. • Contains a precursor miRNA or shRNA meson, which is spliced via ribonucleic acid The mechanism of processing produces a mature miRNA or shRNA gene silencing effector. However, in other embodiments, the first type of ribonucleic acid is transferred to the system via the same spirit and principle ( The ribosomal precursor RNA can also produce a gene siiencing effector with the same function. In addition, the ribonucleic acid transcription molecule can be used to generate a message ribonucleic acid (mRNA), heterogeneous ribonucleic acid. (hnRjsiA), ribosome nucleus @ 糖核核(rRNA), ribonucleic acid _a, snoRNA, small nuclear RNA sRNA, precursor microRNA (pre_mj^NA), viral ribonucleic acid (viral RNA) and the above ribose Derivatives and precursors of nucleic acids. As disclosed in Examples 1 and 2 and Figure 3A, it is known to incorporate red fluorescent protein (red flUOrescent pr〇tein, and GF cadaver) genes (color proteins from ugly HcRedl mutations) And form a recombinant red fluorescent protein-containing SpRNAi (SPmAi-RGFP)-based m-filler. The sputum is SpRNAH, which causes the red fluorescent protein to fail to perform normally, but after the intron is spliced, the red Fluorescent proteins can return to normal performance, and we can use the red fluorescent protein expression detected by the red fluorescent wavelength of 570mn to determine the maturation of the intron message nucleic acid (mRNA). The construction is based on the structural characteristics of the precursor message ribonucleic acid. The main unit of the artificial intron contains several snRNP recognition regions and linkers such as five-terminal splicing, three-terminal splicing, branch point motif (branch point motif (branch point motif) BrP)) (for identification of splices), multiple 38 201009073 Cyclic region ((PPT) for interaction with splices) and linkers for ligation of maternal early and restriction enzyme cleavage sites. As shown in FIG. 3B, the SpRNAi (artificial intron) having a silent effector of the present invention comprises a five-terminal splicing, an anti intronic insert from the five-terminal to the three-terminal (can be spliced or After treatment, a gene silencing effector such as a mir-302-like gene silencing effector, a branch point region (BrP), a multi-mouth bite region (PPT), and a three-terminal splicing site (which can be formed) For the combination of shears). In addition, some translational termination codons (T codon) are located in the sequence of connectors near the three-terminal splicing. Generally, the 'five-terminal splicing site can be a micronucleotide sequence containing or homologous to 5'-GTAAGAGK-3, (SEQ.ID.N0.4) or GU(A/G)AGU sequences (eg ., 5'-GTAAGAGGAT-3, (SEQ. ID. NO. 30), 5'-GTAAGAGT-3', 5'-GTAGAGT-3' and 5'-GTAAGT-3,). The tri-terminal splicing sequence may comprise or be homologous to a GWKSCYRCAG (SEQ. ID. N0.5) or ❹ CT (A/G) A (C/T) NG sequence (eg, 5'-GATATCCTGCAG-3, (SEQ. ID.N0.31), 5, -GGCTGCAG-3, and 5, -CCACAG-3,). Furthermore, the 'branches point sequence is located between the five-terminal and three-terminal splicing, the branch point region sequence contains or is homologous to 5,-TACTWAY-3, (SEQ.ID.N0.6) (eg, 5'-TACTAAC) -3' and 5'-TACTTAT-3'). The branching point nucleotide of the branch point region sequence is an adenosine (A), which can form a set of (a) station intron ribonucleic acid structure (2'-5, mode linkage), By 2,-5, 2'-5'-olig〇adenylate synthetases and splices are formed in most of the splicing 39 201009073. In addition, the multiple region is located between the branch and the three-terminal splicing region, wherein the polypyrimidine region has a plurality of nucleotide sequences of Thymine and Cytosine, and the nucleotide sequence of the polypyrimidine region is homologous to

5 _(TY)m(C/-)(T)nS(C/-)-3'(SEQ.ID.N0.7) and 5'-(TC)nNCTAG (G/-)-3' (SEQ .ID.N0.8). , m" and, n" means a repeating sequence of more than one, preferably the number of sequences of m is between one and three and the number of sequences of n is between seven and twelve. "One" means no nucleotides. The units of all introns are linked by a single nucleotide of the linker, based on the use of symbols and formulas for expression of the nucleotide and amino acid sequence of 37 CFR 1.822, and W is adenine. (adenine (A)) or thymine (T) / uracil (U), K means guanine (G) or thymidine / uracil, s means cytosine (〇 or guanine, Y refers to cytosine or thymidine / uracil, R refers to adenine or guanine and N refers to adenine, cell carcinoma bite, guanine or thymus bite / urinary For the above spliceomomal recognition components, deoxythymidine may be replaced by uridine. ❹ In order to determine the function of the spliced insert, many oligonucleotides. The sequence is genetically engineered into the recombination and intron meson regions of GFP. These anti-intron meson regions contain several restriction enzyme cleavage sites, wherein these restriction enzymes are selected from 乂 plus / /, Jcc /, 4 / ?/// melon, 乂 furnace /, but a / / ZJ,

Asel, Asp7181, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp 1201, BspHI/LUllI/1201, BsrI/BI/GI, BssHII/SI, BstBI/Ul/XI, ClaI,

Csp6I, DpnI, DraI/II, EagI, Eel 13611, EcoRI/RW47III, EheI, 40 201009073

FspI, Haelll, Hhal, HinPI, Hindlll, Hinfl, ΗραΙ/ΙΙ, KasI, Kpnl, Maell/III, Mfel, Mlul, MscI, Msel, NaeI, NarI, NcoI, Ndel, NgoM, NotI, NruI, NsiI, PmlI, PpulOI, PstI, PvuI/II, Rsal, SacI/II, Sail, Sm3AI, Smal, SnaBI, SphI, SspI, StuI, 7^:/, 7^/, Ιδβ/ and a restriction enzyme mixed with the above restriction enzymes. The intronic oligonucleotide insert of these introns is a DNA template which can be transcribed into a transgenic molecule having a considerable number of secondary structures. The above molecule is selected from the group consisting of snail ribose fumarate. Short-temporary ribonucleic acid (stRNA), antisense RNA, small interfering ribonucleic acid (siRNA), small ribonucleic acid (shRNA), microRNA (miRNA), Piwi interaction ribose Nucleic acids (piRNA), ribozymes, and precursors or derivatives of these ribonucleic acids, which may be in the form of either a positive or negative form or in two forms, as well as mixtures of the above ribonucleic acids. For the convenience of gene delivery to induce specific cell activation, the preferred recombinant structure of the present invention is incorporated into an expression vector which may be selected from the group consisting of a DNA transgene, a plasmid, a transposon. (transposon), retrotransposon, jumping, viral vector, and a mixed vector of the above vectors. The vector is introduced into a specific cell or tissue by gene transfection, and the gene transfection method is selected from the group consisting of liposomal transfection, chemical transfection, electroporation, transposon DNA. Reposon-mediated DNA recombination, jumping gene insertion 201009073 (jumping gene insertion), viral infection, micro-injection, gene-gun penetration, and more Method of mixing. The vector further comprises a promoter of at least one virus or a second type ribonucleotide polymerase (Pol-II) or a third type ribonucleic acid polymerase (Pol-ΠΙ) to express the corpse. Furthermore, the vector may comprise a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells, multiple polyadenylation signals of SV40 in the downstream region of * Further processing of the three ends of the recombinant gene transcriptional molecule, pUC origin of replication to a large number of copies in prokaryotic cells, at least two restriction enzyme cleavage sites for conjugation into vectors, selectivity (may or may not The SV40 replication promoter is used to express the SV40 T antigen in mammalian cells, and the selective (may or may not be) early promotor is used to exhibit at least one antibiotic in replicable prokaryotic cells. Resistance. The performance of antibiotic resistance genes can be used to screen for indicators of successful transfection or infection of target cells. The above antibiotic resistance gene can be countered from penicillin G, ampicillin, neomycin, paromycin, kanamycin, streptomycin. , erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamicin (gentamycin), chloramphenicol, cephalothin, and tylosin are antibiotics mixed with antibiotics of 42 201009073 and above. The vector has been tested in the zebrafish of the (Tg (actin-GAL4: UAS-gfj)) strain and has been shown to inhibit the expression of the green fluorescent protein gene. As shown in Examples 3 and 6 and Figure 3C, the anti-green fluorescent protein (the plastid of the anti-five pioneer microribonucleic acid meson, which is displayed after transfection by liposome (lane 4)) A significant gene silencing effect of green fluorescent protein (more than 80% of the protein was inhibited), however, no silent effects were detected in the other experimental groups and the control group; these experimental groups and the control group were The order (from left to right) is 1. control group (blank vector control, Ctl); 2. for AIDS virus protein; the precursor of micro-ribonucleic acid (pre-miRNA) control group. 3. against anti-intentional green fluorescent protein And the anti group of the antisense EGFP insert without the hairpin loop structure and the miR* group of the inverted precursor microRNA sequence, wherein the inverted precursor microRNA sequence is completely complementary to the anti-green fluorescent protein Pioneer micro-nuclear acid ❹ (anti-five GFP pre-miRNA, miR*). For non-specific genes, such as red fluorescent protein and actin, no silent effects were found, indicating introns. Microribonuclease intervention The intrinsic miRNA-mediated RNA interference (RNAi) is highly specific. In addition, as shown in Figure 3D, the Northern blotting analysis shows the effective use of the intron nucleus. Intronic RNA is produced only in the red fluorescent protein carrier group with the intron-resistant green fluorescent protein precursor microRNA, (second column in the middle), in the absence of introns 43 201009073 The fluorescein group (intrcm FP). (left column - column) or lack of five-terminal splicing and GFP group did not occur. At this time, red fluorescent protein exons can be joined to form Mature ribonucleic acid for performance -

Functional red fluorescent protein. _ Previous experiments to establish intronic miRNAs in introns provide a compound and method that can be used to silence genes in vivo. In order to further evaluate the efficiency of the gene silencing effect of the microRNA of the intron and the structure of the micronucleic acid of the preferred intron, the gene silencing effect of the optimal ® is produced. According to the study, the RNA silencing (RNAi)-related gene silencing effect mechanism (such as RISC) in cells has a structural preference for the specific-strand of mature microRNA. RISC is an egg-to-thief complex that affects the degradation of the underlying gene transcription molecule or the translational inhibition of the target gene or the PTGS mechanism via the RNAi mechanism. The zebrafish was chosen as the experimental system. The spot-loop of the lion's nucleus was determined by the actual shot. The combination of the parental complex and the mature microRNA sequence was determined. The complex is different (Lm ei.a/. (2005) Gewe 356: 32-38). The formation of siRNA double strands plays an important role in the siRNA-RISC complex. The double-strand function of siRNA is not the same, only one will be combined with the RISC complex. This preference is due to the thermodynamic stability of each 5'-end base-pairing of the siRNA. Based on siRNA model, 44 201009073 miRNA and its complementary miRNA (also known as miRNA, the double-strand formation between the two is considered to be one of the key steps in the composition of the miRNA-RISC complex. If the pattern is correct then there will be no preference specific A phenomenon occurs in the deformed structure of the pioneer micronuclear acid-cleaving nucleic acid. However, it has been found that the niCpre-m of the blood stasis has an influence on the composition selection of the miRNA-RISC complex. The first and second disclosure methods utilize the constructed cadaveric miRNA vector and miRNA*-stemloop-miRNA ❹ (1) and miRNA-stemloop-miRNA*[2] (miRNA* represents a microRNA complementary to the mature microribonucleic acid sequence The two different precursor microRNAs are 'integrated into a pre-prepared and GFP vector after synthesis by a DNA synthesizer. The precursor microRNAs of these two groups contain the same double-stranded fold structure (double-sttandstem) -arm region), this bent structure can silence the nucleotide sequence of 280th to 302th of the five genes, because the _pvw/can restriction enzyme is cut through the five-terminal and three-terminal The meson is incorporated into the meson. The above meson can be easily replaced by many other mesons that are resistant to different genes (for example, anti-ugly GFP, mir-3〇2 pioneer microRNA), whereby the meson can be replaced. The way of other mesons against different gene transcriptional molecules' microRNA-expressing system of this intron can provide an important tool for the development of microRNA in vivo. In order to determine the structural preference of the pioneer microRNA First, it is separated by mirVana microribonucleic acid separation column (Ambion, Austin, TX), and then with the latex beads, it has the potential of silent effect in the zebrafish. Ribonucleic acid, miR_EGFP (280-302), has been shown to have a silent effect in the miRNA_stemloop_miRNA*[2] structure, as shown in Figure 4A and Figure 4B (gray shaded sequence). Because this effective mature micronucleus is only available in The zebrafish transfected miRKA-stemloop-miRNA* [2] was found to be effective 'so it is speculated that the miRNA-RISC complex has a structural preference for miRNA_ stemloop-miRNA*[2] rather than [1]. As shown in Figure 4C, experiments were performed using zebrafish Γ^ (&(Λίη-ΟΑΙΑυΑ8·^φ)) expressed by the actin-activated promoter to visualize the supervised gene silencing effect and miRNA expression. Relationship, this zebrafish will always express green fluorescent protein in various cells. The zebrafish is transfected with an anti-vector and expresses a red fluorescent protein which can be used as an indicator protein, and can be used as an indicator of the production of microRNA in cells. Use of the above carrier

Pu<3ene eationie liposomal reagent (Roche, In) Transfection Service The vector entered the zebrafish embryo and found that all vectors were able to completely enter the zebrafish embryos after % 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 green fluorescent protein (EGFP) was only in the precursor microRNA which was transfected with miRNA-steml00p_miRNA*[2]. It was observed in the zebrafish group (miR group). As shown in Fig. 4D, Western blot analysis quantitatively confirmed the gene silencing effect, and the zebrafish transfected with the P] structure had greater than 85 percent gene suppression. However, the gene suppression effect in the intestine (〇1) is lower than in other tissues, and this phenomenon may be related to higher RNase activity in the intestine. Since 201009073 is the same as the thermal stability of the five ends of [丨] and [2], it is speculated that this pioneer microribose. The bent structure of nucleic acid is related to the stock selection of mature microribonucleic acid combined with RISC complex. Dicer's cut position in the stem-loop is known to determine the stock selection of mature microRNAs, so the bent structure of the pioneer microRNA may play a role in determining the special cut position. Therefore, based on the diversity of the bending structures of different microribonucleic acids, the present invention utilizes a modified set of pre_mir_3〇2 bends (eg, 5'-GCTAAGCCAGGC-3, (SEQ. ID. N0.1) and 5, -GCCTGGCT ❺ TAGC-3' (SEQ. ID. N0.2)) is optimally combined with the RISC complex to exert the utility of the present invention.

Mir-302 is stained by human primary embryonic cell OidESCV human prostate cancer cell PC3 舆 human primordial melanoma cell Colo cell based on the above experiment, will be designed mir-302a-mir_302b-mir-302c-mir-302d The precursor of the microRNA promoter or redesigned mir-302 precursor microribonucleic acid meson (such as the small clip sequence, which contains 5'-UAAGUGCUUC CAUGUUUUAG UGU-3, (SEQ.ID.N0.9)) Among the vectors, genes related to silent development and differentiation in hpESC, PC3 and Colo cells. The mature sequences of mir-302a, mir-302b, mir-302c and mir-302d are 5,-UAAGUGCUUC CAUGUUUUGG UGA-3, (SEQ.ID.NO.10), 5'-UAAGUGCUUC CAUGUUUUAG UAG-3, (SEQ .ID.NO. 11), 5,-UAAGUGCUUC CAUGUUUCAG UGG-3, 47 201009073 (SEQ.ID.NO.12) and 5,-UAAGUGCUUC CAUGUUUGAG UGU-3' (SEQ.ID.NO.13). These mir-302 gene silencing effector homologs share a consensus five-terminal region in the anterior 17-nucleotide (100% homology), which is a 17-nucleotide with 5'-UAAGUGCUUC CAUGUUU- 3. The sequence of (SEQ. ID. N0.3) is the same. In these mir-302 homologous sequences, the thymus gland (T) can be used to replace uracil (U). In these experiments, the precursor microRNA of mir-302a-mir-302b-mir-302c-mir-302d was transfected into hpESC and PC3 cells, and the redesigned mir-302 homologous sequence (SEQ.ID.NO) .9) Transfected into Colo cells. After mir-302 transfection, the pattern of all cell lines (mojphoiogy (lower surface)) changed, changing from a spindle-shaped or amoeba-like shape to a chubby appearance, indicating that these cells may lose movement ( The ability to migrate and its cell proliferation rate is very low, at this time as the growth rate of stem cells (Fig. 5A to Fig. 5C). The cellular DNA content (Y-axis) of the flow cytometer (upper panel) corresponds to a decrease in the mitotic cell population shown by different cell cycles (X-axis), confirming the rate of cell proliferation of these mir-302 transfected cells. Slow, and the above cell population is determined by the DNA content. The first peak (left) and the second peak (right) are shown as the relative number of g〇/gi and mitotic ticks in the whole cell population. After rmr-302 transfection, the 'mitotic cell population decreased from 36.1% to H in hpES (:6) to 9%; from 38.4% to 12 6% in PC3 cells; from c〇i〇 cells 36.5% is reduced to 1L5%. However, when transfection was not done - Nahu, cell morphology 48 201009073 or cell proliferation did not change significantly. The above mir-gft) precursor microRNA is designed to inhibit the firefly's green fluorescent protein, which has no homology to the human or mouse gene sequence. Based on the above findings, gene transfer of mir_3〇2 homologues can transform human blast cells and cancerous cells into more similar embryonic stem cell types and proliferation rates, as found in previous iPS cells (Okita et al (2007) Nature 448: 313-317; Wemig et al, (2007) 448: 318-324).形成 Embryoid body formation, embryonic stem cell indicator protein expression, genomic DNA demethylation and mir-302 Korean colonization. In CoMColo+mirGCm and PC3 (PC3+mir-302) cells Fine 腧 箄 箄 评估 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了 为了The mir-302 transfected cells have a high density of cell culture confluence (>80%-90%) and tend to form tight cell clusters of the above embryoid bodies like native humans. Embryonic stem cells derived from embryonic stem cells (Fig. 6A). However, without proper hormone or growth factor guidance, these embryoid bodies will disperse and differentiate into neuron-like cells when cultured again (as shown in the lower right panel) in order to confirm the inheritance of embryonic stem cells and embryoid-like somatic cells. Characteristics to further evaluate the indicator protein performance of these embryonic stem cells. As shown in Figure 6B, mir_3〇2 transfected Colo cells (Colo+mir-302) significantly exhibited a series of indicator proteins of embryonic stem 49 201009073 cells, such as 〇ct3/4, SSEA-3, SSEA- 4 and Sox2, etc., however, there is no indicator protein of the above embryonic stem cells in the general C〇l cells or in the colo cells transfected with an empty vector, transfected with mir-g § micro There were no detectable signs in cells such as Colo cells (Colo+mir-gQj) of ribonucleic acid vector or Colo cells (Colo+mir-434-5p) transfected with mir-434-5p microribonucleic acid vector. The sequence of mir-434-5p is not homologous to mir_3〇2. Because the Colo cell line has differentiated human melanoma cell lines, the results show that transfection of 11^-3〇2-run^^·- and GFP vectors can redirect c〇l〇 cells to embryonic stem cells again. The state is similar to real embryonic stem cells. 〇ct3/4 (or 〇ct-3 or Oct-4) is a kind of POU transcription factor, which is highly expressed in differentiated pluripotent embryonic stem cells and germ cells (Sch〇ler deduction α/., (1989) EMBO J. 8: 2543-2550; Rosner et al., (1990) Nature 345: 686-692). The performance of 〇ct3/4 plays an important role in maintaining stem cell self-renewal and differentiation pluripotency. Inhibition regulation of 〇ct3/4 results in the differentiation of embryonic stem cells into different developmental processes. The SSEA protein contains SSEA-1, 3 and 4 initially identified by monoclonal antibodies. This monoclonal antibody recognizes the lactose glycolipid on the surface of the initial implantation stage of murine embryonic cells and on the surface of abnormal cancer cells, but it cannot The differentiated cells after the above cell differentiation are recognized by the antibody (S〇lter β α/., (1978) Proc. Wi/. 知 t· at 75: 5565-5569). Undifferentiated early embryonic stem cells, human embryonic carcinoma cells (hEC) and embryonic stem cells all exhibit SSEA-3 and SSEA-4 but no SSEA-1 (Thomson β 乂, (1998) 282: 50 201009073 1145-1147) . During the production of imprints, SSEA_3 and ssea 4 are produced on the cell membrane surface of _, sac, and early dividing embryonic cells (Shevinsky (1982) & (4): age dog. S()x2 is loose on maintaining differentiation pluripotency The core transcriptional molecule, but this function is not specific to embryonic stem cells (Boyer-, (2005) CW/122: 947-956). Therefore, based on the knowledge of embryonic stem cell indicator proteins, chrom-302 transfected Cok) Cells have the property of expressing all of the above-described embryonic stem cell indicator proteins and embryonic stem cells. ® In addition, differentiated pluripotent stem cells have another unique property of post-modification changes, such as genomic de-mercapitation (Hochedlinger ei or/., (2006) Atowre 441: 1061 -1067). In order to transdifferentiate differentiated somatic cells into the embryonic stem cell stage, many embryonic genes need to be re-intensified to inhibit developmental and differentiation-related signals or genes. DNA methylation regulates the expression of these genes or Does not play an important role because the thiolation of the upstream promoter region often interferes with the combination of numerous transcriptional molecules for gene expression, the demethylation process Must be activated to regenerate newly activated embryonic genes such as Oct3/4, SSEA-3, SSEA-4 and Sox2. To assess the degree of thiolation of Colo+mir-302 cells and Colo cells, first use (DNA isolation kit, Roche, IN) isolating the genome of the above cells, and using the restriction enzyme Hpall, which is a CCGG cleavage, to isolate the genomic body. HpaII is sensitive to CpG thiolation and only cuts the ccGG without methylation, but cannot The thiolated CCGG was cut. As shown in Fig. 6C, the fragment of the gene fragment after excision, the fragment of the embryonic stem cell Colo+mir-302 cell was smaller than that of the general Colo cell, suggesting that the transfected mir_3〇2 The Colo cells do have a de-demethylation of 51 201009073. In addition, the original size of the genomes of Colo cells and Colo+mir-302 cells is almost the same, so the above conclusion can be confirmed. Figure 6D further shows 〇ct3 The morphological changes in the methylation domain of the /4 gene promoter region are particularly evident in the changes between hpESC, PC3 and Colo cells and the above cells transfected with mir-302. To confirm the thiolation of these regions, this experiment utilized Heavy sulfite (bisulfite) to isolate the genomic DNA (CpGenome DNA modification kit, Chemicon, CA), bisulfite can convert unpurified cytosine to uracil, then use two forward primers 5'- GTTGTTTTGT TTTGGTTTTG GATAT-3' (SEQ.ID.N0.36) and 5'-ATTGTTTTGT TTTGGTTTTG GATTTA-3, (SEQ.ID.N0.37) and a reverse primer 5'-GTAGAAGTGC CTCTGCCTTC C- 3' (SEQ. ID. N0.38) and polymerase chain reaction (PCR) (long template PCR extension kit, Roche, IN) were used to isolate the five-terminal upstream promoter region of Oct3/4. The cell genome (100 ng) was first mixed with the primer (150 pmole) and lxPCR buffer, and heated to 94 ° C for four minutes and then placed on ice for cooling. Then, after 25 PCR cycles, the conditions were as follows: 92 ° C, 1 minute; 55 ° C, 1 minute; and 70. (:, 5 minutes. Then, the PCR reaction product (Qiagen, CA) was used to collect the PCR reaction product, the PCR product and the multiple restriction enzymes of the cut ACGT sequence. These restriction enzymes include dc//(AACGTT), knife w/ (CACGTC), Pw//(CACGTG), 5>w5/(TACGTA) and parent tyC^W/RACGT), each 5 units (5U each) are uniformly mixed. Since the ACGT which is not thiolated in this region can be converted to AUGT via the bisulfite reaction, the AUGT cannot be cleaved by the above restriction enzyme. As shown in Figure 6D, at least four thiolated ACGTs were transformed into demethylated ACGTs in hpESC, PC3 and Colo cells transfected with mir-302. The demethylation of the 〇// gene promoter region by mir-302 transfection may be reproduced by mir-302 transfected cells such as c〇l〇+mir-302, and the reactivation of Oct3/4 gene expression. Caused. In addition, inhibition of cell migration is usually observed in cancerated sputum cells transfected with mir-302. In general, embryonic stem cells tend to stay in one place and form an in situ embryoid body (embryoidbody/wszYw), which may explain why PC3 and Colo cells lose mobility after mir-302 transfection. As shown in Fig. 6E, when PC3 cells were placed next to PC3+mir-302 cells, we were able to clearly observe that the cancerated PC3 cells rapidly moved along one side of PC3+mir-302 cells for about 30 seconds. Black arrows indicate the direction in which PC3 cells move. The results of this experiment suggest a potential therapeutic application of mir-302 transfection in cancer cells, which can not only convert cancer cells into useful stem cells, but also reduce the possibility of cancer cell metastasis, and more advantageous because of mir- The 302 transfected cancer cells are still compatible with the patient's immune system. The differentiated pluripotent cells of the embryonic stem cells described above can be used for transplantation therapy without any immune rejection reaction. Identification of the porcine gene mir-302 by miRNA gene crystallization 为了 In order to confirm the amount of mir-302 gene transfected in transfected cells, we used miRNA microarray analysis. Small RNAs isolated from each cell by mz>VanaTM miRNA isolation kit 53 201009073 (Ambion, Inc., Austin, TX) at approximately 70% confluence. The isolated picorucleic acid was purified and quantified using 1% furfural-cabbage electrophoresis and spectrophotometer measurement (Bio-Rad, Hercules, CA) and sent to LC Sciences (San Diego, CA). For further miRNA biochip analysis. In the intensity pictures of Cy3 and Cy5, the intensity of the signal increases from level 1 to level 65535' and the corresponding color change changes from blue to green to yellow to red. In the picture ratio of Cy5/Cy3, when the intensity of Cy3 is higher than that of Cy5, the color is green, when the intensity of Cy3 and Cy5 is the same, the color is yellow, and when the intensity of Cy5 is higher than that of Cy3, the color is red. As shown in Fig. 7A, Cy3 is a cell without any treatment (e.g., Colo), and Cy5 is a cell transfected with mir-302 (e.g., Colo+mir-302). In the picture ratio of Cy5/Cy3 (far right), all of the .mir-302 families (white circles) were highly expressed after transfection with GFP-mirJO. Because the mir-302 family gene and the redesigned mir-302 reagent have about 91% homology, this shows that the redesigned mir-302 reagent acts as a mir-302 member and replaces the original function of mir-302. Figure 7B shows a list of different amounts of miRNA in Colo+mir-302 cells. Based on the results shown in Figure 7A, we can also find that the increase in the amount of mir-302 can increase the performance of the precursor mir-302 as well as other microRNAs, such as mir-92, mir-93, mir-200c, mir-367. , mir-371, mir-372, mir-373, mir-374 and all members of the mir-520 family. The miRBase::Sequence programfhtto://microma.sanger.ac.uk/) and the target gene analysis of these mirs confirmed that the mir-302 family can target more than 400 target genes, suggesting that this 201009073 microRNA may be responsible for maintaining stem cells. Differentiation pluripotency and regenerative play an important role. Their target genes include, but are not limited to, RAB/RAS-associated oncogene members, ECT-related oncogenes, pleiomorphic adenoma genes, E2F transcriptional molecules, Cydin D-binding Myb transcriptional nucleons (cyclin D) Binding of myb-like transeripation factors, HMG-box transcriptional molecules, rushing 3 transcription factors, CP2-like proteins transcription factor, NFkB-activated protein-based genes, cyclin-dpendent kinases (CDK) ), ΜΑΡΚ related kinases, SNF-related kinases, muscle chain kinases, TNF-alpha-induce protein genes, DAZ-associated protein genes LIM-associated homeobox genes, DEAD/H box protein genes, forkhead box protein genes, BMP regulatory scorpions (BMP regulators), Rho/ Rac/Rac guanine nucleotide exchange factor, IGF receptor, endothelin (endothelin receptor), left-right determination factors, cyclins, p53-inducible nuclear protein gens, RB1-like 1 gene, RB-binding protein gene (RB binding protein genes), Max-binding protein genes, c-MIR cellular modulator of immune recognition, Bd2-like fine 55 201009073 Bcl2-like apoptosis facilitator, protocadherins, binding protein (integrin β4/β8), inhibitor _bin), ankyrins, SENP1, NUFIP2, FGF9/19, SMAD2, CXCR4, EIF2C , PCAF, MECP2, histone acetyltransferase MYST3, nuclear RNPH3, and many nuclear receptors and factors. All of these genes are involved in embryonic development and cancer formation. Μ甩 Gene chip analysis identifies embryonic stem cell markers and gauges. ❹ The correlation between the expression of the embryonic stem cell marker protein and the transgenic mir_3〇2 gene has been confirmed. We used gene chip analysis to search for intracellular mir-302 transfection. Before and after, gene expression changes within the genome range between mir_3〇2 transfected cells and other human embryonic stem cells. Afiymetrix gene chips (GeneChip U133A & B arrays, Santa Clara, CA) were used to assess more than 32,668 human gene expression patterns between Colo+mir-302 and other human embryonic stem cells, such as HuEC8 and H9. All ribonucleic acids isolated from each test cell line were collected by RNeasy spin column (Qiagen, Valencia, CA). In order to recognize the different backgrounds of the background, we repeated the biochip test and used the same Colo+mir302 sample and selected two hundred genes, which appeared on one side. As shown in Fig. 8A, the expression changes of these selection genes (white spots) were all less than twice that of Colo+mir-302, and the noise lines showing these background values were quite small. Based on these pre-selected genes and using a one-fold change as a threshold, we can calculate the relative coefficient of the comparison genes in the two groups 56 201009073 (correlation coefficiency (CC)). The 32668 human gene expression pattern has a similar ratio of relative coefficient ratios between the compared samples. At such a relative coefficient ratio, the results shown in Fig. 8A confirmed that the gene expression pattern of Colo+mir-302 cells was 88% and 86% similar to that of hES HuEC8 and H9 cells, whereas only 53% of the relatives were The coefficient is shown between Colo and Col〇+mir-302 cells. This confirms that the relevant mir-302 transfection changes the expression of at least 15354 cellular genes, which may be associated with the transformation of cancerous ❹ Colo cells into embryonic stem cell c〇l〇+mir-302 cells. A list of many of the major differences in performance between Colo and Colo+mir-302 cells is shown in Figure 8B. For example, in H9 and HuEC8 cells, Colo+mir-302 cells express embryonic stem cells and germ cell cell-associated proteins (eg, SSEA-4, Utfl, Oct4, Sox2, Pulimio-2 anti-Nanog'). However, 'Klf4 does not appear in Colo+mir-302 cells, showing a slight difference from our cells (as shown in Figure 6B). In addition, many cancer markers, developmental signals, and cue proliferating facots were found to be significantly inhibited in πώ-302 transfected cells as 'undifferentiated round cells' The pattern and the phenomenon of slower cell replication as shown in Figure 5 were observed. In summary, these findings indicate that the mir-302 transfection method can genetically transform a differentiated c〇1〇 cell line into a Colo+mir-302 cell line with embryonic stem cells, and differentiated pluripotent H9 and HuEC8 stem cells are similar. Using various hormones and growth factors to induce 丨 cell differentiation 57 201009073 By definition, 'differentiated pluripotent stem cells can differentiate into many cell types, which are similar to those derived from embryonic ectoderm, mesoderm and endoderm. . For example, in the absence of mammalian cells (feeder_ftee) using in vitro (沩V), Helmon and growth factors, the present invention can already be achieved - c〇lo+mir-302 Differentiation of cells into three different cell morphology is shown in Figures 9A through 9C. First, in the absence of mammalian cells, the above-mentioned cells were transplanted with androgen and dihydrated sputum _ (dihydrotestosterone (DHT 50 ng/ml>>> after six hours of treatment with Colo+mir-302 cells). 105 cells were inserted into the uterus of a six-week-old female immunodeficient beige mouse. One week later, a type of spermatogonia was produced at the transplant site (in the middle of Figure 9A). The recipient's use of the transforming growth factor (factory31 (TGF-/31 l〇〇ng/mi)) treatment: 2 hours, the transplanted cells entered the six-week-old female immune loss (immunocompromisedpciD beige mice, the cells differentiated into The type of fibroblasts began to secrete collagen, and within a week, these cells occupied most of the uterus in the uterus (Fig. 9B). After using 'bone morphogenetic protein 4 (BMP4 100 ng) /ml)) After treatment of Colo+mir-302 cells for 12 hours, the treated cells were transplanted into the liver of a six-week-old immunodeficient SCID beige mouse by xenograft. Chondrocytes, some around Calcified precipitation (Fig. 9C). By using thymic nude mice as an environment for mimicking colonization therapy in vivo, these experiments confirmed that the mir-302 transfection method of the present invention can produce novel embryos. Stem cells 58 201009073 strain 'These cell lines can be guided and produced in the in vitro environment without mammalian cells. 'No woven cell ray. Shame, this (four) can not only transform or transform to differentiate. Body _ shouting - fetal stem cells Differentiated traits, and can also maintain the differentiation pluripotency and self-replication ability of stem cells in a culture environment without mammalian cells. Therefore, the present invention provides a novel novel by using the mir-302 expression vector of introns. A method for producing stem cells, specifically, a method for producing a cell line derived from an embryo and a cell line of a cancer cell. These mechanisms are coordinated by the silent pathway of the microRNA-related gene of the intron and many intracellular regulatory mechanisms. Contains gene transcription factors, RNA splicing, exosome elimination, and nonsense-mediated degradation. The gene silencing effect of microRNAs in introns is considered to be the most Effective, most specific, and safest for all three known RNAi pathways. Based on all advantages, the use of the intron mir_3〇2 reagent of the present invention provides a simple, effective and safe method of gene use that is not only available Transforming somatic cells into differentiated pluripotent cells of embryonic stem cells can also maintain the differentiation pluripotency of embryonic stem cells in culture conditions without mammalian cells, thus avoiding four large transcription factors as in the previous iPS stem cell method. The gene is transfected into a single cell with an annoying retrovirus. As shown in Figure 10, based on mir-302-induced embryonic stem cells, this suggests that SSEA-1 is present in mir-302-induced cells (Colo+mir-302) cells. Degree of performance; however, there is no such performance in Klf4. Compounds of the invention: 59 201009073 - A recombinant ribozyme nucleic acid compound for inducing a ribonucleic acid-related gene silencing effect of an intron comprises: a) at least an intron, wherein the intron consists of a plurality of exons Surrounded by these exons, the fine-nose RNAsplieing exon is excised and manipulated by a processing mechanism; and b) a plurality of exons, wherein the exons can be joined to form a gene with specific functions. The above recombinant ribonucleic acid compound further comprising a) at least one restriction enzyme cleavage site, wherein the restriction enzyme cleavage site can be used to combine the recombinant ribonucleic acid compound in a expression vector for providing a precursor ribonucleic acid of the recombinant ribonucleic acid compound The transcriptional molecule is in the mammalian cell; and b) a plurality of transcriptional and translational termination sites, wherein the transcription and translation termination can be used to make the correct size of the ribonucleic acid transcription molecule of the recombinant ribonucleic acid compound. The intron of the above recombinant ribonucleic acid compound further comprises: a) a meson of a gene silencing effector complementary or homologous to at least one of the underlying genes; b) a five-terminal splicing; c) a three-terminal splicing; d ) - a branch point region for identification of the splice body; 201009073 e) - a polypyrimidine region for splice engagement; Q a plurality of connectors for connecting the main components. The gene silencing effector meson comprises a nucleotide sequence homologous to 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID.N0.3)'. The five-terminal splicing is a nucleotide sequence and is homologous to 5'-GTAAGAGK-3' (SEQ.ID.N0.4) or GU(A/G)AG U sequence (for example, 5,-GTAAGAGGAT-3, ❹ (SEQ.ID.NO. 30), 5, -GTA AGAGT-3,, 5, -GTAGAGT-3, and 5'-01^01'-3'). At this time, the three-terminal frying joint (3'>^1^3 such as (^3' (; Qiu) sequence is homologous to GWKSCYRCAG (SEQ.ID.N0.5) or CT(A/G)A ( C/T) NG sequence (eg, 5'-GATATCC TGCAG-3, (SEQ.ID.N0.31), 5 '-GGCTGCAG-3 ', 5 '-CCACAG -3 '). In addition, the branch point region sequence The line is located between the five-terminal and the two-terminal splicing region, wherein the branching point region is located in a nucleotide sequence homologous to 5'-TACTWAY_3' (SEQ. ID. N0.6) (eg, 5, -TACTAAC-3, and Φ 5'-TACTTAT-3'). In addition, the 'polypyrimidine region is located between the branch point region and the three-terminal splicing region, wherein the polypyrimidine region is a high density of Thymine and Cytosine. Nucleotide sequence, the nucleotide sequence of the polypyrimidine region is homologous to 5-(TY)m(C/-)(T)nS(C/-)_3' (SEQ.ID.N0.7) and 5' -(TC)nNCTAG (G/-)-3' (SEQ.ID.N0.8), wherein "m" and ", n" refer to a multiple repeat sequence of one or more, preferably the number of sequences of m is Between one and three, and the sequence number of η is a butterfly twelve "" means that there is no nuclear Wei. Some connecting nucleotides can be used to link the Lezi fragment. Based on 37 CFR1 822 regulation 61 2010 09073 (Taiwan) nucleotide and amino acid sequence table format, same as), w refers to adenine (adenine (A)) or thymine (thymine (T)) / uracil twist acil ^ j )), κ refers to guanine (G) or thymus bite (Τ) / urinary sputum (υ), s refers to the cell 'pyridine (C) or guanine (G), Y refers to the cell Pyrimidine (C) or thymine (τ) / uridine (9) 'R refers to adenine (A) or bird sputum (G) and N refers to adenine (4), cytosine (C), guanine ( G) or thymine (T) / uracil (U) or other. For all splicing recognition elements of the upper sputum, the deoxythymidine (T) nucleoside acid system can be replaced by urinary pyridine bite (U). Method of the invention: A method for transgenic genes for inducing intron mir-302-related gene silencing effects in mammalian cells, comprising the steps of: a) constructing a recombinant ribonucleic acid compound comprising mir_3〇2 At least one intron of the related gene silencing effector, the gene silencing effector is surrounded by an exon, wherein the intron can be excised and separated from the exon for further mir-302 gene silencing effect; b) Incorporating the recombinant ribonucleic acid compound into a performance vector; and c) transducing the vector into a plurality of mammalian cells, wherein the cell produces a precursor ribonucleic acid transcription molecule of a plurality of the above ribonucleic acid compounds, wherein the splicing of the cells is within The splicing of the sera from the precursor ribonucleic acid transcriptional molecule provides a mir_3〇2-related gene silencing effect against a sequence gene homologous or complementary to the mir_3〇2 gene silencing effector sequence. 62 201009073 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 are not intended to limit the scope of the invention. On the contrary, modifications and equivalents of the spirit and scope of the invention are included in 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 is a construction of a synthetic nucleic acid sequence comprising a recombinant milk gene for the production of three different introns, the sequence comprising a positive-stranded, anti-sense or small-pin GFP meson (hairpin-EGFP insert), as shown below :N1-sense,5,-GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3' (SEQ.ID.NO.14); N1-reverse, 5'-CGCGTCTTGA AGAAGATGGT GCGCTCCTGC ❹ GATCGGATCC TCTTAC-3, (SEQ. ID.NO.15); N2-positive stock,

5,-GTAAGAGGAT CCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3, (SEQ.ID.NO. 16); N2_

5,-CGCGTCAGGA GCGCACCATC TTCTTCAAGC

GATCGGATCC TCTTAC-3, (SEQ.ID.NO.17); N3-正股, 5,_GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GTTAACTTGA AGAAGATGGT GCGCTCCTGA-3' (SEQ.ID.NO.18); N3-antisense (antisense), 63 201009073

5,-CGCGTCAGGA GCGCACCATC TTCTTCAAGT

TAACTTGAAG AAGATGGTGC GCTCCTGCGA

TCGGATCCTC TTAC-3, (SEQ.ID.NO. 19); N4-sense, 5,-CGCGTTACTA ACTGGTACCT CTTCTTTTTT

TTTTTGATAT CCTGCAG-3' (SEQ.ID.NO.20); N4-reverse, 5,-GTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3' (SEQ.ID.NO.21). All sequences from SEQ. ID. NO. 14 to SEQ. ID. NO. 21 were acidified at the five ends of the sequences. In addition, the two exon fragments were generated by Dra// restriction enzyme cleavage of red fluorescence and the 208th nucleotide of the GPP gene (SEQ.ID.NO.22), and the five ends of the fragment were further passed through T4 DNA. The polymerase acts to generate a blunt end. As used herein, and GFP is a novel red-shiited fluorescent protein gene (HcRedl) that is embedded with an additional aspartate at the 69th amino acid. Derived from Hai Cai//e wonderful Biosciences, CA). The modified red fluorescent protein has less denaturation and nearly twice the far-infrared fluorescence intensity of the red fluorescent 57 〇 nm wavelength ray. The present invention utilizes a carrier (BE) Bi〇sciences, CA) to cut with a melon and a restriction enzyme and purify a full-length 769 base pair with a 2% agarose (g) electrophoresis and a GFP gene fragment and a 3934 bp corpse. The vector is empty and purified (gel extracti〇nkit, gamma, CA). Mixing Nl-positive strands with veins, Ν2·positive strands and N2_reverse, N3_positive strands with 64 201009073 N3_ anti-sense and N4-positive strands with N4-reverse sequence (1:1) Individually heat each complementary sequence to 94 ° C (2 minutes), heat 70 ° C in lxPCR buffer (such as 50 mM Tris-HCl, pH 9.2 at 25 ° C, 16 mM (NH4) 2S04, 1.75 mM MgCl2) 〇 minutes). Immediately afterwards, individual cooling (one-hour period from 50 ° C to 10 ° C), hybridization of N1+N4, N2+N4, Ν3+Ν4 (1··1), and m, N2, N3 hybridization to N4 Sequential ligation. Then, the T4 DNA ligase and the corresponding buffer were incubated at 12 ° C for 12 hours to obtain a cardiac intron for insertion of the πσπ exon.

Dra / / cut position. After the GF_P exon fragment was added to the reaction (1:1:1), the Τ4 DNA ligase and the corresponding buffer were further adjusted for the corresponding reaction for 12 hours (12°〇). In order to construct the correct size Recombination cake/access and GFP gene (recombinant 5)? and A^-inserted and GFP gene), 10 ng of ligated nucleotide sequence by PCR technology and a pair of specific primers 5,-CTCGAGCATG GTGAGCGGCC ❿ TGCTGAA-3, (SEQ.ID.NO.23) and 5,-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3' (SEQ.ID.NO.24) under PCR conditions at 94 ° C (1 min), The reaction was repeated 30 times at 52 ° C (1 minute) and 68 ° C (2 minutes). Then, the PCR product was isolated using 2% agarose gel, and the nucleotide sequence of about 900 bp was purified by Gel Extraction kit (Qiagen, CA). This 900 bp size "m^inserted gene" can be further confirmed by sequence sequencing. Preferably, in the absence of an intron meson, the positive strand sequence of the intron is 65 201009073

5'-GTAAGTGGTC CGATCGTCGC GACGCGTCAT

TACTAACTAT CAATATCTTA ATCCTGTCCC TTTTTTTTCC ACAGTAGGAC CTTCGTGCA-3' (SEQ.ID.NO.25), and the reverse sequence of the toilet/intron is 5'-TGCACGAAGG TCCTACTGTG GAAAAAAAAG GGACAGGATT AAGATATTGA TAGTTAGTAA TGACGCGTCG CGACGATCGG ACCACTTACJ (SEQ.ID.NO. 26). In another embodiment, the recombinant transgenic gene can be fused to and/or restricted by Dra// of the exon and (except for SEQ.ID.NO.25 and SEQ.ID.NO.26) Enzyme cleavage position. The subsequent experimental process is as described above. The mir-302 pioneer miRNA meson used to test the redesign and the transfer gene construct vector were formed in this manner. Since the recombinant SpRNAi-RGFP gene has a cleavage at the five-terminal and three-terminal ends, respectively, and restriction enzyme cleavage, it can be easily incorporated into the vector via restriction enzyme restriction. The vector must be an organic tissue or a suborganism selected from the group consisting of DNA transgenes, plasmids, jumping genes, genes, transposons (tramp〇s〇ns), and viral vectors ( Retroviral vectors). In addition, since the meson located in the intron is incorporated into the intron at the five-terminal and three-terminal translocations, respectively, and the restriction enzyme sites, the present invention can also use the same restriction enzyme cleavage site to access other different mesons. The carrier is replaced. U(inserteci sequenee insert) is preferably a hairpin-like gene silencing effector, which has a highly complementary sequence capable of targeting a specific gene, and these specific genes are selected. Sequences are silenced from the sequence of green fluorescent protein gene, luciferasegenes, lactose control group (lac_z) gene, viral gene, bacterial gene, plant gene, animal and human gene. These silent effector mesons have a complementarity or homology rate of about 3〇% to 100% with their target gene sequence, and preferably 35% to 49% for hairpin-shRNA mesons. For RNA and anti-intention-RNA mesons, the preferred sequence complementarity ranges from 90% to 100%. Recombination Example 2 __Construction includes the reorganization of the island see \%·(SvRNAi-RGFP) into the expression-competent Vftntnr because the recombination corpse gene has ^ and the fear at the five-terminal and three-terminal A restriction enzyme cleavage site that allows the recombinant gene to be introduced into the steroid using these cleavage sites. The present invention mixes the recombinant gene with a 3934 bp vector at a ratio of 1:16 (w/w) and from 65. (: Cool down to 15 ° C for more than 50 minutes, then add T4 ligase and its buffer to mix it (12 hours) for bonding. The resulting performance vector can utilize five.co" DH5aLB (50ug/ml Mass (kanamycin XSigma Chemical, St. Louis, Mo)) was subjected to large-scale replication using PCR technology and its GFP-specific primers (SEQ.ID.NO.23) and (SEQ.ID.NO.24) at 94 °C ( 1 minute), 68 °C (2 minutes) and 30 cycles of reaction were sequenced to confirm that it was a positive clone with a specific 67 201009073 recombinant gene. In order to incorporate the recombinant gene into the viral vector, the same ligation procedure It can also be carried out on a vector other than the guar/ZY2>a/-linearized/7ZjVCZ2 retroviral vector (BD). Since the mesons are respectively ligated at the five-terminal and three-terminal positions by the Pvw/ and Mm/restriction sites, The present invention can utilize these cleavage removals or replace the anktEGFP shRNA meson with a redesigned mir-302 meson. The redesigned mir-302 meson sequence contains the homologous 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID.N0). .3) Area, such as similar to 5'-UAAGUGCUUC CAUGUUUUAG UGU_3, (SEQ.ID.N0.9 ), 5'_UAAGUGCUUC CAUGUUUUGGUGA-3, (SEQ.ID.NO.10) '5?-UAAGUGCUUC CAUGUUUUAG UAG-35 (SEQ.ID.NO.ll), 5'-UAAGUGCUUC CAUGUUUCAG UGG-3, (SEQ.ID .NO.12) or · 5,-UAAGUGCUUC CAUGUUUGAG UGU-3, (SEQ.ID.NO.13) 〇❹ Synthetic nucleic acid sequences can be used to generate a number of different introns containing mir-302 family precursor microribonucleic acid clusters ( Mir_3〇2 familial pre-miRNA cluster) or redesigned mir_302 meson, examples are as follows: mir-302a-positive strand,

5,-GTCCGATCGT

GTACTTGCTT

CTTCCATGTT C-3' (SEQ.ID.N0.39);

TCCATCACCA

CTTTAGTTTC

CCCACCACTT TGAAACTAAA TTGGTGATGG mir-302a-Reverse, AAACATGGAA AAAGCAAGTA

AAACGTGGAT

GAAGTAAGTG

ATCTCGAGCT

5,-GAGCTCGAGA

GCACTTACTT

CATCCACGTT 68 201009073

TAAGTGGTGG mir-302b-正股 AACTTTAACA TTGAAAGTAA

GACGATCGGA C-3 ' (SEQ.ID.NO.40); , 5'_ATCTCGAGCT CGCTCCCTTC TGGAAGTGCT TTCTGTGACT

GTGCTTCCAT GTTTTAGTAG GAGTCGCTAG CGCTA-3' (SEQ.ID.N0.41); mir-302b-reverse,

GAGAT-3' (SEQ.ID.N0.42); mir-302c-positive strand,

5,-TAGCGCTAGC

AGCACTTACT

TTCCATGTTA

ACCTTTGCTT

TGTGAAACAG

GACTCCTACT

TTCAAAGTCA

AAGTTGAAGG

TAACATGGAG

AAGTAAGTCG

AAAACATGGA

CAGAAAGCAC

GAGCGAGCTC

5,-CGCTAGCGCT

GTACCTGCTG

TTCATGTTTC

AGTGGAGGCG TCTAGACAT-3, (SEQ.ID.NO.43); mir_302c_, 5'-ATGTCTAGAC GCCTCCACTG AAACATGAAC TC TCGACGCGTC AT_3' (SEQ.ID.NO.45);

GACTTACTTC

TCCATGTTAA (SEQ.ID.NO.44);

TAACACTCAA

GCCAGGCTAA 5,-ATGACGCGTC

ACTTAGCCTG

TGTTTCACAC AGCAAAGGTA mir-302d-正股, ACATGGAAGC GTGCTTCCAT

GAACACTCAA

GCTTAGCTAA

AGCAGGTACC GCGCTAGCG-3, 5, _CGTCTAGACA ACTTAGCTAA GTTTGAGTGT mir-302d-reverse, ACATGGAAGC GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3, (SEQ.ID.NO.46); and 69 201009073

miR-302s-正股,5'-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA GCCAGGCACA

CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3, (SEQ.ID.NO.27); mir-302s-reverse, 5,-ATGACGCGTC

GATAAGTGCT TCCATGTTTT AGTGTGCCTG

GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGGAC-3' (SEQ. ID. N0.28).

Mir-302 family precursor microribonucleic acid clusters can be mir_3〇2a_positive strands and mir-302a-anti-hybrid, mir-302b-positive strands and mir-302b-converse hybrids, mir-302c-positive The stock is formed by hybridization with mir-302c-anti-hybrid and mir-302d-positive strands and mir-302d-antisense. Then, mir_302a, mir_3〇2b, and mir-302c were respectively cut by pvui/XhoI, Xhol/Nhel, Nhel/Xbal reverse-restriction enzyme and extracted by gd extraction filter column (Qiagen, CA). ) Extracted in 35 μl of autoclaved ddHe. Immediately thereafter, the collected hybrids are joined to each other by T4 DNA-binding (Roche, 20U) to form a mir-302 family of microribonuclear meson clusters, and these hybrid ribonucleic acids can be further regulated by PVW//MM/etc. The cleavage is incorporated into the expression vector. The recombinant gene comprising the mir_302 family of microribonucleic acid mesoplexes is incorporated into a retroviral vector having a surface antigen which is useful for transgenic infection of hpESC and PC3 cells. In order to form an artificially redesigned mir-302 precursor microribonucleic acid meson, the present invention hybridizes two synthetic sequences of 201009073 (SEQ.ID.NO.27) and (SEQJD.NO.28), and then Pvw//Mw/etc. The restriction enzyme was cleaved and the hybrid sequence was ligated to the expression vector with T4 DNA ligase (20 U). This recombinant gene contains a redesigned mir-302 pioneer microRNA that is available for recombinant DNA recombination of Colo cells. Successfully transfected cells can be isolated and collected for subculture 24 hours later and collected using a single antibody against RGFP and a cell flow meter (Clontech, Palo Alto, CA). The mir-302 precursor microribonucleic acid clustering and mir-302 expression vector can be mass-produced in the plastid vector at the 5.co/z·DH5aLB (50 μg/ml povidine) as described above. 〇〇μg/ml of ampicillin was subjected to extensive replication of the 〆M:Z2 reverse transcription vector. The large number of replicated expression vectors were isolated and purified by mini-prep or maxi-prep plasmid extraction kit (Qiagen, CA). The detailed procedure is described in the instructions. For retroviral vectors, the present invention utilizes a packaging cell line GP2-293 (Qiagen, CA) for the manufacture of a virus that is infectious but not replicable. Infected GP2-293 cells in lxDMEM (carbon-adsorbed 10% fetal bovine serum (FBS), 4 mM L-face valeric acid, 1 mM sodium pyruvate, 100 μg/ml sulphate, 50 μg/ The milliliter neomycin (sigma Chemical, MO) medium was cultured at 37 ° C and 5% carbon dioxide. The amount of virus produced by GP2-293 cells was determined to be at least 1 〇 6 cfil/ml prior to transfection. 71 201009073 Example 3 沅V 〇 〇 〇 忖 忖 对 以 以 以 以 以 以 以 以 以 反 反 反 反 反 反 反 反 反 反 hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp hp The ribonucleic acid mesons were divided into (4) and (7) ϊρ. /?ΖΛ02 reverse transcription expression vectors were co-transfected with pVSV_G Gp2 293 cells (Clontech, CA). After culturing at 37 ° C, 5% carbon dioxide for 36 hours, the broth of GP2-293 cells (per 1 ml) was filtered and directly transferred to hpESC and PC3 cell culture medium by filtration (0.25 μm pore size). . Since the culture medium contains a high dose of the retroviral vector, all of the test cells are transfected with the vector and start to exhibit intron meson and RGFP within 24 hours. In order to transfer the redesigned artificial mir_3〇2 precursor microRNA into Colo cells, the present invention first mixes the prepared pre-prepared cake and the cadaver transfer gene (60 micrograms (pg) per cell culture dish). In 1 ml, the transgenic gene comprises the pre-designed mir-302 precursor microribonucleic acid meson of Example 2, mixed with FuGene reagent (Roche, IN) and operated according to the manual. Next, the mixture was used for Colo cell culture for 24 hours. Because the transgenic gene also contains a transposon-like sequence, which is homologous to a specific human genome region that does not have a codon, and then successfully transfected through a cell flow meter and anti-RGFP monoclonal antibody (Clontech, CA). Dyed cells for subculture. Example 4 Northern Blot Analysis RNA (20pg of total RNA or 2ug of poly[A+]RNA) was separated by electrophoresis using 1% Methyl-72 201009073 (Fonnaidehyde-agarose gels) RNA was adsorbed onto a nylon membrane (Schleicher & Schuell, Keene, NH). A synthetic probe that is complementary to a 75 bp junction sequence between the ligated 5-terminal exon and the previously designed precursor micro-ribonucleotide pre-miRNA insert is subjected to a Prime-It II kit ( Stratagene, La Jolla, CA) Labeled ' and by [jom primer extension] technique, using [32P]-dATP (>3000 Ci/mM, Amersham International, ❹ Arlington Heights, IL) And the purified i〇bp-cutoff Micro Bio-Spin chromatography columns (Bio-Rad, Hercules, CA) to calibrate the sequence. The hybridization of the sequence is by mixing 50% deionized formamide (pH) 7.0, 5x Denhardfs solution, 0.5% SDS, 4 x SSPE and 250 mg/mL denatured salmon sperm DNA fragments (18 hr, 42 ° C)). Nylon membrane at 2x SSC, 0.1% SDS (15 min, 25°) After washing twice under conditions of C) and washing the strip once with 0.2 x SSC, 0.1% SDS (45 min, 37 ° C), (automatic) radiography (Example). Sodium citrate polyacrylamide colloidal electrophoresis (SDS-PAGE) and Western special point method (West Ern Blot Analysis) Immunoblotting of specific proteins, after removal of the cultured cells, the cells are lysed by ice-saturated phosphate buffered saline (PBS) and dissolved in CelLytic-M cells. Extractant (lysis/extraction. reagent) (Sigma, MO) and supplemented with protein 73 201009073 Protease inhibitors such as Leupeptin, TLCK, TAME and PMSF according to the instructions. The cells are then shaken at room temperature. After shaking for 15 minutes, the cells were scraped into a test tube, and the cells were sacrificed by centrifugation at a speed of 12000 x g for 5 minutes, and the cell extract containing the protein was collected and stored at _7 〇〇 c for use. The protein was quantified using an SOFmax software package on an E-max microplate reader (Molecular Devices, Sunny vale, CA). Each 30 pg of cell extract was added to SDS_PAGE sample buffer (with or without reducing 50 mM DTT) and the sample was boiled for 3 minutes before injecting the sample into 8% polyacrylamide gels. A 2~3 μΐ protein marker (Bio-Rad) was injected into it. SDS-polyacrylamide gel electrophoresis was performed according to a standard procedure (Molecular Cloning, 3rdED). After protein chromatography, the protein was adsorbed on a nitrocellulose membrane by electroblotting, and treated with Odyssey blocking reagent (Li-C〇r Biosciences, Lincoln, NB) at room temperature for 1 to 2 hours. The present invention utilizes a primary antibody to directly target a specific protein (EGFP (1: 5,000; JL-8, BD), RGFP (1: 10,000; BD), Oct3/4 (1:500; Santa Crutz), SSEA-3 (1:500; Santa Crutz), SSEA-4 (1:500; Santa Crutz), Sox2 (l:1000; Santa Crutz), Klf4 (l:200; Santa Crutz)), calibrated at 4 ° C all night , for evaluation of protein expression. Then, it was washed three times with TBS-T buffer, and then the nitrocellulose membrane (goat anti-mouse IgG conjugate with Alexa Fluor 680 reactive dye 201009073 (1:2,000; Molecular Probes)) was applied as a secondary antibody. After an hour, the cells were washed three times with TBS-Τ buffer and imaged and images were recorded with Li-Cor Odyssey Infrared Imager and Odyssey Softearev.10 (Li-Cor). Example 6 Intronic RNA-mediated Gene Silencing of the zebrafish In this Τ§(ύ^/«-ΟΑ]ϋ4:υΑ8$φ) zebrafish juvenile 〇mi ❹ 0·2χ serum-free (semm-jfree) RPMI 1640 medium in a container for transfection. A transfection pre-mix was prepared by lightly dissolving 60 μl of FuGene liposomal transfection reagent (Roche Biochemicals, Indianapolis, IN) in lml lx serum-free RPM 1640 medium. As described in Examples 1 and 2, with and without the anti-five GFP precursor microribonucleic acid meson, the GF cadaver and the GF cadaver (20 μδ) were mixed with the above-mentioned transfection pre-agent and placed on ice for 30 minutes and then directly introduced. The zebrafish juvenile fish is containerd for transfection. All three doses (all 6 ug) were injected over a 12 hour interval and samples were collected and observed after 60 hours of the first transfection. Example 7 Flow Cytometry Assay Cells were trypsinized, collected by centrifugation and re-mixed with 1 ml of PBS (pre-cooled 70% methanol) and placed at _2 °C for 15 hours. The cells were then collected by centrifugation and washed once with 1 ml of PBS. The cells were again collected by centrifugation and re-mixed with 1 mi of PBS (1 mg/ml of pr0pidium iodide and 0.5 mg/ml 75 201009073 nuclease) and placed at 3TC for 3G minutes. Approximately 15 cells were analyzed by BD FACSCalibur (San J0se, CA). The cell doublet (Cell doublet) sets the gate diameter by a single cell by the relative value of the pause zone width and the pause domain 2 (10) width versus pulse area. The collected data was analyzed by Flowjo software and the Watson Pragmatic algorithm (alg〇rithm).魅例八-圭oxyribose nuclear deuteration test DNA Metlwlatimi ❹

Assay The present invention first isolates the genome of a cell by a DNA isolation kit (R〇che, IN). The genomic DNA sample was cultured in test cells at 1〇 jjjjw Tris_HC1

8.0), 10 mM EDTA, and 0.2 mg/ml Protease K (proteinase K) Prepared by ethanol precipitation at 55 ° C for 3 hours. The isolated gene body was then allowed to act individually at 37 ° C for 4 hours via (^(^(^) restriction enzyme Hpall (lOU). The final DNA fragment was then separated by 1% acacia gel electrophoresis. The thiolated region of the promoter region, the present invention treats the isolated genomic DNA with a bisulfite (CpGenome DNA modification kit, Chemicon, CA), followed by PCR (long template PCR extension kit, Roche, IN) and two Forward primer 5'-GAGGAGTTGA GGGTACTGTG-3, (SEQ.ID.N0.47) and 5,-GAGGAGCTGA GGGCACTGTG-3' (SEQ.ID.N0.48) and a reverse primer (reverse primer) 5'-GTAGAAGTGC CTCTGCCTTC C-3, (SEQ. ID. NO. 76 201009073 49) to isolate the OdW five-terminal upstream promoter region. The cell genome (loo Nike (ng)) was first introduced (all 150 pmole) Mix in lx PCR buffer and heat to 94 ° C for 4 minutes and then directly on ice. Then carry out 25 cycles of PCR 'reaction, the reaction conditions are as follows 92 (: 1 minute; 55. (: 1 minute; 5 minutes at 70 ° C. The final PCR product was collected via PCR purification kit (Qiagen, CA) and equal ACGT cleavage restriction enzymes (5U each) were mixed and reacted. The restriction enzymes contained jc// (AACGTT), (CACGTC), cadaver m/J φ (CACGTG), -5/ (TACGTA) and substance C Γ (ACGT) The final DNA fragment was separated by 3% acacia gel electrophoresis. Example 9 Micronucleus riding nucleic acid biochip analysis (MicroRNA Microarray Analysis, human PC3 and Colo cell strains by American Type Culture Collection (ATCC) , Rockville, MD)) obtained ❹ and hpESC cells were isolated from the skin of the inventor via trypsin. Under 70% correlation (confluenCy), small RNA was passed through the miRNA isolation kit (Ambion, Inc" Austin, TX) was isolated from each cell line and tested as described by the user. The purity and quality of the small RNA was assessed by 1% furfural gel electrophoresis and spectrometer (Bio-Rad, Hercules, CA). Sent to LC Sciences (San Diego, CA) for microRNA biochip analysis. Each biochip is hybridized via a single sample that calibrates Cy3 or Cy5 or individually calibrates a sample of Cy3 or Cy5. Eliminate the background and 77 201009073 normalization. For the dual sample assay, the cadaver-value calculation showed a transcriptional molecule with a difference of more than 3 fold. Example 10 Gene Biocrystallization Synthetic Analysis In order to prepare a calibrated probe for hybridization with a gene on a biochip, the entire extracted ribonucleic acid (2jig) was converted into a double-stranded DNA, and Superscript was used. Choice system kit (Gibco/BRL, Gaithersburg, MD) and modified oligh(dT)24_T7 promoter primer such as ❿ 5'-GGCCAGTGAA TTGTAATACG ACTCACTATA GGGAGGCGG-(dT)24_3' (SEQJD.NO.50) The subsequent steps are as described by their user. The double-stranded cDNAs were extracted with phenol/chloroform, and then precipitated with ethanol and re-dissolved to 0.5 μδ/μ1 diethyl pyrocarbonate (DEPC)-treated ddH20. Phase-Lock

Gel (5 Trime-> 3' Prime, Inc., Boulder, CO) can be used to increase the extraction rate. While transcription can be used with T7 RNA polymerase with 10f 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) were reacted at 37 ° C for 4 hours to generate cRNA, followed by cRNA was purified using RNeasy spin columns (Qiagen, CA). The sample was confirmed by 1% acacia gum, and then cRNA was randomly divided into about 50 nuclei by heating to 94 ° C for 35 minutes in Tris_acetate, pH 8.0, 100 mM KOAc/30 mM MgOAc buffer. Large 78 of the nucleotides 201009073 Biochips of four nucleotide sets (GeneChip U133A & B arrays, Affymetrix, Santa Clara ^ CA) and U133 plus 2 human genome biofilm (Affymetrix, Santa Clara, CA), A total of 32668 genes were included for hybridization. The heterozygous reaction was carried out in 2 μl of AFFY buffer (Affymetrix) at 40 ° C for 16 hours with continuous agitation. After the hybridization reaction was completed, the biochip was rinsed 3 times with 200 μL of 6x SSPE-T buffer (lx 0.25 ❹ sodium citrate/15 mM sodium phosphate, pH 7.6/1 mM EDTA/0.005% Triton) and then Wash with 200 μL of 6x SSPE-T buffer at 5 (TC for one hour. Then wipe with 0.5X SSPE-T twice and 〇.5x SSPE-T at 50. (: rinse for 15 minutes. Then use 2 pg/ Molstreptavidin-phycoerytiirin (Molecular Probes) and 1 mg/ml acetylated BSA (Sigma) were stained in 6x SSPE-T (pH 7.6) buffer, and the biochip was placed in a confocal scanner (Molecular Dynamics) for interpretation at 7.5 μηη. The analysis was performed with Afifymetrix Microarray Suite ❹ version 4.0 software. After the samples were normalized by the average difference between the perfectly matched probe and the mismatched probe, the difference in the collected signal was more than twice. Signals. Cell Differentiation Assay All cell lines of the present invention were cultured in phenol red-free DMEM medium (10% charcoal-adsorbed fetal bovine serum (1% charcoal_stripped fetal bovine) Semm(FBS))) Under 70% confluency, no 79 201009073 hormones and growth factors were added separately to the cell culture medium, such as 50 ng/ml DHT, 100 ng/ml TGF-βΙ And / or 100 ng / ml BMP4. After 6 to 12 hours of culture, the treated cells were collected by trypsin in 4 parts (aliquot) of 200 μΐ lx PBS solution, and then immediately diluted into 6 weeks of athymic gland. Immunodeficient SCID beige mice in the neck skin, tail vein, uterus and liver. Example 12 Statistical tiller biochip results are presented as mean soil SE. Statistical analysis of these sample data by One-way ANOVA method calculation. When there is a statistically significant difference, 'D_ett's post-hoc test is used to identify the sample group that differs from the standard. In order to compare _ rows between the two groups, SU(jent (test system is use. For comparison, the ANOVA was compared and then compared by post_h〇c multiple range test. The probability value p < 〇.〇5 is considered to be statistically significant. 'All p values are determined by a two-tailed test. BRIEF DESCRIPTION OF THE DRAWINGS The present invention or application slot contains at least the drawings in color. The invention or the patent application and the pure drawing are submitted to the relevant unit to record the relevant fees. Figure 1 shows the cell-containing micronucleic acid (IV)-mirc_A, miRNA). The helper contains the money and the precursor message, and the nucleic acid is shared with RNAsplieing_, (10)-aged and connected to the message nucleus to produce synthetic eggs (4). After the miscellaneous _ _ sub-nuclear 201009073 nucleus S 夂 is treated by it step into a mature micro-nucleic acid, for the production of Chi ^. Thus, our mt-human inclusions contain at least a precursor micro-nucleic acid structure (or fine) for the synthesis of microRNAs that mimic the introns of the cells themselves (Fig. 3A and Fig. 3B). You are introduced into the cell or New Zealand by a second type polymerase promoter or viral promoter and are expressed by a first-type polymerase system. After the fine transcription-promoting production, after the RNA splidng treatment, the nucleic acid of the time is designed to be ❹+. In other real cases, only the lion-reducing molecule can be an anti-intentional ribonucleic acid structure, and the above-mentioned anti-intentional structure can be used for gene knocking. In other embodiments, the 'predesigned intron ribonucleic acid molecule can comprise a partial antisense and a positive ribonucleic acid fragment for use in forming a double stranded siRNA and generating a J^Ai mechanism. In other embodiments, the pre-designed intron ribonucleic acid molecule can be a small ribonucleic acid structure' for generating an RNAi-related gene silencing effect. Figure 1 shows the mechanism of micronucleic acid (in vivo, such as coffee mirc〇RNA, 10 miRNA) in the intracellular intron. Figure 2 shows the siRNA, the exonie (intergenic microRNA) of the exon and the intronic microRNA pathway mechanism of the intron. 3A to 3D show a φ-turbine recombinant gene structural compound (Fig. 3A) (SEQ. ID. N0.32) and its principle (Fig. 3A) of a preferred embodiment of the present invention to produce a microRNA which mimics an intron (Fig. 3A). Artificial microRNA of intronic miRNA). Tests of intracellular expression of the expression vector showed that the inhibition of green fluorescent protein in zebrafish was more than 85% of the gene inhibition (kn〇ckd〇wn) effect directly in 81 201009073 and was confirmed by Western blotting, as shown in Fig. 3C. The iconic miRNA with the intron of the green fluorescent protein and its fried precursor can be electrophoresed by l%f〇rmaldhyde agar and observed after the northern dot method (Fig. 3D). . Figures 4A through 4C show the evaluation of the utility of the intronic insert of the intron of the different designs of the G/rp structure of the present invention. Efficient miRNA biosynthesis and efficient generation of green fluorescent protein silencing in two-week zebrafish juveniles confirms that the ribonuclease-induced silent complex is in 5'-miRNA*-stemloop-miRNA-3, [l There is a tendency to asymmetry between the two small ribonucleic acids with 5,-miRNA-stemloop-miRNA*-3' [2] (Fig. 4A). The gene silencing of green fluorescent protein was only found in the transfection of the precursor microRNA structure [2], but not in the transfected structure [1:|, but confirmed its 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. Therefore, the amount of green fluorescent egg white is much lower than that of red fluorescent protein in the pioneer microRNA. Structure [2] transfection group, at this time the indicator of the vector red fluorescent protein is the average performance (Fig. 4B) (SEQ. ID. N0.33-35). Analysis of the amount of green fluorescent protein by Western blotting confirmed 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 the liposome group (Lip〇s〇me only (Lipo)), the empty vector group without any meson (Vctr), and the siRNA (siR) group. 82 201009073 Figures 5A to 5C show that mir-302-like ribonucleic acid molecules are transfected in human epithelial cells (hpESC), human prostate cancer cell line (PC3) and human primary melanoma cell line (Colo), resulting in cell type State and changes in cell proliferation. After mir-302 transfection, the cells expressing mir-302 were individually called hpESC+mir-302, PC3+ mir-302 and C0I0+ mir-302, after mir-g^ transfection (mir-gfp is silent green fluorescence) Sequence of protein)

The cells are individually referred to as hpESC+mir-g^), PC3+mir-gj53 and Colo+mir-g^D. Figures 6A to 6E show the characteristics of embryonic stem cells transfected with mir-302 by Colo and PC3 cell lines. These characteristics include the formation of embryoid bodies (embry〇id b〇dy) as shown in Figure 6A. Western blot analysis of proteins such as 〇ct3/4, SSEA-3 and SSEA-4 is shown in Fig. 6B. De-mercaptolysis analysis of the gene body, as shown in Figure 6C. CpG demethylation analysis, as shown in Figure 6D. And cell displacement, as shown in Figure 6E. Figures 7A through 7B show microRNA biochip analysis confirming that the mir_3〇2 family is all highly expressed in Colo+ mir_3〇2 cells. Figures 8A to 8B show the results of analysis of gene chips in c〇I〇+ mir-3〇2 cells (Affymetrix human GeneChip U133A & B, CA), and the results show that 'the gene expression status of human embryonic stem cells HuEC8 and H9 cells In contrast, there are many embryonic stem cell marker genes that have risen significantly, while cancer and developmental genes have decreased significantly. ~ Figures 9A to 9C show c〇i〇+ ^ viewing cells with red glory protein

St many fine 峨 'such as 'primitive chondrocytes, fibroblasts and after treatment of the developmental signaling material (eg, different hormones and / 83 201009073 or growth factors) 'proven that their differentiation is similar to those embryos stem cell. Figure 10 is a data of a biochip (Affymetrix U133 plus 2 human genome gene chips) showing that it is expressed in c〇1〇+mir_3〇2 cells, whereas the Klf4 gene does not. In addition, many standard human embryonic stem cell markers are highly expressed in Colo+mir-302 cells rather than native c〇1〇 cells such as '0ct4, Sox2, Nanog, Utfl, Rexl, SALL2, and SALL4, and this result echoes Figure 8B. Experimental results. ❹ 84 201009073 Sequence Listing

<110> LIN, Shi-Lung, et al. Lin Xilong et al. <120> Using an intron nuclear St gene in Generation Embryonic Stem-Like Cells Using Intronic RNA <130> 5199- 0138PUS1 <140> 098114452 <141> 2009-04-30 <160> 50 ❹

<170> Patentln version 3.5 <210> 1 <211> 12 <212>DNA<213> artificial sequence<220><223> chemical synthesis oligonucleotide<400> 1 gctaagccag gc 12 <210> 2 <211> 12 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotide

<400> 2 gcctggctta gc <210> 3 <211> 17 <212> RNA 85 12 201009073 <213> Artificial sequence <220><223> Chemical synthesis oligonucleotide <400> Uaagugcuuc cauguuu <210> 4 <211> 8 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 4 gtaagagk <210> 5 < 211 > 10 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 5 gwkscyrcag <210> 6 <211> 7 <212> DNA <;213> artificial sequence <220> <223> chemical synthesis nucleotides <400> 6 tactway 201009073 <210> 7 <211> 17 <212> DNA <213>artificial sequence <220>;<223>Chemical Synthesis Oligonucleotide<400> 7 tytycttttt tttttts <210> 8 <211> 19

10 <212>DNA<213> artificial sequence <220><223> chemical synthesis oligonucleotide <220>221> misc_feature <222>(15)..(15) <223 〉n is a,c,g,ort <400〉 8 tctctctctc tctcnctag ❿ <210> 9 <211> 23 <212> RNA <213> artificial sequence <220><223> Nucleotide <400> 9 uaagugcuuc cauguuuuag ugu <210> 10 201009073 <211> 23 <212> RNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400 〉 10 uaagugcuuc cauguuuugg uga 23 23

<210> 11 <211> 23 <212> RNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide <400> 11 uaagugcuuc cauguuuuag uag <210> 12 < 211 > 23 <212> RNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 12 uaagugcuuc cauguuucag ugg 23 <210> 13 <211> 23 <212&gt RNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide <400> 13 88 201009073 uaagugcuuc cauguuugag ugu <210> 14 <211> 42 <212> DNA <213> Artificial sequence <220><223> chemical synthesis oligonucleotide <400> 14 gtaagaggat ccgatcgcag gagcgcacca tcttcttcaa ga Q <210> 15 <211> 46 <212> DNA <213> artificial sequence< 220> <223>Chemical Synthesis Oligonucleotide <400> 15 cgcgtcttga agaagatggt gcgctcctgc gatcggatcc tcttac <210> 16 <211>

❹ <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 16 gtaagaggat ccgatcgctt gaagaagatg gtgcgctcct ga <210> 17 <211> 46 <212> DNA <213> artificial sequence ❿ 60 70 60 〇201009073 <220><223> chemical synthesis nucleotides <400> 17 cgcgtcagga gcgcaccatc ttcttcaagc gatcggatcc tcttac 46 <210> 18 <211> 70 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 18 gtaagaggat ccgatcgcag gagcgcacca tcttcttcaa gttaacttga agaagatggt gcgctcctga <210> 19 <211> 74 <212> DNA <213> artificial sequence <220><223> chemical synthetic nucleotide <400> 19 cgcgtcagga gcgcaccatc ttcttcaagt taacttgaag aagatggtgc gctcctgcga tcggatcctc ttac <210> 20 <211> 47 <212> DNA < 213 > artificial sequence <220><223> chemical synthesis oligonucleotide <400> 20 cgcgttacta actggtacct cttctttttt tttttgatat cctgcag 47 90 201009073 <210&g t; 21 <211> 45 <212>DNA<213> artificial sequence <220><223> chemical synthesis nucleotides <400> 21 gtcctgcagg atatcaaaaa aaaaagaaga ggtaccagtt agtaa 45

<210> 22 <211> 689 <212>DNA<213> artificial sequence <220><223> by HcRedl color protein gene derived from sea anemone (i/eiemcri·? crfjpa) Chromoprotein gene) The mutant red fluorescent protein gene generated by the insertion of aspartate (Asp) in the 69th amino acid <400> 22 atggtgagcg gcctgctgaa gg£^agtatg cgcatcaaga tgtacatgga gggcaccgtg 60 aacggccact acttcaagtg cgagggcgag ggcgacggca accccttcgc cggcacccag 120 agcatgagaa tccacgtgac cgagggcgcc cccctgccct tcgccttcga catcctggcc 180 ccctgctgcg agtacggcag caggacgacc ttcgtgcacc acaccgccga gatccccgac 240 ttcttcaagc agagcttccc cgagggcttc acctgggaga gaaccaccac ctacgaggac 300 ggcggcatcc tgaccgccca ccaggacacc agcctggagg gcaactgcct gatctacaag 360 gtgaaggtgc acggcaccaa cttccccgcc gacggccccg tgatgaagaa caagagcggc 420 ggctgggagc ccagcaccga ggtggtgtac cccgagaacg gcgtgctgtg cggccggaac 480 gtgatggccc tgaaggtggg cgaccggcac ctgatctgcc accactacac cagctaccgg 540 agcaagaagg ccgtgcgcgc cctgaccatg cccggcttcc acttcaccga catccggctc 600 cagatgctgc ggaagaagaa ggacgagtac ttcgagctgt acgaggccag cgtggcccgg 660 tacagcgacc tgcccgagaa ggccaactg 689 <210> 23 <211> 27 <212> DNA <213> Artificial sequence 91 201009073 <220><223>Chemical synthesis of nucleotides <223> 400> 23 ctcgagcatg gtgagcggcc tgctgaa 27 <210> 24 <211> 27 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 24 tctagaagtt ggccttctcg Ggcaggt G 27 <210> 25 <211> 89 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 25 gtaagtggtc cgatcgtcgc gacgcgtcat tactaactat caatatctta atcctgtccc ttttttttcc Acagtaggac cttcgtgca 60 89 ❹ <210> 26 <211> 89 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 26 tgcacgaagg tcctactgtg gaaaaaaaag ggacaggatt aagatattga Tagttagtaa 60 tgacgcgtcg cgacgatcgg accacttac 89 92 201009073 <210> 27 <211> 82 <212> DNA < 213 > artificial sequence <220><223> chemical synthetic nucleotide <400> 27 gtccgatcgt cataagtgct tccatgtttt agtgtgctaa gccaggcaca ctaaaacatg gaagcactta tcgacgcgtc at <210> 28 0 <211> 82 <212> DNA < 213> artificial sequence <220><223> chemical synthesis oligonucleotide <400> 28 atgacgcgtc gataagtgct tccatgtttt agtgtgcctg gcttagcaca ctaaaacatg gaagcactta tgacgatcgg ac <210> 29 <211>

❹ <212> RNA <213> artificial sequence <220><223> chemical synthesis nucleoside 睃 <400> 29 wuccaagggg g <210> 30 <211> 10 <212> DNA < 213> artificial sequence 201009073 <220><223> chemical synthesis nucleotides <400> 30 gtaagaggat 10 <210> 31 <211> 12 <212> DNA <213> artificial sequence <220>;<223> chemical synthesis of nucleotides 12 60 65

<400> 31 gatatcctgc ag <210> 32 <211> 65 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 32 aggtaagagt cgatcgacgc gttactaact Ggtacctctt cttttttttt tgatatcctg caggc <210> 33 <211> 53 <212> DNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide <400> 33 aagaagatgg tgcgctcctg gatcaagaga ttccaggagc gcaccatctt ctt 53 94 201009073 <210> 34 <211> 54 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 34 caagaagatg gtgcgctcct ggatcaagag attccaggag cgcaccatct tctt 54

<210> 35 <211> 53 <212> DNA Q < 213 > artificial sequence <220 < 223 > chemical synthetic nucleotides <400> 35 aagaagatgg tgcgctcctg gatcaagaga ttccaggagc gcaccatctt ctt 53 <210> 36 <211> 25 <212> DNA <213> artificial sequence 〇<220><223> chemical synthesis oligonucleotide <400> 36 gttgttttgt tttggttttg gatat 25 <210> 37 < 211 > 26 <212> DNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide 95 201009073 <400> 37 attgttttgt tttggttttg gattta <210> 38 <211> 21 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 38 gtagaagtgc ctctgccttc c <210> 39 <211> 91 <212>DNA <213> Artificial sequence <220><223> chemical synthetic nucleotide <400> 39 gtccgatcgt cccaccactt aaacgtggat gtacttgctt tgaaactaaa gaagtaagtg cttccatgtt ttggtgatgg atctcgagct c <210> 40 <211> 91 <212> DNA <213> Artificial sequence ≪220><223>Chemical Synthetic Nucleotide<400> 40 gagctcgaga tccatcacca aaacatggaa gcacttactt ctttagtttc aaagcaagta catccacgtt taagtggtgg gacgatcgga c <210> 41 <211> 95 201009073 <212> DNA <213> Sequence • <220〉 ' <223>Chemical Synthetic Nucleotide' <400> 41 atctcgagct cgctcccttc aactttaaca tggaagtgct ttctgtgact ttgaaagtaa gtgcttccat gttttagtag gagtcgctag cgcta <210> 42 <211> 95 <212> DNA < 213 > artificial sequence ❹ <220><223> chemical synthesis oligonucleotide <400> 42 tagcgctagc gactcctact aaaacatgga agcacttact ttcaaagtca cagaaagcac ttccatgtta aagttgaagg gagcgagctc gagat <210> 43 <211> 89 <212> DNA <;213> artificial sequence © <220> <223> chemical synthesis nucleotides <400> 43 cgctagcgct acctttgctt taacatggag gtacctgctg tgtgaaacag aagtaagtcg ttcatgtttc agtggaggcg tctagacat <210> 44 <211> 89 <212> DNA <;213>artificialsequence<220><223> chemical combination Raised nucleotides 6082

60 82

201009073 <400> 44 atgtctagac gcctccactg aaacatgaac gacttacttc tgtttcacac agcaggtacc 60 tccatgttaa agcaaaggta gcgctagcg 89 <210> 45 <211> 82 <212> DNA <213> artificial sequence <220><223> Glycoside <400> 45 cgtctagaca taacactcaa acatggaagc acttagctaa gccaggctaa gtgcttccat gtttgagtgt tcgacgcgtc at <210> 46 <211> 82 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleoside Acid <400> 46 atgacgcgtc gaacactcaa acatggaagc acttagcctg gcttagctaa gtgcttccat gtttgagtgt tatgtctaga eg <210> 47 <211> 20 <212> DNA <213> artificial sequence <220><223><4〇〇> 47 gaggagttga gggtactgtg 20 98 201009073 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <220><223> Chemical synthesis oligonucleotide <400> 48 gaggagctga gggcactgtg 20

<210> 49 <211> 21 <212> DNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide <400> 49 gtagaagtgc ctctgccttc c <210> 50 <211> 63 <212> DNA <213> artificial sequence

<220〉 <223>Chemical Synthesis Oligonucleotide <400> 50 60 63 ggccagtgaa ttgtaatacg actcactata gggaggcggt tttttttttt tttttttttt ttt 99

Claims (1)

201009073 VII. Patent application scope: 1. A method for transgenic gene for inducing mir_3〇2-mediated gene silencing effect of intron in mammalian cells, comprising the following steps: a. constructing a recombinant nucleic acid compound (rec) 〇mbinant nucieic acid composition), the genetic recombinant nucleic acid compound comprises at least one intron (intr〇n) having one type of mir-302 gene silencing effector (mir_3〇2-like gene-silencing effector), the inclusion Is ligated by at least one exon, wherein the intron can be excised and separated from the exon for inducing a mir-302-mediated gene silencing; b. Cloning the gene recombinant nucleic acid compound into a performance vector; and c. transfecting the expression vector into a plurality of mammalian cells, wherein the hairy mammal cells produce a plurality of recombinant nucleic acid compounds prior to the transcription of the light nucleic acid nucleic acid Primary RNA transcript, at which time the mammalian cells splice the intron and isolate it from the precursor ribonucleic acid transcription molecule Generating the mir-302-mediated gene silencing effect on a plurality of genes comprising a sequence complementary to a gene silencing effector of the mir-302, thereby causing the mammalian cells to reprogram into a plurality of stem cells Stem cell-like pluripotent cells. 2. The method of transferring a gene according to claim 1, wherein the mammalian cell comprises a human 100 201009073 type cell. The method of transgenic according to claim 1, wherein the gamma cell comprises a somatic cell. 4. The method according to claim 1, wherein the mammalian cell comprises a cancerous cell. 5. The gene silencing effector of the mir 302 of the intron comprises a synthetic excitation sequence as claimed in claim 1. . 6. The method of transferring a gene according to claim 1, further comprising the step of synthesizing the intron or the exon and a part of the nucleic acid phases of the two. 7. The method according to claim 1, wherein the gene nucleic acid compound comprises a rec〇mbinant cdlular gene. 8. The method according to claim 1, wherein the recombinant nucleic acid compound comprises a regombinant gene derived from a green fluorescent protein gene (GFPgene), a viral gene, Mammalian genes, jumping genes, transposons, and mixed genes of the above genes. 9. The method of claim 1, wherein the intron is constructed by a genetic engineering method selected from the group consisting of DNA restriction and ligation, homologous recombination (h〇m〇i〇guous recombination), transgene incorportation, transposon insertion, jumping gene integration, retroviral infection, and the above methods The method of mixing. 101 201009073 ίο. The method of transgenic gene according to claim 1, wherein the intron package comprises a nucleic acid sequence comprising an intronic insulator, the meson comprising the mir-302 Gene silencing effector, a branching point region, a multi-cancerous sputum region, a donor splice site, and an acceptor sp sp site. 11. As described in claim 10 Colony method 'where the intron meson comprises a hairpin-like nucleic acid sequence comprising a hinged structure homologous to SEQ.ID.N0.1 or SEQ.ID.N0.2 (stem Loop structure). The method of claim 10, wherein the intron comprises a nucleic acid sequence comprising a homologous or complementary or both in the manner of SEQ. ID. N0.3. 13. The method according to claim 10, wherein the intron comprises a type of small hairpin-like precursor microRNA' (pre-miRNA) sequence, the sequence comprising a nucleic acid sequence It is selected from the group consisting of SEQ.ID.N0.9, SEQ.ID.NO.10, SEQ.ID.NO.il, SEQ.ID.N0.12❹ and SEQ.ID.N0.13. 14. The method of claim 1 as claimed in claim 1 , wherein the intron is incorporated into the intron via at least one restriction enzyme cleavage site selected from the group consisting of 乂, //, AccI, Aflll/III, Agel, Apal/LI, Asel, Asp718I, BamHI, Bbel, BclI/II, BglII, BsmI, Bspl20I, BspHI/LUllI/120I, BsrI/BI/GI, BssHII/SI, BstBI/XJl/XI, Clal, Csp6I, Dpnl, Dral/II, EagI, Ecll36II, EcoRl/RJI/47III, Ehel, FspI, Haelll, Hhal, HinPI, 102 201009073 Hindlll, Hinfl, Hpal/II, KasI, ΚρηΙ, MaeII/ΙΙΙ, Mfel, Mlul, MscI, Msel, Nael, Narl, Ncol, Ndel, NgoMI, Notl, Nrul, Nsil, PmlI, PpulOI, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, Smal, SnaBI, SphI, SspI, StuI, Tail, TaqI, Xbal, Xhol, Xmal and the tangent of the above-mentioned tangent. 15. The method of claim 10, wherein the branching point region comprises a branching point and the branching point is an adenosine (A) and is located in a nucleic acid sequence, the nucleic acid sequence comprising Or homologous to SEQ. ID. N0.6. 16. The method of claim 10, wherein the branching point region comprises a branching point, and the branching point is an adenosine (A) and located in a nucleic acid sequence comprising at least one An oligonucleotide region (oligonucleotide motif) homologous to 5'-TACTAAC_3. 17. The method of claim 10, wherein the bite region comprises a nucleic acid sequence having a high density of Thymine and Cytosine, the nucleic acid sequence comprising or Originated from SEQ. ID. N0.7 or SEQ. ID. N0.8. 18. The method of transferring a gene according to claim 1 wherein the five-terminal splicing comprises a nucleic acid sequence comprising or homologous to SEQ. ID. N0.4. 19. The method of claim 1, wherein the five-terminal splicing comprises a nucleic acid sequence comprising or homologous to 5'-GTAAG-3. 20. The method of claim 1, wherein the three-terminal splicing comprises a nucleic acid sequence comprising or homologous to SEQ. ID. N0.5. 21. The method of transgenic gene according to claim 1 wherein the three-terminal splicing comprises 103 201009073 nucleic acid sequence' which comprises or is homologous to 5,-CTGCAG-3. 22. The method according to claim 1, wherein the π ώ · 302 base g]. the silent effector comprises a nucleic acid sequence comprising homologous or complementary or both in a manner .ID.N0.3. 23. The method of transgenic gene according to claim 1, wherein the gene silencing effect of the mk-302 gene comprises a SEQ ID NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12 and/or SEQ.ID.NO.13. 24. The method of transgenic according to claim i, wherein the mir_3〇2 gene 静 silent effector comprises a nucleic acid sequence comprising SEQ.ID.N0.9. 25. The method according to claim 1, wherein the expression vector is selected from the group consisting of a DNA transgene, a plasmid, a transposon, a retrotransposon, a jumping gene, A vector in which the viral vector and the above vector are mixed. 26. The method of claim 1, wherein the expression vector comprises a viral promoter or a second RNA polymerase (Pol-II) promoter or both, a Kozak translation start, Multiple adenylation signals and a plurality of restriction enzyme cleavage sites. 27. The method of claim 2, wherein the restriction enzyme cleavage site comprises an oligonucleotide cleavage site for endonuclease activity, the cleavage site being selected from the group consisting of d, //, AflII/III , AgeI, ApaI/LI, AseI, Asp718I, BctmHI, BbeI, BcU/II, BgUI, BsmI, Bspl20I, BspHI/LUUI/120I, BsrI/BI/GI, BssHII/SI, BstBI/Ul/XI, Clal, Csp6I , Dpnl, Dral/II, EagI, Ecll36II, EcoRI/RII/47III, Ehel, FspI, Haelll, Hhal, HinPI, Hindlll, 104 201009073 Hinfl, ΗραΙ/ΙΙ, KasI, ΚρηΙ, MaeII/ΙΠ, Mfel, Mlul, MscI , Msel, Nael, Narl, Ncol, Ndel, NgoMI, Notl, Nrul, Nsil, Pmll, PpulOI, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, Tail , TaqI, Xbal, Xhol, Xmal, and the inverse of the tangent of the tangent bits. . 28. The method according to claim 26, wherein the vector further comprises a pUC origin of replication, and the prokaryotic cell has at least one antibiotic resistance gene. An SV40 early promoter and an optional S V40 origin for replication in the mammalian cell. The method of transgenic gene according to claim 28, wherein the antibiotic resistance gene is resistant to a drug selected from the group consisting of penicillin G, ampicillin, neomycin, and paromycin. , kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, rifapicin ), amphotericin B (amphotericin B), gentamycin (chloramphenicol), cephalosin (cephalothin), tylosin (tylosin), G418 and antibiotics mixed with the above antibiotics. 30. The method of claim 1, wherein the expression vector is delivered to the mammalian cell by a gene transfection method selected from the group consisting of liposomal transfection, chemical transfection. Chemical transfection, recombinant DNA recombination, viral infection, transposition 105 201009073 transposon insertion, skip gene transfection ϋιιιηρώ§ gene transfection, microinjection ^ (micro- Injection), electroporation, gene grabbing (gene_gun penetrati〇n) and methods of mixing the above methods. The method of claim 1, wherein the precursor nucleic acid transcript of the recombinant nucleic acid compound is produced via a transcriptional mechanism selected from the group consisting of a second type of ribose Nucleic acid polymerase transcription mechanism, third-type ribonucleic acid polymerase transcription mechanism, first-type ribonucleic acid polymerization 0 enzyme transcription mechanism and viral ribonucleic acid polymerase transcription mechanism. 32. The method of claim 1, wherein the precursor nucleic acid transcription molecule of the recombinant nucleic acid compound comprises a nuclear nucleotide sequence selected from the group consisting of a message ribonucleic acid ( mRNA), heteronuclear ribonucleic acid (hnRNA), ribonucleotide ribonucleic acid (rRNA), transduction ribonucleic acid (tRNA), (snoRNA), small nuclear ribozyme nucleic acid, pre-microRNA, viral ribozyme Nucleic acid (virai r)s|A) and the above-mentioned ribonucleic acid derivatives and precursors. 33. The method according to claim 1, wherein the mir_3〇2 gene silencing effector is capped from the intron via an intron resection mechanism (in1r〇n excisi〇n meehanism) Axillary splicing system (RNA splicing), exosome digesti〇n and non-sense-mediated degradation (NMD processing) systems. 34. The method of transferring a gene according to claim 1, wherein the mk_3〇2 mediated group 106 201009073 is caused by intracellular posttranscriptional gene silencing, translational suppression, Caused by mechanisms such as RNA interference and/or nonsense-mediated degradation. 35. The method according to claim 1, wherein the differentiated pluripotent cells of the stem cells exhibit mir-302 microRNA. 36. The method according to claim 1, wherein the differentiated multicellular cells of the stem cells exhibit embryonic stem cell marker proteins OciW, cargo £-3 and SSEA-4. 37. The method of transferring a gene according to claim 1, wherein the differentiated cells of the stem cells are cultured in a feeder-free culture environment. 38. The method of transgenic gene according to claim 1, wherein the differentiated pluripotent cells of the stem cells are cultured in a culture environment of DMEM (10% carbon adsorbed calf serum (FBS)). The method of claim 1, wherein the differentiated pluripotent cells of the stem cells are capable of differentiating into germ line-like cells. 40. The method of transgenic gene according to claim 1, wherein the differentiation of the stem cells is multi-monthly, and the 'cell is transformed into a spermatogonial cell (sp-cheer ^(10)匕-this e ceu) . 41. The method according to claim 1, wherein the differentiated pluripotent cells of the stem cells are capable of differentiating into fibroblasts (10) (4) staining e ceU). 42' The method of transgenic as described in the claim, wherein the differentiated pluripotent cells of the stem cells are capable of differentiating into a ytele cell. The method of transgenic gene according to claim 1, wherein the differentiated multi-month b-cell of the stem cell differentiates into an embjyoid b〇dy-like colony. 44. The method of transferring a gene according to claim 1, wherein the gene silencing effector of the mir_3〇2 is homologous to mir-93, mh>367, mir-371 'mir-372' mir-373 and mir- 520 and specific calibration genes. 45. The method according to claim 1, wherein the differentiated pluripotent cells of the stem cells are selectively isolated by using mir-302 microribonucleic acid as a screening target. 46. A gene-recombinant nucleic acid compound for inducing mir_3〇2-mediated gene silencing effect of an intron comprises: at least one intron comprising a gene silencing effector of a class mir_3〇2, such a mir -302 gene silencing effector is ligated by at least one exon, wherein the intron can be excised and isolated from the exon for inducing a mir-302 mediated gene silencing effect, the exons They are linked to each other to form a gene having a protein expression function. 47. The recombinant nucleic acid compound according to claim 46, wherein the intron comprises: (a) an intron meson having a gene silencing effector of the mir_3〇2; (b) a five-terminal splicing site And a three-terminal splicing; (c) a branch point zone; and (d) at least one bite zone. The recombinant nucleic acid compound according to claim 47, wherein the intron 201009073 comprises a hairpin-like nucleic acid sequence, the sequence is homologous to SEQ.ID.N0 .1 or the bent structure of SEQ.ID.N0.2. 49. The recombinant nucleic acid compound of claim 47, wherein the intron comprises a nucleic acid sequence comprising a homologous or complementary or both in the manner of SEQ. ID. N0.3. 50. The recombinant nucleic acid compound of claim 47, wherein the intron comprises a hairpin-like precursor microRNA (pre-miRNA) sequence comprising a nucleic acid sequence And is selected from the group consisting of SEQ.ID.N0.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, and SEQ.ID.N0.13 〇51. a recombinant nucleic acid compound, wherein the intron is incorporated into the intron via at least one restriction enzyme cleavage site selected from the group consisting of Aatll, AccI, AflII/III, Agel 'Apal/LI, Asel, Asp7181, BamHI, Bbel, BcWII, Bglll, BsmI, Bsp 1201, BspHI/LU111/1201, Q BsrI/BI/GI, BssHII/SI, BstBI/Ul/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecll36II, EcoRI/RII/47III, Ehel, FspI, Haelll, Hhal, HinPI, Hindlll, Hinfl, Hpal/II, KasI, Kpnl, Maell/III, Mfel, Mlul, MscI, Msel, Nael, Narl, Ncol, Ndel, NgoMI, Notl, Nrul, Nsil, Pmll, PpulOI, PstI, PvuI/II, Rsal, SacI/II, Sail, Sau3AI, Smal, SnaBI, SphI, SspI, .StuI, Tail, TaqI, Xbal, melon/, JTma/ and the cut position of the above cut. The recombinant nucleic acid compound according to claim 47, wherein the branch point 109 201009073 region comprises a branch point, and the branch point is an adenosine (A) and is located in a nucleic acid sequence 'the nucleic acid sequence Contains or is homologous to SEQ. ID. N0.6. The recombinant nucleic acid compound according to claim 47, wherein the branch point region comprises a branch point ' and the branch point is an adenosine (A) and is located in a nucleic acid sequence comprising at least one It is homologous to 5'-TACTAAC-3, an oligonucleotide motif. The genetically modified nucleic acid compound according to claim 47, wherein the multi-sucking region comprises a high-density Thymine and Cyt〇sine nucleic acid sequence, the nucleic acid sequence comprising Or homologous to SEQ. ID. N0.7 or SEQ. ID. N0.8. The recombinant nucleic acid compound according to claim 47, wherein the five-terminal splicing comprises a nucleic acid sequence comprising or homologous to SEQ.ID.N0.4. 56. The recombinant nucleic acid compound of claim 47, wherein the five-terminal splicing comprises a nucleic acid sequence' which comprises or is homologous to 5'-GTAAG-3. 57. The recombinant nucleic acid compound of claim 47, wherein the three-terminal splicing comprises a nucleic acid sequence comprising or homologous to SEQ. ID. N0.5. The genetically modified nucleic acid compound according to claim 47, wherein the three-terminal splicing comprises a nucleic acid sequence comprising or homologous to 5'-CTGCAG-3. 59. The recombinant nucleic acid compound according to claim 46, wherein the gene silencing effector of the mir_3〇2 comprises a nucleic acid sequence comprising homologous or complementary or both in a manner of SEQ.ID. N0.3. The genetically modified nucleic acid compound according to claim 46, wherein the gene silencing effector of the mir_3〇2 comprises a nucleic acid sequence comprising 110 201009073 SEQ.ID.NO.10, SEQ.ID.NO. Ll, SEQ.ID.NO.12 and / or SEQ.ID.NO.13. The genetically modified nucleic acid compound according to claim 46, wherein the gene silencing effector of the mir_3〇2 comprises a nucleic acid sequence comprising SEQ.ID.N0.9. 62. At least one embryo-like differentiation pluripotent stem cell is produced by the method of claim 1. 63. The embryo-differentiated pluripotent stem cell of claim 62, wherein the ovation differentiates the pluripotent stem cell as an integral cell or a portion of the organelle, and the donor cell nuclear transfer technique (SCNT) is applied. 64. The embryo-differentiated pluripotent stem cell of claim 62, wherein the embryonic differentiation pluripotent stem cell comprises a cell extract comprising at least one organ of the embryonic differentiation pluripotent stem cell Or cytoplasm. 111