TW201009074A - Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents - Google Patents

Abstract

The present invention generally relates to a method for developing, generating and selecting tumor-free embryonic stem (ES)-like pluripotent cells using electroporation delivery of an inducible tumor suppressor mir-302 agent into mammalian cells. More particularly, the present invention relates to a method and composition for generating a Tet-On/Off recombinant transgene capable of expressing a manually re-designed mir-302 microRNA (miRNA)/shRNA agent under the control of doxycyclin (Dox) in human somatic/cancer cells and thus inducing certain specific gene silencing effects on the differentiation-associated genes and oncogenes of the cells, resulting in reprogramming the cells into an ES-like pluripotent state.

Description

201009074 VI. Description of the Invention: [Technical Field of the Invention] The present invention generally relates to a method and method for developing, generating and screening embryonic stem (ES) pluripotent cells free of tumors, which are used in Gene transfer performance of recombinant tumor suppressor microRNA (miRNA) or small hairpin RNA (shRNA) reagents in one of these cells of interest. More specifically, the present invention relates to a gene transfer method for generating a non-naturally occurring recombinant intron and a composition thereof, and consisting of an inducible nucleic acid which can be spliced and processed in a mammalian cell. Formation of small ribonucleic acid _ VIII) gene silencing effectors (such as miRNA precursors (pre-miRNA) and / or shRNA), and then induce specific gene silencing effects in genes involved in development and cell differentiation and oncogenes 'leaving to convert these cells into an ES-like pluripotent state. Preferably, the mini-RNA gene silencing effectors include miRNA-like tumor suppressor miRNAs such as: mir-302a, mir-302b, mir-302c, mir-302d, and their artificially reprogrammed shRNA homologous derivatives The object is combined with it. In other words, the pluripotent stem cells thus produced can be cultured under cell culture conditions without feeder cells (feeder_ftee). Such cells of interest include somatic or cancer cells isolated in vitro (ex vivo, ex Wvo, and/or in vivo v/vo). [Prior Art] Recent studies in human stem cells have been shown in One of the highly desirable potentials of transplantation therapy. However, such sources for the selection of human stem cells are limited and extremely difficult to control their purity and quality. In 1998, James Thomson et al. (eg: 201009074 US Patent No. 5,843,780) No. 6,200,806, 7,029,913, and 7,220,584) isolate the first human embryonic stem (ES) cell line from late blastocysts of human embryos (Thomson et al., (1998) 282: 1145-1147 The HI and H9 cell lines were obtained from two typical cell lines of such isolated human ES cells. Two years later, Gearhart et al. (e.g., U.S. Patent Nos. 6,090,622, 6,245,566, and 6,331,406) A method for isolating ES-derived progenitor cells from post-blastocyst human embryos has also been developed. Because of the manner in which these ES cell isolation methods must destroy the original embryo, many ethical and human concerns have arisen. It is suspected that these batches of cells are used for clinical treatment. In recent years, 'treatment safety and the use of informed consent have also received attention. For example, because ES cell growth needs to be surrounded by surrounding "lacking cells" fibroblasts Some of the undetermined factors of release, all human ES cells are preferably grown on a layer of mouse or human fibroblasts (Reubin ff et al., U.S. Patent No. 6,875,607). However, these fibroblasts have different surface antigens that generally contaminate the purity of the ES cell antigen and can cause immune rejection in patients. Although some culture conditions for non-mammalian cells have been developed, none of these non-dairy cell methods maintain this undifferentiated Es cell state for a prolonged period of time. Unfortunately, human ES cell lines do not achieve 100% population purity under any of the currently available culture conditions. Even under optimal conditions of mammalian cell culture, some (about 5% - 1 〇 / / or more) ES cells tend to differentiate into other tissue cell types and lose their stem cell properties. One of the most commonly observed cell types from 201009074 of these ES cells is the ® teratoma. Teratoma is a tumor commonly obtained from human germline cells that contains multiple tumor cell types like embryonic endoderm, mesoderm and ectoderm. Therefore, how to avoid cell contamination of mammalian cells, increase stem cell purity and reduce the risk of tumor formation are the three main subjects of stem cell research. In 2006, Takahashi and Yamanaka introduced induced pluripotent stem cells (iPS cells) (Ce//126: 663-676). By using four transcription factor genes (〇咖), c-Myc and 尤/月] retroviruses into mouse fibroblasts, Takahashi and Yamanaka successfully transformed these fibroblasts in vitro and Transformation into an ES-like iPS cell line. The success rate of this method is estimated to be less than 0.002% _2% in all of the tested cell populations. At 2 and 7 years, the genetic and behavioral characteristics of these ips cells were observed to be similar to those of these mouse ES cells (Okita et al., (2007) Ato/彤 448: 313-317; Wemig et al. 2007) Garten 448: 318-324). At the same time, Yu et al. used a similar method, but in another group of parts, the novel iPS cell line of human-maternal cells from Weilin, including: (10), s〇x2, running, etc. , (2007) 3⁄4 (10) 318: 1917_192 〇). However, the method of Yu is mixed.

The Takahas (4) method is inefficient. The advantages of these sputum cell applications show that the technique of co-nuclear nuclear transfer (axis a-udear transfer, SCNT) not only solves the ethical problem of the original ES cell method, but also provides a potential therapy for the patient" 201009074 (Meissner et al. (2006) iVaiWre 439: 212-215). This iPS type SCNT treatment has been tested to treat squamous cell anemia in a gene transfer mouse model (Hanna et al., (2007) * SW (9) ce 318: 1920-1923). However, there are still two unresolved problems; the transcription of a line of retroviruses uses the use of its second lineage oncogenes (eg c-field ^ and especially). Retroviral infection is the only effective means to transfer four large transcription factor genes into the target cell; however, random insertion of multiple retroviral vectors into the cell genome of the G may also affect other non-target genes. . This is questionable because retroviral insertions due to uncertainty often cause cell variability, especially when one or more transgenic lines are oncogenes. How to avoid the formation of tumors by iPS cells induced by these transcription factors is currently incomprehensible. Another disadvantage of lPS cells is their heterogeneity. To generate an iPS cell, at least three or more different transcripts must be inserted into the mono-cell genome by anti-viral infection. However, because of the different ratios of delivery efficiency and insertional variation of different retroviral transfection genes, multiple retroviral insertions typically result in a diverse combination of transgenic genes in the host cell genome. Only cells with appropriate ratios and numbers of these transcript gene genes can become iPS cells with good pluripotency. Why does this iPS cell represent only 2%-2% of the entire cell population after retroviral insertion, while other cell lines that account for more than 98% of these cells are complex with uncertain combinations of transgenic cells. Transformed. In order to collect pure-post cells with the correct combination of transgenic genes, a series of cell screening programs (ShinyaYam domain a, No. 7, No. 255) are required. 201009074 Although the mechanism of cell rafting is not clear, this technique does not require multiple transcriptional regulators to hybridize the gene and to deactivate the 〇ct4~Sox2~c-Myc~Klf4 4 〇ct4~Sox2 The combined effect of ~Nanog-~LIN28 m is in direct contact with the secret gene; the fine, solution effect, the overall developmental message necessary to eliminate somatic differentiation has not been determined. In addition to the gene. (10), all other transgenic genes used in the iPS method are actually included in some of the initial acid wipes. The cell is transformed into an ES-like state by placing the material development message at the 'coordination or disruption of such developmental messages to somehow terminate cell ❿ differentiation'. This is an unnatural mechanism and can involve uncertainties like cell variability and tumor formation. Experiments on somatic cell age (SCNT) have been shown (4) The cytoplasmic heterozygosity of the scorpion blast cells can form pluripotent stem cells, which indicate some maternal elements in the cytoplasm of the cells, but not in the nucleus. Transcription factors play an important role in nuclear transformation (Simc) nssc)n (IV) Gurdon, (2004) 〇// Ship 6 : 984-990). The natural fertilization before the mulberry embryo and the early mating scorpion' maternal elements are responsible for the regulation and maintenance of normal stem cell regeneration and pluripotency, which is completely free of tumor formation risk. This is why the embryonic cell lines of the 32_64 cells (mulberry embryos) are all identical and versatile. Maternal elements are produced during oogenesis and are present in the mature oocytes required during the initial embryogenesis. A mouse oocyte lacking Dicer (a ribonuclease required for microribonuclear biosynthesis) stops the meiotic division of meiosis, which is indicated in the microribonucleotide oocyte One of the main maternal elements 201009074 (Murchison et al. (2007) Gewej Dev. 21: 682-693). In murine oocytes, ribonucleic acid occupies a large part of the maternal element, which is equivalent to about 45% of all transcripts of the gene (Stitzel et al., (2007) 3⁄4ce 316: 407-408). During maternal-germline transformation, these maternal RNAs degrade rapidly, and transcription of the germline gene is initiated very early in the 2-4 cell phase and produces messages for embryonic development (O'Farrell et al. Person, (2004) Cwr·〇〇/. 14 : R35-45). It is conceivable that many of these maternal rna are inhibitors of these germline genes to synchronize developmental messages during the earliest stages of embryonic development and to maintain pluripotent/pluripotent ES cell regeneration. Therefore, the 'parental version' may be one of the main maternal elements necessary for ES cell maintenance and regeneration. In summary, the natural mode of ES cell maintenance and regeneration relies on certain maternal RNAs (as inhibitors) rather than the four transcription factors (as activators) used in ips technology. In order to generate ES-like pluripotent cells that mimic the natural mechanisms of Es cell maintenance and regeneration, we are in desperate need of a new strategy to identify and evaluate the function of these 母 maternal catchers. The identified maternal RNA can be delivered into human adult stem cells or somatic cells to maintain the characteristics of the stem cells or to convert the somatic cells into a class of ES states, or both. Therefore, it is desirable for us to use an effective, simple and safe method and reagent composition for the production of ES-like pluripotent cells. SUMMARY OF THE INVENTION The present invention provides a method for converting at least - (iv) milk animal cells into at least one pluripotent stem cell. The Finance Act contains _: provides at least one cell 201009074 matrix, which represents a plurality of mir-302 target cell genes; provides at least one recombinant nucleic acid composition that can be transmitted, transcribed, and processed into mir- in the cell matrix 302 homologous at least one gene silencing effector; and in the case where the cellular genes indicated by mir_3〇2 are inhibited, the cellular matrix is treated with the recombinant nucleic acid composition. In other words, the present invention provides a method for developing, producing, and screening embryonic stem (ES)-like sputum, which allows ectopic expression of recombinant micronuclease nucleic acid (miRNA) reagents, such as mir-302a. , mir-302b, mir-302c, mir-302d, and their artificially relocated miRNA precursors (pre-miRNA) and/or small hairpin homologous derivatives and compositions thereof. The design of the non-naturally occurring/artificial/artificial mir_3〇2 reagent includes mismatched and perfectly matched (small-hairpin-type RNA (shRNA) and/or small interfering RNA (siRNA) homologous derivatives or gene groups (permatched) ) constructs 'all of which can improve target specificity and reduce the number of mir-302 required for gene transfer and gene silencing (copy number (copy_beri〇 natural microribonucleic acid (miRNA) usually is about 18-27 Nucleotides (nt), and can directly degrade their target message RNA (messenger RNA, _Α) according to their mutual complementarity or inhibit the translation of the target _ 八八. mir-302 family (mir-302s) A group of highly homologous intergenic miRNAs that share more than 89% of the same heterogeneity and are consistent among all mammals. The Mir-302 family consists of four members, which are shared as ## (non-coding) The RNA gene group, including: mir_3〇2b, her cum c, mir-302a, mir-302d, and mir-367, which are connected in a five-terminal to three-terminal direction (Suh 201009074 et al. (2004) Dev· and ·〇/· 270 : 488-498). Although the mir-367 and mir-302 families are common Now, because of its distinctly different genetic systems against different groups of seed motifs, the seed motifs are actually different from each other. They are found in many mammalian early zygote and embryonic stem (ES) cells. The performance of the mir-302 family is extremely high (Tang et al., (2007) Dev. 21: 644_648; Suh et al., (2004) Z) ev···〇/· 270: 488-498). The ES cells are the most abundant in expression and rapidly decrease after cell differentiation and/or proliferation. Mouse ❹ oocytes lack the consistency required for Dicer's miRNA biosynthesis. Ribonuclease, therefore, these oocytes stay in the first stage of meiosis (meiosis I), indicating that these miRNAs play a decisive role in egg production (Murchison et al., (2007) Gewes Dev. 21: 682-693). In addition, miRNAs have a small RNA-inhibiting property that inhibits translation of a gene with a high complementarity (Bartd, DJP. (20()4) Ce//116 _· 281- 297), the mir-302 family may be responsible for avoiding ES fine cells in early embryonic form Main matrilineal of any possible premature differentiation during suppressor. These findings suggest that the mir-302 family plays an important role in the maintenance and regeneration of normal ES cells. All mir-302 members share an identical (100%) sequence (including the complete seed motif) in the first seventeen (17) nucleotides of its five ends, as well as its 23th nucleotide acid mature miRNA sequence. It has an overall: 83% _96% homology. The seed motif is located in the first eight nucleotides of the five ends of the mature miRNA sequence, which determines the binding specificity and efficiency between the miRNA and its target gene. According to the link to 201009074

Sanger miRBase::Sequences website (http://microma. sanger.ac.uk/) database "TARGETSCAN" (http://www.targetscan.org/vert 42/) and "HCTAR-VERT" (http: //pictar.bio.nyu.edu/cgi-bm/PicTar-vertebrate.cgi?) The program's predictions are directed against almost the same cellular genes, including more than 445 identical genes in humans and mice. In addition, mir-302 shares certain overlapping genes with mir-93, mir-367, mir-37, mir-372, mir-373, and mir-520 family members. Most of these target gene developmental messages and transcription factors are involved in the initiation and/or promotion of lineage-specific cell differentiation during early embryogenesis (Lin et al. (2008b) Xiao Yu 14: 2115-2124 ). Many of these genes are also well known to be oncogenes. Thus, the function of the 'mir-3〇2 family is more likely to inhibit the development of developmental messages and differentiation-associated transcription factors, rather than establishing transcriptional stimuli on certain embryonic message transmission pathways as in the previous iPS method. In addition, due to the developmental messages of these targets and the transcription factor-related oncogenes of differentiation, the mir-302 family can also act like a tumor suppressor to prevent normal 0 ES cell regeneration bias and form tumors. In other words, the present invention provides a method for generating ES pluripotent cells free of tumors. For example, insulin-like growth factors (IGF) are used for potential developmental messages specific to the differentiation of neuronal cell lineages. These messages are based on Ras/Ra/, '''''' The mitogen-activated protein kinase (MAPK) pathway or the signal transduction pathway via phospholipidlinosital 3-kinase (PI3K)/Akt. The same message transmission path is also associated with many tumor/cancer transformations, such as brain tumors, breast cancer, lung cancer, prostate cancer, and cutaneous melanoma. The present inventors have found that the IGF receptor (IGFR>Ras/PI3K message transmission pathway has a strong target of more than eighteen member lines of the mir_3〇2 family, indicating that there is a very tight blockade in mammalian oocytes and ES cells to prevent Neuronal cell differentiation. Similar mir-302 family inhibitory effects were observed in many other various cell lineages. Based on the above evidence, the inventors of the present invention, the mir-302 family, are the major regulators of maintenance and regeneration of normal ES cells, It can convert differentiated somatic cells into a homogeneous ES-like state. To test the function of the mir-302 family, the inventors have developed a second type based on the natural intron miRNA biosynthesis mechanism (Fig. 1A). _ polymerase-driven (Pol-11-driven) miRNA expression system' and successfully use this system to generate natural, miRNA families and artificial shRNAs in vitro and in vivo. In a broad sense, this intron is a A non-coding sequence of a gene, including the same in-firame intron, a five-terminal non-translated region (5, _UTR; ^ three-terminal untranslated region © (3 -UTR). Our previous studies have confirmed Effective mature miR NA can be derived from these intron regions of the mammalian gene and is referred to as the intronic miRNA (Lin et al., (2003) by the coffee company. Commun. 310.754-760; Lin # A »(2005) Gene 356 : 32-38) (Lin et al. (2003) ga c/ze/« 〇/%^ and from (^〇 and 7« muscle 310: 754-760; Lin et al (2005) Ge 356: 32-38). Since about 50 〇/〇 of mammalian miRNAs are present in these introns of protein-coding genes, intron miRNAs are a common phenomenon in mammals. You are a general practitioner, (2〇〇4) 13 201009074, 14 : 1902-1910). As shown in Figure 1A, the intron-claw A biosynthesis system is mediated by a second-type RNA polymerase. The binding interaction between pre-mRNA transcription and intron splicing/resection (nascent Pd-II-mediated) occurs in certain nuclear regions of the sylvestre-stained cytoplasm (Ghosh et al. (2000)^4 6 : 1325_1334 ; Lin et al., (2008a) price fusion - Noga (10) fine ce 13 : 2216-2230). These miRNAs are in the precursor transcriptional molecules of their host genes (pre-mRNAs). Grab the second type Octazyme (Ροΐ-Π) is transcribed and spliced with a splicing and other endonuclease such as RNasein to form mature mjRNA (Lin et al., 2003;

Danin-Kreiselman et al. (2003) Mo/ Ce//11: 1279-1289); however, such a procedure may not be required (Ruby et al., (2〇〇7) 448: 83-86) ° The miRNA biosynthesis system is tightly regulated by multiple intracellular monitoring systems, including: second RNA polymerase (Pol-II) transcription, RNA splicing, exosome treatment, and nonsense-mediated RNA decay. 'NMD). In other words, the miRNA gene silencing effector is released by an intracellular mechanism. The mechanism is selected from the group consisting of RNA splicing, exosome processing, nonsense mediated RNA degradation, and combinations thereof. Due to this highly intracellular monitoring, RNA oversaturation problems found in other shRNA/siRNA expression systems can be avoided', resulting in a more efficient, target-specific and safer gene silencing effect on the target gene (Lin et al., 2008a) ). By simulating the natural intrinsic pathway (Fig. 1A), the inventors invented a novel intron expression system to transcribe a recombinant transgene of one of red-shifted fluorescent protein (2010) (red-shifted fluorescent protein, and GFP), It is called a splicing-competent intron (§?^4ζ·) that contains a silent effector that produces an intron miRNA and/or shRNA-like gene. The SpRNAi is transcribed together with Pol-II in the pre_mRNA of the §? Xiao gene, and is cleaved by RNA splicing. Then, the splicing process is further processed into mature gene silencing effectors, such as natural miRNAs and artificial shRNAs, which cause a specific posttranscriptional gene silencing (PTGS) effect on the target gene. At the same time, after intron splicing, the exon of the known gene transcriptional molecule is linked together to form a mature mRNA for translation of the RGFP-tagged protein, which can be used to recognize miRNA/shRNA expression. In other embodiments, some functional protein exons can be used in place of RGFP to provide additional gene functions, such as embryonic stem (ES) gene markers for somatic cell transformation. In other words, the 〇 gene silencing effector can induce an intracellular gene silencing effect via post-transcriptional gene silencing, translational repression, RNA interference', and/or nonsense-mediated degradation. Since more than 1000 natural miRNA species are currently found in vertebrates, their functions are not yet known, and more new miRNAs are still being recognized, the intron miRNA expression system of the present invention can be used both in vivo and in vitro. A powerful tool for testing the function of these miRNAs. The intron comprises a plurality of identical nucleotide components comprising: a five-terminal splice position (5'-31) 1 to 3 also; 8 £ (^10 to 0.4), a branch point motif 15 201009074 (branch-point motif 'BrP; SEQ.ID.NO.6), a poly-pyrimidine tract (ΡΡΤ; SEQ.ID.N0.7 and SEQ.ID.N0 8), and a three-terminal Splice position (3'-3|51 to 3 also; 8 £(^10.]^0.5). In addition, a small hairy miRNA or s_A precursor is inserted in the five-terminal splice site and the branch point motif The intron of this part usually forms a set of lariat structures during RNA splicing and processing. In addition, the three ends contain a multiple translation stop codon region (T codon) to increase the inclusion. Correction of daughter RNA splicing and nmd treatment. When this T codon appears in a cytoplasmic mRNA, it emits a message that activates the NMD system to degrade any unconstructed RNA accumulated in the cell. However, it retains a high degree of construction. Sh]RNA and precursor miRNA (pre-miRNA) are further digested by gamma (9) to form mature siRNA and miRNA, respectively. For gene transfer performance, we are in the gene (SEQ .ID.NO.22) artificially merged with the restriction bit (208th nucleotide). This forms a recombination of the transgenic gene. The enzyme cuts the rupture of the Drall cleavage at each end. The AG_GN nucleus with three concave nucleotides is micro-cut π, which will be divided into a five-terminal splice position and a two-terminal splice position after insertion, because this intron insertion interrupts the 70 integrity of the RGFp protein. It can be restored by intron splicing, so we can determine the release of the intron miRNA/s_ and the maturation of the capture mRNA via the red RGFP present in the transfected cells. It also includes a number of exonic splicing enhancers (ESEs) to increase the correctness and efficiency of rka splicing. 201009074 In the specific embodiment of the 杈 之 , , , , — — — — — mi mi mi mi mi mi mi mi mi mi 2A and 2B), which improves the control of the degree of secretness of the melons in the body and in vitro. This view is not only better than the safe electroporation/microinjection method (4), the sword (4) (10) Thin gene delivery' to replace the tumor-prone retrovirus infection The possibility of excessive accumulation of (10) eight in these transfected cells was also avoided. According to this improvement, the inventors have succeeded in generating various mir_3〇2 transducing pluripotent stem pluripotent stem, mirPS) cell lines, It is derived from normal epidermal skin cells (mirPS-hpESC), normal hair follicle cells (mirPS-hHFC), human primary culture ❻ & machi and cancer breast cancer MCF7 (mirPS-MCF7), prostate cancer pc3 (mirPS-PC3) And skin melanoma Colo 829 (mirPS-Colo) cells. As shown in Figures 2A and 2B, the inventors first incorporated the artificial mir-302 family pre-miRNA/s_A structure (Fig. 3B) into the intron insertion site of the cadaveric gene (ie: pVM/limitation/10 colonization). And then insert the transgenic gene into the deoxygenated hydroxyl (D〇x) _ to induce the multiple-selection of the vector--(3)- still* vector (ie: C7a/restriction), thus forming 〆<^- 〇 «-ir*S-mz> 30Zs transgenic gene vector (Fig. 3A). In other words, the recombinant nucleic acid composition (e.g., * and the like) comprises a drug-evokable gene expression vector. In addition, the GFi3 can also be incorporated into a gene expression vector selected from the group consisting of a plastid, a viral vector, an inverted vector, and combinations thereof. The (4) jGFP-transgenic gene line is flanked by a 370 base pair bp homologous region for recombinant insertion into the target position of the 17 201009074 host cell genome. In terms of gene transfer delivery, the vector (10_3〇μβ) is mixed with the host cells (2〇〇2〇(8)) in a hypotonic buffer (400 μΐ; covered tube is hungry)*, and Electroporation was performed at 400-450 volts for 1 〇〇 microsecond (ah) to deliver the transgene into the host cell genes. After 72 hours, both flow cytometry and anti-RGFP and anti-Oct3/4 monoclonal antibodies were used for isolation and collection of cells successfully transfected with mirPS (Fig. 3C). The success rate of this novel mir_3〇2 family gene transfer method is over 91%, which is much higher than the success rate of 0.002%-2% found in the original milk method 该. All sequences of the artificial mir_3〇2 family are based on Sanger miRBase: : Sequence synthesis of the Sequences program and chemical synthesis. /?7^-〇7^ Vector encodes the CMV-driven (TSV-driven tTS inhibitor gene) to deactivate the transgenic gene-CW promoter. When deoxyhydroxyl (Dox) occurs, The suppression function of tTS is

Dox neutralizes, thus exhibiting a transgenic gene and its coding as a family. In another preferred embodiment, the present invention provides a genetic engineering method for constructing a human jade/artificial & 胤^ intron comprising at least one desired meson Generating miRNA, shRNA and/or antisense RNA gene silencing effectors of the mir_3〇2 family or mir_3〇2. The thinning system is formed by synthesizing the link of oligonucleotide elements for RNA splicing, and the synthetic acid-like elements are: five-terminal splice sites, BrP, PPT, three-terminal splice sites, and some Link the oligonucleotide. An oligonucleotide synthesizer can chemically synthesize and link these elements. For the change of 2010 20107474, the intron (such as: the cerebral palsy is synthesized by a chemical synthesis method. In its tamping of the thin towel, the fresh slave chain can be transferred by the transfer and joints. In other words, the intron (eg, Liu can also be formed by recombination of nucleophilic acid. The resulting intron (eg, can be transfected into the cells of interest or Further incorporated in the host gene for expression in conjunction with the gene transcription molecule (p_RNA). Thus, the recombinant nucleotide composition of the present invention further comprises a recombinant intron encoding at least one such as mir_3〇2 ❹ Gene silencing effectors. In general, such intron insertion methods include enzyme restriction/selection, homologous DNA recombination, transposon merging, skip gene δ, retroviral infection, and combinations thereof. It is selected from the group consisting of fluorescent protein (GFP) marker gene, embryonic stem (ES) marker gene, luciferase, _2 lactose expression gene, viral gene, transposon, sea hopping gene, artificial recombinant gene and natural cell gene. In other words, the recombinant nucleic acid The adult further comprises a plurality of exons selected from the group consisting of a fluorescent protein marker gene, a luciferase gene, a lac_z ® lactose expression gene, an embryonic stem cell marker gene, a viral gene, a bacterial gene, a cell marker gene, a skip gene, and a transgene. The position, and combinations thereof. Without limitation, the present invention preferably uses a modified red fluorescent protein (RGFP) gene for indicating the expression of the mir-302 family. In one aspect, the intron comprises a five-terminal Splice site, which is associated with 5'-GTAAGAGK-3, (SEQ.ID.N0.4) or GU(A/G)AGU motif (SEQ.ID.NO.38) (ie: 5,-GTAAGAGGAT-3' (SEQ.ID.N0.37), 5 '-GTAAGAGT-3 '(SEQ.ID.NO.39), 201009074 and 5 '-GTAGAGT-3 '(SEQ.ID.NO.40) S^GTAAGTMSEQ.ID .NOA)) is a homologous, and its three-terminal three-terminal acceptor splice site, the three-terminal receptor splice site and GWKSCYRCAG (SEQ.ID.N0.5) or CT (A/G)A(C/T)NG base; (ie: 5'-gatatcctgc Α(}_3,(8Εαιο.Ν〇42),

Any of 5'-GGCTGCAG-3' and 5'-CCACAG-3')) is homologous. In other words, the intron of the recombinant nucleic acid composition comprises a five-terminal donor splice site (5, _d〇nOT

Splice site), intron insertion, branch point motif, polypyrimidine segment, and tri-terminal acceptor splice. In addition, the branch point sequence is located between the five-terminal and three-terminal splice sites, and comprises a motif homologous to the 5'-TACTWAY-3, (SEQ.ID.N0.6) motif, like: 5 '-TACTAAC-3, and 5,-TACTTAT-3,. In other words, the branch point motif comprises or is homologous to the SEQ.ID.N0.6 sequence. The glandular nucleus "A" nucleotide in this branch point sequence forms part of the intron in almost all potential introns, by adenosine synthase and splicing of cells (2, -5)

(2'-5') Linked to the horseshoe intron RNA. In addition, the polypyrimidine moiety is located near the branch point and the three-terminal splice site, and comprises 5,-(TY)m(C/-)(T)nS(C/-)-3, (SEQ. ID.N0.7) or a high T or C content sequence homologous to either the 5'-(TC)nNCTAG(G/-)-3' (SEQ.ID.N0.8) motif. The symbols "m" and "n" indicate a plurality of repetitions, and ^ is preferably equal to 1 to 3 and the number of n is equal to 7 to 12. The symbol "-" refers to a nucleotide that can be skipped in this sequence. There are also linked nucleotide sequences that are used to link all of these synthetic intron elements. According to 37 CFR 1.822, use 20 201009074 for fresh acid and /1⁄2 acid phase (4) and pick _, symbol ^ refers to adenine (4) or sakisaki) / 屎 u u), symbol κ refers to bird material (6) - or chest ♦ (7) / uracil (υ), symbol s refers to cell sensitivity (c) or guanine (9), symbol Y refers to cell money (c) or chest T) / urine side U), symbol R refers to adenine (A) or guani G), and symbol N refers to adenine (4), cytosine), bird 11 (G) or thymidine (T) / uridine (9). The multi-valve 因 因 因 / / 表现 表现 表现 在 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 In other embodiments' multiple gene silencing effectors can be produced by intron mesons. In other words, the intron insertion site contains at least one gene silencing effector homologous to Qin 3〇2. For example, it has been reported that the ectopic expression of the anti-Qing P-containing pre-miRNA intron in zebrafish produces two different lengths of Tibetan dishes, called miR-EGFP (282/3〇〇) and 疋疋卿(9) (iv) 2), which indicates that the known meson can produce multiple gene silencing effectors (Lin et al., 2005). ® is heterozygous for the fine transcriptional molecule (i.e., mRNA) in which the β-intron-based β-subunit is silent to form a double-stranded siRNA for inducing a secondary muscle dry effect (IV) Ai). Because these gene silencing effects are continually produced by the gene transfer vector, they will alleviate concerns about rapid degradation in vivo. The advantage of this strategy is the stable transmission of its long-term gene silencing efficacy via the vector-type gene transfection or viral infection. In addition, the present invention can produce related gene silencing effectors under the control of a specific RNA promoter, including: miRNA, S_A and siRNA, the specific RNA promoter is selected from the 21st 201009074 type 2 RNA polymerase (Ρ〇1- ΙΙ), viral polymerase, type III RNA polymerase (Pol-III)' and tetracycline responsive element-controlled RNA polymerase (TRE) promoter. These viral promoters are Pol-II RNA promoters from cytomegalovirus (CMV), retroviral long-terminal region (LTR), hepatitis B virus (hepatitis B virus). , HBV), adenovirus (AMV) and adeno-associated virus

Separated from (adeno-assodated virus, AAV). For example, the lentiviral LTR promoter is sufficient to provide more than 5 copies of pre-mRNA transcriptional molecules per cell. A drug-sensitive inhibitor can also be inserted prior to the viral polymerase promoter (ie, to control the transcription rate of the gene's silent effector. The inhibitor can be inhibited by a chemical agent or an antibiotic selected from the group consisting of chemical agents or antibiotics selected from the group consisting of Amino vinegar antibiotics (G418), tetracycline (tetraCyCiine), deoxycydin, neomycin, ampicillin, kanamycin, and derivatives thereof In other words, the performance of the recombinant nucleic acid composition of the present invention can be regulated by an antibiotic touch, such as a tetracycline derivative such as a deoxytetracycline derivative. Inventively, the desired intron meson meson is cut and released by an intracellular mechanism and then elicits a desired gene silencing effect on a particular gene target, the specific gene target having high complementarity with the meson; The squamous exon of the yang recording molecule is connected to the can-like shape to produce the white-skinned splicing mRNA. This can be selected from the red wheel 22 201009074 photoprotein (RGFP/EGFP), embryonic gene marker, Fluorescent , beta-galactosidase (lac-Z) and derivatives thereof. The expression of the reporter/marker protein can be used to identify the extent and location of the silent effector of the intron genes in the transfected cells, and It helps to confirm the resulting gene silencing effect. In other embodiments, mature mRNAs formed by the exon linkage can be used in conventional gene therapy to replace impaired or lost gene function, or to increase specific gene expression. These gene silencing effectors (homologous to mir-302) may also include antisense rna, ribozyme, short temporary RNA (stRNA), fine non-coding RNA (tiny non) -coding RNA, tneRNA;), Piwi-interacting RNA (piRNA), double-stranded RNA (dsRNA), siRNA, shRNA, miRNA, and its precursors (ie: pri-/pre - miRNA). The use of these intron gene silencing effectors is a powerful tool for quantifying unwanted genes, selected from exogenous genes, pathogenic transgenes, viral genes, mutations. Gene, oncogene, Disease-related non-coding RNA genes encode many other types of proteins as well as non-coding cellular genes. Because some natural pre-miRNA stem-l〇op structures are too large and/or too complex 'cannot match the *5 continent And GFP transgenes, the inventors typically used artificially reprogrammed tRNAmet loops (i.e., 5'_(A/U)UCCAAGGGGG_3,) (SEQ. ID. NO. 43) in place of the native pre-miRNA loops. The tRNAmet loop has been shown to efficiently assist in the export of artificially reprogrammed miRNAs from the nucleus to the fine cytoplasm of the 201009074 cytoplasm via the same Ran-GTP and Exp〇rtin-5 delivery mechanisms (Lin et al., (2005) Gewe356: 32-38). Advantageously, the present invention currently employs a pair of artificially improved pre-mir-302 loops, including: 5,-GCTAAGCCAG GC-3' (SEQ. ID. N0.1) and 5,-GCCTGGCTTA GC-3, (SEQ. ID.N0.2) 'It provides the same nuclear export efficiency as the native pre-miRNA' but does not interfere with tRNA output. Moreover, this improvement enhances the formation of mir-302a-mir-302a* and mir-302c-mir-302c* duplexes, which increase the overall functionality and stability of the mir-302 family. The design of these novel pre-miRNA loops is modified by the combination of the tRNAmet loop of mir_3〇2b/mir-302a and the short stem-loop, which is highly expressed in embryonic stem cells and not in other differentiation. In tissue cells. Therefore, the use of these recombinant/artificial/artificial pre-miRNA/shRNA loops in the mir_3〇2 family will not interfere with the natural miRNA pathways in our bodies and is therefore less cytotoxic and safer. The mir-302 pre-miRNA family of genes (cius (4) is formed by the synthesis and chaining/joining of synthetic mir-302 homologous derivatives, which consists of four parts in the five-terminal to three-terminal direction: mir_3〇 2a, mir-302b, mir-302c and mir-302d pre-miRNA (Fig. 3B). All of these artificially-reset mir_3〇2 miRNA/shRNA molecules have identical five in their first seventeen nucleotides.

Terminal I (eg 5'-UAAGUGCUUC CAUGUUU-3, (SEQ. ID. N0.3)). The synthetic oligonucleotides for DNA recombination of the mir-302 pre-miRNA gene group are listed below.

Acidic: including: synonymous mir-302a (mir-302a-sense), 5,-GTCCGATC (JT

CCCACCACTT AAACGTGGAT GTACTTGCTT 24 201009074

TGAAACTAAA

GAAGTAAGTG

CTTCCATGTT

TTGGTGATGG ATCTCGAGCT C-3, (SEQ.ID.NO.29); antisense mir-302a(mir-302a-antisense) , 55 -GAGCT CGAGA

TCCATCACCA

CTTTAGTTTC

AAACATGGAA

AAAGCAAGTA

GCACTTACTT

CATCCACGTT

TAAGTGGTGG GACGATCGGA C-3' (SEQ.ID.NO.30); synonymous mir-302b,5,-ATCTCGAGCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTAG CGCTA-35 (SEQ.ID.NO.31); antisense mir- 302b,5,_TAGCGCTAGC

GACTCCTACT

TTCAAAGTCA

AAAACATGGA

CAGAAAGCAC

AGCACTTACT

TTCCATGTTA

AAGTTGAAGG

GAGCGAGCTC GAGAT-3,

(SEQ.ID.NO.32); synonymous mir-302c, 5'-CGCTAGCGCT

ACCTTTGCTT

TGTGAAACAG

TAACATGGAG

AAGTAAGTGC

GTACCTGCTG

TTCCATGTTT

CAGTGGAGGC GTCTAGACAT-3, (SEQ.ID.NO.33); antisense mir-302c,5,-ATGTCTAGAC GCCTCCACTG AAACATGGAA

GCACTTACTT

CTGTTTCACA

CAGCAGGTAC

CTCCATGTTA

AAGCAAAGGT AGCGCTAGCG-3,

(SEQ.ro.NO.34); synonymous mir-302d, 5’-CGTCTAGACA

TAACACTCAA

ACATGGAAGC

ACTTAGCTAA 25 201009074

GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT

TCGACGCGTC AT-3' (SEQ.ID.NO.35); and antisense mir-302d, 5,-ATGACGCGTC GAACACTCAA ACATGGAAGC

ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT

GTTTGAGTGT TATGTCTAGA CG-3, (SEQ.ID.NO.36) (Sigma-Genosys, St. Louis, MO). In other embodiments, we can use artificially reprogrammed shRNAs to synthesize the synonymous mir-302 family, 5,-GTCCGATCGT CATAAGTGCT TCCATGTTTT

AGTGTGCTAA GCCAGGCACA CTAAAACATG

Hybridization of GAAGCACTTA TCGACGCGTC AT-3' (SEQ.ID.NO.27) with antisense mir-302 family, 5,-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3, (SEQ.ID.NO.28) To form a mir-302 pre-miRNA gene group instead of a simple intron insertion. In other words, one of the preferred gene silencing effectors is a recombinant nucleic acid sequence formed by the hybridization of SEQ.ID.NO.27 and 8(3.10.:^0.28. The mir-302 shRNA is directed to all Natural mir-302 members share more than 91% homology and label the same cellular genes in humans. About the insertion of the mir-302 pre-miRNA/s_A intron, due to the insertion of the recombination gene The cleavage position is defined by /W and Mw/restriction/selection at its five-terminal and three-terminal, respectively. The precursor meson can be simply removed and replaced by various pre-miRNA/s_A mesons (eg mir_3〇2 26 201009074

pre-miRNA/shRNA) with a cohesive end that matches the /w and Mw/restriction sites. The intron mir_3〇2 family expression system of the present invention can be used as a powerful tool for inducing target gene silencing in vitro and in vivo by altering the mesons of such introns for various gene transcriptional molecules. For length confirmation and purification of the transgenic gene, the mir-302 insertion structure (10 ng) was polymerase chain reaction (PCR) with a pair of oligonucleotide primers (ie: 5'-CTCGAGCATG) GTGAGCGGCC 〇TGCTGAA-3' (SEQ.ID.NO.23) and 5,-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3' (SEQ.ID.NO.24) were replicated at 94. (:, 52-57 ° C and then at 68 ° C for one minute each, a total of 25-30 cycles were performed. The resulting PCR product (~900-1100 bp) was chromatographed on 2% acacia, then A gel extraction kit is used for extraction and purification (Qiagen, CA). After confirming the DNA sequence, the purified transgenic gene of 5^\冰- and GF corps inserted into mir-302 is further inserted into a vector. Restriction/selection of colonies (ie, knife w/-C7a/position) to form a transgenic gene expression vector for intracellular expression (Fig. 3A). One of the following methods can be used to achieve this ; 77^-〇77-和>3like y transgene vector delivery into mammalian cells: liposomal/chemical transfection, electroporation, gene gun Gene gun penetration, transposon/retrotransposon insertion, jumping gene integration, microinjection (micr〇-injection), and retrovirus/slow 27 201009074 Viral infection (retr〇Viral/lentiviral ώΜ〇η). To avoid random transfer of gene insertion The risk of entry and cellular variation' The present inventors prefer to use an electroporation method in conjunction with homologous recombination to deliver the transgenic gene vector into the host cell of interest. For example, the Φ solution The transgenic gene line is flanked by the - 370 genomic pair (bp) homologous region, and is used to recombine the human chromophore 6 in the vicinity of the region where l〇C727977 is located. Known gene coding. It is detected that it is accurately inserted in this single position.

SpRNAi-RGFP (Wind 4A). In this case, when the d〇x is present, the SpmAi-RGFP φ turn wire and the Wei code mir_3G2 胄 械 全 全 全 杂 杂 杂 C C C C C C C C C C C C C C C C C C C C This (4) person & tested this novel method that can induce her family to be arrested in both human normal and cancer somatic cells. 'The new powder includes: normal epidermal cells, normal light hair follicle cells, cancer breast cancer MCF7, Prostate cancer PC3, and black _ c〇1〇 cells. In other words, the mammalian cell can be transformed into a pluripotent dry state selected from the group consisting of a human cell, a positively inclined cell, a human _, a sputum tumor or a cancer cell, a human hair follicle cell, Human skin cells and combinations thereof. The resulting mir-302 transduced pluripotent stem 'mirPS cells all carry only the transgenic gene or two coexisting repeat sequences at the same locus position (hid omitant) C_s) (Fig. 4A), which indicates that the entire concentration of -3〇2 can affect the survival of these mirPS cells. We have also observed that the degree of expression of the mk_3〇2 family and its marker RGFP mRNA will increase as the concentration of D〇x increases (Fig. 28 201009074 4B). The entire mu>302 concentration must be expressed more than 3 fold (10) but less than 5 fold' to trigger the conversion of the somatic cells to ES-like pluripotent stem cells. The currently available direct (exo) siRNA/s_A expression system driven by the Pol-III or CMV promoter is inoperable due to its extremely low rate of performance in hair follicles and skin cells (only a few fold increase). This may be because the template of the native mir-302 gene group is too short and structural so that it cannot be directly transcribed by the ρ〇ι_πι or CMV promoter. Accordingly, the present invention provides an inducible mechanism for controlling the degree of expression of the mir_3〇2 family in vitro and in vivo by a specific drug (ie, D〇x), which can be used as a system other than such intracellular monitoring systems. Two lines of defense. Cytotoxicity due to accumulation or supersaturation of j^a is not detected in the mirPS cells of the present invention. The present invention has employed this novel design and strategy for inducing a transgenic gene expression system, which is referred to as the mir_3〇2 family, which is expressed in human somatic/cancer cells. Such members or homologous derivatives of ❹ (mir-302s) can thus convert such somatic/cancer cells into a pluripotent state of embryonic stem-like (ES). In a preferred embodiment, the invention provides a method for inducing a heavy nucleic acid composition using a drug that can be transmitted, transcribed, and processed into a mir-302 miRNA/shKNA in a human cell. Molecules/homologous derivatives, therefore, the developmental and differentiation-related genes of mir-302 in these cells induce specific gene silencing effects, and the method comprises the steps of: a) providing: a cell matrix, which exhibits mir_3〇2 a plurality of developmental and differentiation-related genes of the family target, and u) a recombinant nucleic acid composition 29 201009074, which is a transcribed RNA encoding a plurality of non-coding mir_302 miRNAs/shRNAs or homologous derivatives thereof, the plurality of non-coding mir_3〇2 The miRNAs/shRNAs or homologous derivatives thereof are sequentially processed into mature mir-302 miRNAs/s_As or a homologous derivative thereof by an intracellular mechanism, thereby inhibiting the function of the target genes in the cell matrix; b) treating the cell matrix with the recombinant nucleic acid composition, which is treated under conditions which inhibit the function of the target genes in the cell matrix. More preferably, the drug induces a recombinant nucleic acid composition plus a 0-2 vector comprising a recombinant mir-302 family-based quinone group (mir_3〇2s; SEQ.ID.N0.29-SEQ.ID. Any hybridization of the mir-302 shRNA homologous derivative (ie, heterozygous for SEq.id N〇27 and SEQ.ID.NO.28) in the sub-transgenic gene. The cell substrate can express the mir-302 miRNA/shRNA and its underlying gene in any manner, in vitro, ex vivo, and/or in vivo. Thereafter, the cell matrix is transformed or transformed into an ES-like state, which not only presents standard ES fine paper, such as · Oct3/4, SSEA3, SSEA4, Sox2, Nanog, Liang LIN-28, and contains a high degree of desulfonyl group. A genomic body resembling a zygote genome transformed by stem cells. Applying this invention, the inventors have succeeded in achieving mir_3〇2 in seven aspects.

Pluripotent stem (mirPS) cell production: First, mirPS cell lines derived from two homologous lines of the same cell type have been generated; one line is derived from human normal hair follicle cells (hHFC) and the other is from Cancer melanoma c〇l〇 cells, both of which can form embryoid bodies in vitro (Fig. 5A-5C). Second, microRNAs (miRNA) microarrays and northern blot analyses of these mirPS 30 201009074 transcriptional molecules have been used to demonstrate improved performance in the mir_302 family (Figures 6A-6B). The third 'has been detected to improve the performance of the standard embryonic stem (ES) cell marker' includes: Oct3/4, SSEA-3, SSEA-4, Sox2 & N_g (Fig. 6B, 8B-8C and Fig. 9B). Fourth, overall genomic DNA demethylation has been observed, similar to the state of the zygote genotype following this transformation procedure (Fig. 7A-7C). Fifth, the gene expression patterns of the miRNAs of these mirPS cells have been shown to have a high similarity to the patterns of the human ES WA01 and WA09 (H9) cells (Fig. 8A and Fig. 9A). Sixth, transplanting these mirps cell-derived embryoid bodies (EB) into SCID-beige mice with insufficient immune function can form teratoma-like cysts, which contain all three embryonic germ layers. (ectoderm, mesoderm, and definitive endoderm) (Fig. 1〇). However, unlike teratomas, these tissue cysts form an excellent and clear boundary with their surrounding tissue. Moreover, the growth of these cysts in mice was stopped at about 25 weeks after transplantation. This appears to be a self-regulating mechanism that limits the random growth of these mirPS-derived EB cells in vivo. Finally, the differentiation of mirPS cells can be guided to form various body systems and germline tissue cell types, such as: neuron progenitor, chondrocytes (Cyte), fibroblasts, and fibroblasts. Spermat〇g〇nia_like prim()]: dial cell), which is treated in vitro using various hormones and/or growth factors (Fig. 11A-110). Furthermore, the present inventors have successfully used electroporation-type gene transfection to form mirPs-induced mirPs cell lines, which avoid the risk of reverse transcription 31 201009074 disease infection and cell canine changes (Fig. 2A and 2B). These findings provide strong evidence of micronuclear nucleic acid (miRNA)-induced stem cell production, in which the ectopic manifestation of the mir-30 family not only converts both adult body cells and cancer cells into an ES-like pluripotent state, but The regeneration and pluripotency of these ES mirPS cells can be maintained in the absence of mammalian cell culture conditions. Since the mir_3〇2 family can use both 91%-93% of the success rate to convert both normal and cancer tissue cells into ES-like pluripotent stem cells, the findings of this novel invention can be treated in stem cells and cancer. Both provide advantageous applications. From the above findings, the present inventors have also learned that mir_3〇2 not only greatly inhibits the expression of cydin-dependent kinase 2 (CDK^, cyclin D1 and D2) to attenuate cells. Proliferation and migration, and inhibiting the activity of MECP2 and MECP1-p66 to induce global demethylation of genomic DNA (Figures 8B-8C and Figure 9B). It is well known that the cyclin cyciin E-related CDK2 line enters the cell cycle § It is necessary, and the inhibition of CDK2 can lead to the arrest of G1 phase checkpoint, in which cyclin D1 can surpass G1 arrest in response to ❹ DNA damage. According to this principle, mk_3〇2 is used to inhibit CDK2 and cyclin CyCiinDl. Both suggest a fact that mirPS cells have a very slow rate of division. As shown in Figures 5A and 5C, the average cell cycle of mirPS cells is about 20-24 hours, which is much slower than their body/cancer equivalence. The average cell cycle of the material (about 4-6 hours per cell). Tumor/cancer cells cannot survive at such a low cell proliferation rate. By controlling the cell cycle rate, mir 302 can also affect cell survival. Thus, the results of this cancer_stem cell cycle conversion 32 201009074 have great benefits for cancer treatment. Furthermore, inhibition of [VIECP2 and MECP1-p66 activity] is consistent with the results of Figures 7A-7C, which show that malignant cancer cells are transformed into acquired Benign mirPS cells. It is conceivable that these mirPS cells obtained from patients can further help repair tissue damage in cancer. Cell genes necessary for inhibition of genomic imprinting and cell fate determination The mir_3〇2 family not only converts differentiated somatic/cancer cells into ES-like pluripotent states, but also maintains such ES status in the absence of mammalian cell culture conditions. In addition, due to CDK2, cyclin cyclin Inhibition of D1 and D2 also attenuates the cell proliferation and migration of tumor/cancer cells. The mir-302 family plays a role as a potent tumor suppressor against tumor cell growth and formation (Lin et al., 2008b). This tumor suppressor characteristic of the 302 family can assist in the generation of tumor-free induced pluripotency for clinical transplantation, stem cell and cancer therapy. According to our current findings, the mechanism by which mir_3〇2 induces mirPS cell transformation is more safe, clear and understandable than the mechanism by which these transcription factors induce cells. In summary, the novel mir_302 family expression system provides a safe and A powerful tool for the generation of novel ES pluripotent stem cells, especially native cultures derived from hair follicle cells of the body, as hair is easier to obtain. Since the intron miRNA pathway is tightly regulated by multiple intracellular monitoring systems (eg, transcribed RNA splicing, exosome processing, and nmd), this pathway is more efficient and safer than the well-known siRNA/shRNA pathways. 2〇·). The present invention has at least five beneficial breakthroughs. First, mir_3〇2 expresses the transgene 33. 201009074 can replace all four large transcription factor genes used in these prior iPS methods, which are used to generate more heterogeneous classes derived only from some patient somatic cells. ES eve month bf dry, field cell 'this can improve the patient's immune system stem cell purity and compatibility. Second, because the total length of the mir_3〇2 training lean gene is relatively small (about a thousand test bases), compared with the maximum 2% of the IPS methods, the method-transfer gene transfer Very high (the success rate is over 91%). Third, the production and culture of the mirps cells are completely carried out without the presence of mammalian cells, thereby avoiding the risk of breast cancer cell antigen contamination. Fine, this (4) does not make the ship silk, this avoids the test of cell variability and tumor formation. Finally, the present Fortune · f perforation method replaces the retroviral thorn to transmit the single-Qin 3G2 gene, which avoids the risk of insertion of the retrovirus into the host cell genome, which often leads to insertional mutations (msertionalmutagenesis). In fact, mir_3〇2 has been shown to be a potent tumor suppressor, which can even convert various tumor/cancer cells into ES-like multi-stomach stem cells (U.S. Patent Application Serial No. 12/149,725, issued to Lin et al. have). In summary, these advantages address the two main problems of the postal method, which avoids the risk of retroviral infection, oncogene mutations, and uncertain tumors. In addition to the general functions of the ES-like pluripotent cell line, the present invention includes the following methods: maintaining the culture state of no τ-free breast cells and tumor-free ES cells, avoiding cancer cell differentiation and transformation, and separating Pure or homologous stem cell population, in vitro selection and purification of adult stem cell lines, differentiation of stem cells into pure body tissues in vitro, and the production of _s pluripotent stem cells for 34 201009074 transplantation and stem cell therapy development. The present invention can also be used as a work and bean to study stem cell functions and mechanisms, or to provide compositions and methods for the specific use of the stem cells. In other embodiments, these types of ES pluripotent stem cells can be used by normal and cancer somatic cells as well as adult stem cells of mammals (such as humans, monkeys, dogs, juveniles, rats, and mice). generate. [Embodiment] ® 胄 现 现 现 现 现 现 现 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , It may represent the principles of the invention: Application. It will be apparent to those skilled in the art that various changes and modifications are within the spirit, scope, and concept of the invention as defined by the P4 claim patent. The present invention provides a novel nucleic acid composition and gene transfer method, which uses a small micro hairpin type ribonucleic acid (shRNA) which induces recombinant microRNA (miRNA) to transform genes and behaviors of mammalian somatic cells/cancer cells. The characteristics are transformed into a type of embryonic stem (ES) pluripotent state. In other words, the invention provides a method of transforming at least one mammalian cell into at least one pluripotent stem cell. The method comprises the steps of providing at least one cellular matrix 'which exhibits a plurality of cellular genes designated by mir-302 to provide at least one recombinant nucleic acid composition that can be transmitted, transcribed, and processed into a cytoplasmic matrix At least one gene homologous to mirjo is said to be treated with the recombinant nucleic acid composition under conditions in which the cellular genes are inhibited by mir_3〇2. Unlike the design of the original shRNA, the shRNA of the present invention may comprise a mismatched stem-arm similar to the natural mir-302 precursor (pre-mir-302). In addition, the shRNA of the present invention may also comprise a modified pre-mir-302 stem-loop 'e.g., 5,-GCTAAGCCAG GC-3, (SEQ.ID.N0.1) and 5,-GCCTGGCTTA GC -3, (SEQ.ID.N0.2) 'which provides the same nuclear export efficiency as the native pre_miRNA' and does not interfere with tRNA output. In other words, the gene silencing effecter comprises a sequence homologous to either the SEQ.ID.N0.1 sequence or the SEQ.ID.N0.2 sequence. Without being bound by any particular theory, 'this transformation is applied to a newly discovered mir-302-mediated gene silencing mechanism, which can be expressed by a mir-3Q2 family gene group (mir_3〇2s) or a mir- Transfection of one of the 302-homologous shRNAs is triggered by transfection of the recombinant transgene. In a preferred embodiment, the design of the gene transfer performance of the present invention is based on the pathway of natural intron miRNA biosynthesis (Fig. 1A). The present inventors have devised a novel nucleic acid composition which expresses a recombinant transgenic gene encoding a red-colored fluorescent protein (RGFP), namely: broth/V^-ieGFP, which comprises splicing via intracellular RNA and Processing mechanism to create an artificial/artificial splicing competent intron of intron miRNA and/or shRNA-like gene silencing effectors (see Lin et al., 2003, 2006a, 2006b). The design program for designing and constructing the intron and anti-SpRNAURGFP transfectase. The SpRNAi is in the pro-transcriptional molecule (pre-mRNA) of the GFP gene as 201009074

Mammalian type 2 RNA polymerase (Pol-ΙΙ) is co-transcribed and cut by RNA splicing/treatment. The spliced is then further processed into mature gene silencing effectors, such as natural miRNAs and artificial shRNAs, to trigger specific posttranscriptional gene silencing (PTGS) effects on the target gene. In this case, the invention produces a recombinant mir-302s and/or a shRNA homologous to the mir-302s. At the same time, after intron splicing, the exons of the gene transcriptional molecule are linked together to form a mature GFP mRNA, which is used to translate into a red fluorescent marker that is useful for identifying the desired miRNA/shRNA expression. protein. In other embodiments, some functional protein exons may be used in place of RGFP to provide additional gene function ', such as .ES cell marker genes Oct4, Sox2, Nanog, LJN-2.8, SSEA3 anti-SSE44. In another preferred embodiment, the present invention provides a novel method for transforming somatic animal somatic cells/cancer cells into ES-like pluripotent stem cells (Fig. 2A and Fig. 2B) using a separate drug Inducing a nucleic acid composition that can be transmitted, transcribed, and processed into mir-302 miRNA/shRNA molecules/homologues in mammalian cells; thus, the mir-302 is labeled in such cells Inducing specific gene silencing effects in genes involved in development and differentiation, the method comprises the steps of: a) providing a cell matrix that exhibits a plurality of developmental and differentiation genes identified by the mir-302 family; a recombinant nucleic acid composition capable of transcribed: a plurality of non-coding mir-302 miRNAs/shRNAs or a homologous RNA of a homolog thereof, which can be sequentially processed into mature melons by intracellular mechanisms such as ' 37 201009074 miRNAs/shRNAs or homologs thereof' are therefore capable of inhibiting the function of the target gene in the cell matrix; c) consisting of the recombinant nucleic acid under conditions in which the function of the target gene in the cell matrix is inhibited Object processing Cellular substance. More preferably, the drug induces a recombinant nucleic acid composition system-a vector comprising a recombinant mir_3〇2 family gene group (mir_3〇2s; SEQ.ID.NO.29-SEQ.ID.NO.36) Compound or a human-reset mir-302 shRNA homolog (ie, a hybrid of SEQ.ID.NO.27 and SEQ.ID.NO.28) inserted into the co-transgenic gene. In other words, the recombinant nucleic acid composition comprises a drug-evokable gene expression vector. Furthermore, the recombinant nucleic acid composition may comprise a gene expression vector selected from the group consisting of a plastid, a viral vector, an inverted position, and combinations thereof. In addition, the recombinant nucleic acid composition comprises a gene expression vector. The stromal matrix can express the mir-302 miRNA/shRNA and its target gene in any manner in vitro, in vitro, in vitro, or in vivo (ZW Wvo). Examples 2 and 3 describe the procedures used for mir-3〇2 miRNA/shRNA construction and gene transfer delivery. The present invention utilizes the advantages of intracellular splices, exosomes, and NMD systems to catalyze the release of the intron mir_3 〇2 miRNA/shRNA agent from the transgene. In the first embodiment and FIG. 2A and FIG. 2B, a sequence of DNA recombination via intracellular splice components on several snRNp recognition sites in the process is described [eg: snRNPs Ul, U2 and U4/U6 .U5 The binding motif required for tri-snRNP, including: a five-terminal splice site (SEQ.ID.N0.4), a branch point motif (BrP; SEQ.ID.N0.6), a polypyrimidine segment (ρΡτ ; SEQ.ID.N0.7 or 38 201009074 SEQ.ID.NO.8), and the three-terminal splice position (SEQ.ID.NO.5)], used to combine the synthetic snRNP identification elements into one m4z· The inclusion of the subunit and the incorporation of this artificial recombination into a single method that induces the RGFP gene to form a *like and GFi3 transgenic gene. In addition, the program and the A%-in-step include an intron insertion position between the five-terminal splice site and the Brp motif for the selection and expression of a heavy mir-302 miRNA/shRNA agent. . Example 2 and Figure 3B depict the construction of a recombinant mir-302 family gene group (mir-302s) or an artificially reprogrammed mir-302 shRNA homolog. Example 3 and Figure 3C depict the transfection of the recombinant mir_302s miRNA/shRNA into cells of interest, as well as screening of successful gene transfer cells. In other words, the pluripotent stem cell line is selectively isolated using mir-302 miniribonucleic acid or Oct3/4 as a marker. Examples 4-12 describe assays for assessing the conversion of mammalian somatic/cancer cells into ES-like pluripotent cells. Figures 4 through 11 show the results of these tests. One of the miRNA-shRNA expression systems that can express the mir-302 miRNA shRNA can be used to induce an intron miRNA/shRNA expression system, ie; 2s (Fig. 3A) The gene transfer performance of the mir-302-like gene silencing effector was triggered by the mechanism of intracellular intron miRNA biosynthesis under the control of deoxyhydroxyl induction (Fig. 1A). Description of Embodiments 1 and 2; construction. When the expression vector is transfected into the cell of interest, it transcribes a TRE-Pol-II-driven recombinant transgene, 39 201009074, which contains one of the silent effectors of the intron gene that can be generated. Sub-segments (Fig. 3A and Fig. 3B), such as hairpin-like miRNAs and shRNAs. The genetic engineering of joining several synthetic DNA sequences by sequence is shown in Example 1, and the chimeric/merged into a red-colored fluorescent protein gene CRGFP).办和#乂/ contains a precursor miRNA or shRNA meson (insert position), which can be released by intracellular RNA splicing and processing mechanisms such as: splice, exon and NMD system components, and then trigger an intron RNA - mediates the mechanism of gene silencing. Other RNA transcriptional molecules that can be used to carry and generate include: hnRNA, mRNA, rRNA, tRNA, snoRNA, snRNA, smnRNA, viral RNA, pre-microRNA, and precursors and derivatives thereof. As shown in Example 1, the synthesis was combined and incorporated into a red-colored fluorescent protein gene (and or rGF cadaver) containing no introns to form *5^ and cultivating? Transgenic gene, which is a variant of HcRedl pigment protein (chr〇moproteins) from the purple sea anemone (the sputum along the cn·print alpha). Since the inserted formula ^^\(4)· interrupts the functional fluorescent protein structure of RGFP, it can be irradiated by the red fluorescent light at a wavelength of 57〇_nm in a successfully transfected cell or organism. Appear to determine intron removal and GF_P-mRNA variation (Fig. 1B). This recombination allows the construction of a transgenic gene to be based on one of the precursor RNAs (pre_mRNA) to cleave the native structure of the intron. The main components of the fraction and the number include several snRNP ages and linds, such as: the five-terminal and three-terminal splice sites in the terminal end, which are used for splicing and splicing, and the branch point motif (Brp). The interaction of the interacting body - the multi-shot (ρρτ), the link used to connect each of the 201009074 components, and some limit bits for the desired intron insertion. The SpJiNAi of the present invention comprises: a five-terminal to three-terminal direction according to a structure: a five-terminal cut and a homologous one of the mir_302-like gene silencing effector, a branch point motif (BrP) , _ polypyrimidine segment (ΡΡΤ), and a two-terminal splice site. In addition, some translation stop codons (D. codon) can be located in the linker sequence close to the three-terminal splice site. In general, the five-terminal splice site contains 5,-GTAAGAGKX3, (SEQ.ID. Any one of the N0.4) or GU (A/G) AGU motifs or a nucleotide sequence homologous thereto (eg, 5'-GTAAGAGGAT-3, (SEQ.ID.NO.37), 5'- GTAAGAGT-3', 5'-GTAGAGT-3, and 5,-GTAAGT-3,), and the three-terminal splice site contains GW犮SCYRCAG (SEQ.ID.N0.5) or CT(A/G)A (C/T) a nucleotide sequence of either or a homologous sequence of the NG motif (eg, 5, _GATATCCTGC AG-3, (SEQ. ID. NO. 42), 5 - GGCTGCAG-3, and 5' -CCACAG-3'). Moreover, a branching point motif is located between the sig-terminal splicing site and the three-terminal splicing site, and comprises a homologous sequence to the 5'-TACTWAY-3' (SEQ.ID.NO.6) motif. Homologs such as: 5'-TACTAAC-3, and 5,-TACTTAT-3,. The adenosine "A" nucleotide of the branch point sequence forms part of the cell (2,_5,) oligoadenylate synthetase and the splice form in almost all of the spliced introns (2, a 5,) Linked to the horseshoe intron RNA. In addition, a polypyrimidine moiety is located near the branch point and the three-terminal splice site, and comprises 5,-(TY)m(C/-)(T)nS(C/-)-3, ( SEQ.ID.N0.7) or 5'-(TC)nNCTAG(G/-)-3, (SEQ.ID.N0.8) One of the homologs of one of the homologs of high T or C content Glycosidic acid sequence. Symbol 201009074 Two? And "n" indicates a plurality of repetitions, and (1) is more preferably 1 to 3 and the number of η is equal to 7 to 12. The symbol "^ can be skipped by one 枋: y: 醯, .L". Privacy is used in this sequence to connect the linked sub-acid phase system to have such intron components. According to the criteria for the symbol and format of the acid amino acid sequence (four) in 37 CFR U22, the symbol w ^ 曰 - gland 呤 (A) or thymus ♦ (τ) / urine money (u), symbol K refers to a bird嗓吟 (9) or thymus tilting τ) / urinary ejaculation (9), symbol s refers to one cell bite (C) or guanine (G), symbol γ refers to - cell money (〇 or thymus (7) / urine (9), The symbol R refers to an adenine (4) or a bird sigh (9), and the symbol N refers to - glandular noise (4), cell money (〇, bird (6) or thymus (7) / uracil (U). The splice identification component, deoxythymidine (T) nucleotides can be replaced with uridine (uridine).

In order to test the function of the VA. meson, a variety of gene silencing effectors can be constructed and inserted into the intron insertion site of the recombination gene. The intron insertion site comprises a plurality of restriction and selection sites, which can be identified by restriction enzymes. The restriction enzymes are selected from the group consisting of 乂 plus π,

AflII/III, Agel, Apal/LI, Asel, Asp718I, BamHI, Bbel, BclI/II, BgUI, BsmI, Bspl20I, BspHI/LUllI/1201

BsrI/BI/GI, BssHII/SI, BstBI/Ul/XI, CM, Csp6I, Dpnl, Dral/II, EagI, Ecll36II, EcoRI/RlI/47III, Ehel, FspI,

Haelll, Hhal, HinPI, Hindlll, Hinf!, 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, 42 201009074

SspI, StuI, Tail, TaqI, Xbal, xhoI, XmaI, post-incision and combinations thereof. The intron meson DNA template, which can be transcribed into a highly secondary structure selected from the group consisting of: lariat-form RNA, short temporary RNA (stRNA), anti- ' (10) VIII, small interfering RNA (siRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi interaction RNA (piRNA), ribozyme, and synonymous or anti Any of a group of precursors and derivatives, or both, and combinations thereof. In order to facilitate gene transfer into a cell or organism of interest, the transgenic gene of the present invention is preferably ligated into a competent vector selected from the group consisting of a DNA transgene, a plastid, an inverted position, and a transposon. , a group of jumping genes, viral vectors, and combinations thereof. The resulting gene transfer expression vector is preferably introduced into the cell or organism by a high efficiency gene delivery method. The gene delivery method is selected from the group consisting of chemical/❹ liposome transfection, electroporation, transposition Mediating DNA recombination, skip gene insertion, viral infection, microinjection, gene grab penetration, and combinations thereof. In other words, the recombinant nucleic acid composition is introduced into the mammalian cell by a gene delivery method. The gene delivery method is selected from the group consisting of microlipids.

Liposomemal transfection, chemical transfection, transgenic DNA recombination, viral infection, transposon insertion, jumping gene insertion , micro-injection, electroporation 43 201009074 (electroporation), gene-gun penetration, and a combination thereof. The vector may further comprise at least one virus, Pol-II, or Pol-III promoter, or a combination thereof' to express the cadaveric gene. More preferably, the transgenic gene is combined into a 7^-0«/0# vector by electroporation and the gene is transfected into the target cells as shown in Example 3. In addition, the vector may comprise: a Kozak consensus translation initiation site for increasing the translation efficiency of the eukaryotic cell, and a plurality of SV40 polyadenylation messages downstream of the coding-like gene. (SV40 polyadenylation signal), a pUC origin of replication for propagation in prokaryotic cells, at least two restriction positions for merging the construct in the vector, for expressing sy4〇τ antigen An optional sV40 replication source for replication in mammalian cells, and an optional SV40 early promoter for expression of an antibiotic resistance gene in a competent prokaryotic cell. In other words, the recombinant nucleic acid composition is selected from the group consisting of a tetracycline reaction element, a virus or a second type of polymerase (p〇l_H) promoter, or both, and a Kozak-translated method. A group consisting of an onset, a polyadenylation message, a plurality of restriction/selection sites, and combinations thereof. In addition, the recombinant nucleic acid composition is derived from a source of pUC replication, an SV40 precocious promoter which exhibits at least one antibiotic resistance gene in a competent prokaryotic cell, a replication surface for replication in mammalian cells, and The combination is _. The performance of the miscellaneous her drug gene can be used as a -selectable marker, which can be used to isolate successful colonized cells with the gene transfer to 201009074. These antibiotics are selected from penicillin G, ampicillin, neomy Cin, paromycin, kanamycin, streptomycin, and red mold. Erythromycin, sPectromyein, phophomycin, tetracycline, rifapicin, amphotericin B, gentamicin A group consisting of gentamycin, chloramphenicol, cephalothin, tylosin, and combinations thereof. The performance strategy of using the intron miRNA/shRNA of the transgenic gene has been tested in vivo in a cultivar zebrafish in a Dinglu 〇>2-〇8 1<4:118 八??) Gene expression is marked. As shown in Example 6 and Figure 1B, one of the artificially recombinant anti-five GFP pre-miRNA mesons (fourth column) was transfected with a liposome, which showed a very strong gene silencing effect. (> 80% gene knockdown), however, no other silent effects were observed in the above-mentioned mesons from φ left column to right column: (1) blank vector control (Ctl); 2) the target pre_miRNA meson (mock), (3) the antisense meson (sister) without a hairpin-type loop structure; and (5) the reverse pre-miRNA sequence, which is associated with the anti_£(^pp pre - miRNA is fully complementary (10) R*). Moreover, no effects were detected on the non-target (〇ff_target) gene, such as the marker rgfp and the house-keeping actin (p_actin) gene, suggesting that this intron miRNA-mediated Gene silencing has a high degree of target-specificity. In addition, by Northern blotting analysis (Fig. 1β, right), 45 201009074, we observed that the generation of small intron gene silencing effectors originated only from the design and GFi3 gene transcription molecules (left column), not From a natural transcription molecule containing no introns (middle column) or a transcriptional molecule (right column) constructed without defective SpRNAi-RGFP, which is one of the functional five-terminal 笏 junctions, The lines are linked together to form mature RNA for translating the labeled red fluorescent protein. Design and construction of a recombinant mir-302 family gene group and a class of mir_302 shRNA homologs ❿ Because some natural pre-miRNA hairpin-type loop structures are too large and/or too complex to match the 汾4ζ·- and GFP The gene was transduced, so the inventors designed a modified tRNAmet loop (i.e., 5'-(A/U)UCCAAGGGGG-3') (SEQ. ID. NO. 43) in place of the natural pre-miRNA loops. This tRNAmet loop has been shown to efficiently promote the export of artificially reprogrammed miRNAs from the nucleus to the cytoplasm via the same Ran-GTP and Exportin-5 delivery mechanisms as native miRNAs (Lin et al., 2005). Advantageously, the present invention currently employs a pair of artificially modified 〇pre-mir-302 rings, including: 5'_GCTAAGCCAG GC-3, (SEQ. ID. NO. 1) and 5, -GCCTGGCTTA GC-35 (SEQ. ID.NO.2), which provides the same nuclear export efficiency as native pre-miRNAs without interfering with tRNA output. Moreover, this modification enhances the formation of mir-302a-mir-302a* and mir-302c-mir-302c* double bonds, which stabilize the overall function of the mir-302 family. The design of these novel pre-miRNA loops is modified by the binding of the mir-302b/mir-302a tRNAmet loop and the short stem loop 46 201009074, which is highly expressed in embryonic stem cells but not in other differentiated tissue cells. Therefore, the use of these artificial/artificial pre-miRNA loops in the mir-302 family will not interfere with the natural miRNA pathways in our body and is therefore less cytotoxic and safer. The mature sequences of these mir-302a, mir-302b, mir-302c and mir-3O2d are '-UAAGUGCUUC CAUGUUUUGG UGA-3, (SEQ.ID.NO. 10), 5,-UAAGUGCUUC cauguuuuag UAG-3' ( SEQ.ID.NO.il), 5,-UAAGUGCUUC ® CAUGUUUCAG UGG-3' (SEQ.ID.NO.12), and 5'-UAAGUGCUUC CAUGUUUGAG \JG\J-3' (SEQ.ID.NO.13 ). These mir-3〇2 family gene silencing effectors share a highly consistent five-terminal region (1% homology) in the first seventeen nucleotides, which is associated with 5'_UAAGUGCUUC CAUGUUU-3, (SEQ.ID.N0.3) is identical. In other words, the gene silencing effector comprises a sequence homologous or complementary to SEQ. ID. N0.3, or both. In the sequence designed to be homologous to the 5 荨 mir-302 sequence, the thymus shunt (丁) can be used to replace uracil (U). As described in Example 2, the gene group of the family mir_3〇2 pre_miRNAs is formed by the hybridization and linkage/joining of synthetic mir-302 homologs, which consists of four parts in a five-terminal to three-terminal direction: Mir_3〇2a, (5) 卜川沘, mine and mir-3〇2d pre_m_A (Fig. 3B). All of these artificially-reset mir-302 miRNA/shRNA homologues are in the first seventeen nucleotides/, with the same five-terminal end of 70 kings [eg: 5, _uaagugcuuc 201009074

CAUGUUU-3' (SEQ. ID. N0.3)]. In other embodiments, an artificially reprogrammed mir-302 shRNA can be used in place of the mir-302 pre-miRNA gene population for intron insertion. The reset mir-302 shRNA is synthesized by synthesizing the synonymous mir-302 family (mir-302s-sense), 5,-GTCCGATCGT CATAAGTGCT TCCATGTTTT 'AGTGTGCTAA GCCAGGCACA CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3, (SEQ.ID.NO.27) and Antisense mir-302 family (mir-302s-antisense), 5,-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG ❹

A heterozygosity of GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3' (SEQ, ID. N0.28) was formed. This reset mir-302 shRNA shares more than 91% homology to all native mir-302 members, and the same mir-30> target gene in humans. Will be able to express mir-302-expressing Sp and any of the stagnation of a certain indigo human hair hair follicle (hHFC) and cancer melanoma Colo cells in the peak of the touch ❹ due to the intron insertion of the recombinant gene The five-terminal and three-terminal are flanked by a PvwJ and a M/w/restriction/selection, respectively, so the native meson can be simply a variety of pre-miRNA/shRNA mesons (eg, mir-302pre-miRNA/shRNA) Removed and replaced, it has a matching point that matches the /W and an M/wJ limit. By changing the mesons of these introns for a variety of gene transcriptional molecules, the intron miRNA/shRNA expression system can be used as an inducing target in vitro and in vivo 48 201009074 A powerful tool for silence. In the experiment, the mir-302 pre-miRNA/shRNA is first inserted into the transcript and the drain/jGF/5 transgene, and then the transgene is incorporated into the selection of the vector (ie, • XhoI) In the -C!aI position, the western-like fluoro-pTet-On-tTS-miR302s transgene expression vector (Fig. 3A). Thereafter, the vector (10_3〇pg) was mixed with the host cells (2〇〇2〇〇〇) in a hypotonic pH buffer (400 μΐ; Eppendorf) at 400-450 volts. An electroporation method of 100 microseconds (psec) was performed to deliver the transgene into the host cell gene. Cells successfully isolated and collected after 72 hours were screened by anti-RGFP and anti-〇ct3/4 monoclonal antibodies using a FACS flow cytometer (Fig. 3C). The success rate of this novel mir_3〇2s gene transfer method was measured over 91%. Because the scorpion _ _ and the transgenic gene are flanked by a homologous region and recombined into a specific gene position that does not contain the gene (Fig. 4Α), the program and the 4/_ and the coding gene are encoded. The expression of the mir-302 miRNA/shRNA effector is based on the Dox-induced activation of the 77^-CMF promoter of the vector. The cockroach vector contains a CMV-driven still inhibitor gene to deactivate the 77; £: _CMF promoter of the transgene. The function of tTS was inhibited by Dox when deoxyhydroxyl (Dox) was present, thus exhibiting the GFP transgene and its coding mir_3〇2 family (Fig. 4b). 49 201009074 Transformation of human normal and cancer sputum cells into a sea ES state in a cell containing no lactating cells As described in Example 1-2 and Figures 3A-3B, we have designed and constructed a priming gene. , which encodes an artificial link _ mir-302a-mir-302b-mir-302c-mir-302d (mir-302s) pre-miRNA or a reset mir_302 shRNA [eg, a type of hairpin sequence containing 5 '-UAAGUGCUUC CAUGUUUUAGUGU-3' (SEQ.ID.N0.9)], and then tested the transgenic gene to silence various body cells and cancer cells (such as human normal hair follicle cells (hHF C) and cancer melanoma) Target genes related to development and differentiation in Colo cells). Following the procedures shown in Figures 2A and 2B, the inventors separately transfected the recombinant mir-302s pre-miRNA gene into hHFC cells' and the reset mir-302 shRNA homolog (SEQ .ID.N0.9) Genes are transferred into Colo cells. In other words, the gene silencing effect is a recombinant hairpin RNA comprising a sequence homologous to SEQ. ID. N0.9. After the expression of ectopic mir-302 induced by Dox, the morphology of all such 〇mir-302 transduced/induced pluripotent stem (mirPS) cell lines was transformed from spindle to round (roun(j) (Bottom), which means that it is not only likely to lose the ability to migrate 'may also have a very slow cell regeneration rate similar to ES cell growth (Figure 5A). Comparison of DNA content and flow cytometry analysis at a cell cycle stage (Overview; Example 7) further shows that there is a 67% reduction in the mitotic cell population of mirPS cells, suggesting that these cell proliferation rates are much lower than their body/cancer source proliferation rate of 50 201009074. Said cell populations are split once every 20-24 hours in a culture condition without mammalian cells, which comprises DMEM/F12 or RPMI 1640/B27 medium at 37 ° C and 5% C〇2, Supplementary ingredients are 10% charcoal-stripped fetal bovine serum (FBS), 4 mML ghtamine, 1 mM sodium pyruvate, 5 ng/ml activin, 5 ng /ml Noggin protein, 3 ng/mi bFGF, and 0·5 μΜ Y-27632 and 0.5 μΜ GSK-3 One of the preparations xv is an equal mixture (ie: mirPS cell culture medium). In other words, the pluripotent stem cells can be cultured in the DMEM/F12 〇 or RPMI 1640/B27 culture medium without the mammalian cells, the culture solution The supplement is 1% carbon adsorption FBS, 4 mM L-face decylamine, 1 mM sodium pyruvate, 5 ng/ml activin, 3 ng/ml

bFGF, and an equal mixture of 0.5 μΜ Y-27632 and 0.5 μΜ GSK-3 inhibitor XV. The first (left) and second (right) peaks of the flow fine code counter round table indicate the content of the cell population in the G0/G1 and mitotic stage of dormancy in the entire cell population subjected to the test. After the πήΓ-3023 transfection, the mitotic cell population (pupling stage) decreased from 41% to 11% in hHFC and from 36% to 11% in Colo cells, but it was allowed to be treated in one empty space. After transfection with the vector and Dox (+Dox) or one of the Dox-free mir-302 expression vectors (+mir-302s-Dox), the cell morphology or cell proliferation rate did not change significantly. Based on these findings, we demonstrate that ectopic mir-302s exhibit a morphological and cell division rate that converts normal and cancer human somatic cells into a class of ES cells. 51 201009074 Formation of embryonic thin mites such as mirPS. All of these mirPS cells are capable of forming compact cell clusters derived from embryonic bodies (EB) such as human embryonic stem (ES) cells (Fig. 5B). In other words, this demonstrates that the pluripotent stem cells transformed by the present invention can form embryoid bodies. When EB cells were isolated by a mixture of trypsin EDTA (trypsin-EDTA) and collagenase IV (collagenase IV), and then cultured in RPMI 1640 medium with only 10% FBS supplement, These EB cells differentiate into neuronal progenitor cells, many of which have neuronal markers Tujl and/or ABCA2. In the limiting dilution method and further in the DMEM/F12 culture medium containing no lactating cells (the supplement is 1% carbon adsorption FBS, 4 mM L-glutamate, 1 mM sodium pyruvate, 5 ng/ml activin) After culturing in 3 ng/ml bFGF and 0.5 μΜ Y-27632 in an equal mixture with 0.5 μΜ GSK-3 inhibitor XV, each mirps cell can continue to be formed for sub-culturing and / or a pure embryonic body of the transplant / implant test (Figure 5C). In view of these ES stem cell characteristics, we then tested the expression of mir-302s and ES cell markers in these mirPS cells and compared them with human ES WA01-H1 and WA09_H9 cells. Micro-nucleic acid (miRNA) microarray analysis of mir_302s expressed in giirPS to confirm the gene transfer mir_3〇2 expression in these mirPS cells, we carried out the micronucleic acid (miRNA) described in Example 9. Microarray analysis. As shown in Figure 6A, miRNA microarray analysis showed the performance of all mir-302 members after Dox treatment (100 μΜ) compared to the original body 52 201009074 cells (control group) (bottom right, white box) The circle) is markedly increased in these mirPS-hHFC cells. The degree of expression of mir-302s in mirPS cells was proportional to the Dox-induced concentration (Fig. 4B), as determined by the Northern blotting method. The same results were also observed in mirPS-Colo cells (Lin et al., 2008b). In the early EB stage, multiple small RNAs were isolated from each cell line using a one-m>VanaTM miRNA separation tool (Ambion Inc., Austin, TX). The purity and quality of these isolated small RNAs were assessed using 3.5% furfural-cabbage electrophoresis and spectrometer measurements (Bio-Rad, Hercules, CA) and immediately sent to LC Sciences (San Diego 'CA). Microarray analysis. In the Cy3 and Cy5 intensity images (blue background), when the signal intensity increases from level 1 to level 65,535, the corresponding color changes from blue to green, yellow, and then red. In Cy5/Cy3 than in images (black background), when Cy3 is higher than Cy5, the color is green; when Cy3 is equal to Cy5, the color is yellow; and when Cy5 is higher than Cy3, the color is red . Since the high homology (>91%) is shared between the mature miRNA sequences of the natural mir-302 family members and the artificially-reset mir-302 pre-miRNA/shRNA agents of the present invention, this result indicates These reset mir-302 agents can replace the function of natural mir-302s. Based on this result, we also found that the improvement of mir-302 performance can further increase the performance of some other miRNAs, such as: mir-92, mir-93, mir-200c, mir-367, mir-37 mir-372, mir -373, mir-374, and members of the entire mir-520 family. Based on the analysis of the predicted target genes of these miRNAs, use "TARGET SCAN" (http://www.http://www.microfar.com/sanger.ac.uk/) linked to Sanger miRBase::Sequences Network 53 201009074 (http://www. Targetscan.org/vert_42/) and the "PICTAR-VERT" (http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi?) program show that mir-302 shares more than 400 target genes with these miRNAs. This indicates that these miRNAs may also play an important role in maintaining stem cell pluripotency and regeneration. Such conformable targets include, but are not limited to, members of the RAB/RAS-related oncogene, ECT-related oncogenes, pleiomorphic adenoma genes, E2F transgenic factors, cyclins Cyclin D binds to Myb transcription factor, HMG-box transcription factor, Sp3 transcription factor, transcription factor CP2 protein, NFkB-activated protein gene, cyclin-related kinase (CDK), MAPK-related kinase, SNF-related kinase, and myosin Myosin light ch in kinases, TNF-α-inducible protein gene, DAZ-related protein gene, LIM-related homologous gene, DEAD/H box protein gene, forkhead box protein gene, BMP regulator ,

Rho/Rac guanine nucleotide exchange factor, IGF receptor, endothelin receptor, left and right determinant, cyclin, p53 can induce nuclear protein gene, RB-1, RB binding protein gene, maximal Max-binding protein genes, c-MIR cellular modulator of immune recognition, apoptosis-like facilitator, protocadherins, integrin Integrin)fl4/fl8, inhibin, ankyrins, SENP1, NUFIP2, FGF9/19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, tissue protein B 54 201009074 stone converting enzyme (histone acetyltransferase) MYST3 , nuclear nucleocapsid protein H3 (nuclear RNP H3), and many nuclear receptors and factors. Most of these genes are related to the embryonic development and/or tumorigenesis of tumors/cancers. Identification of standard human ES markers, namely: Oct3/4, SSEA-3, SSEA-4, Sox2, and Nanog As shown in Figures 6B and 9B, these mirPS cells strongly express many of the $ standard human ES cell markers, such as :0ct3/4, SSEA-3, SSEA-4,

Sox2 and Nanog 'However, none of these markers were detected in the original somatic cells (hHFC control group), and the somatic cells were isolated and deoxygenated (hHFC+Dox) or deoxygenated. Transfected with one of the mir-302s vectors (mirPS-Dox). The mRNA and protein content determined by the Northern and Western blotting assays are very similar to those of human ES WA01-H1 and WA09-H9 cells. These scabs indicate that ectopic performance of mir-302s can convert adult somatic/cancer cells into ES-like pluripotent stem cells, which present many standard human ES markers. The same results were also observed in mirPS-Colo cells (Fig. 8C).

Oct3/4 (also known as 〇ct-3 or 〇ct-4) is one of these POU transcription factors that is predominantly and highly expressed in pluripotent embryonic stem and germ cells (Scholer et al. (1989) V. · 8:2543-2550; Rosner et al. (1990) 345:686-692). We need a critical amount of 〇ct3/4 to maintain stem cell self-regeneration and pluripotency. Down-regulation of 〇ct3/4 leads to differentiation of embryonic stem cells into divergent developmental pathways. SSEA protein, SSEA], SSEA-3, and SSEA-4 were originally identified by monoclonal antibodies, which were found on the surface of mouse embryos and teratocarcinoma stem cells at the pre-implantation stage. Lacto- and globo-glycolipids 'but do not recognize lact〇_- and globo-glycolipids on their differentiated derivatives (Solter et al. (1978) Proc. 々Μ · C/& 4 75:5565 -5569). Undifferentiated primate embryonic stem (ES) cells, human embryonic carcinoma (EC) and ES cells all exhibit SSEA-3 and SSEA-4' but do not exhibit sSEA-1 (Thomson et al., (1998) > Foot 282: 1145-1147). SSEA-3 and SSEA-4 are synthesized during _ ❹ ❹ formation and are mainly present in oocytes, zygote and early cleavage stage embryos (Shevinsky et al., (1982) CeiY 30: 697- 705). In terms of maintaining pluripotency, s〇x2 has the function of a nuclear transcription factor, but this function is not specific to embryonic stem cells (BQyer et al. (2005) Ce//122: 947-956). Therefore, based on this recognition, these mirp cells are likely to exhibit all of the characteristics of these human ES markers.

Evaluation of Keel Difficult DNA Demethylation (Kanhua) The change of the day after day modification emphasizes another unique function of ES cells. The genome is demethylated (Hochedlinger et al., (2006) Shi (10) 441:1061- 1067). In order to convert a cell into its ES state, many embryonic genes need to be reactivated by DNA demethylation, such as: (9 to 3/4. In order to assess this acquired effect in these mirPS cells, we first In order to eliminate the entire genomic elimination, rush to / a pair of CpG thiolation-sensitive restriction enzymes, and cut only one undenidized CCGG position instead of one A 56 5609009074

The CCGG bit of the base. Figure 7A shows the DNA fragment from the somatic cell control group which is more than twice as large as the depleted DNA fragment from the mirps cells. This indicates that the entire mirPS cell gene system is highly deaminated. Further evaluation in the OdW• gene promoter region was performed using disulfite-genosome PCR and nucleic acid sequencing (Takahashi and Yamanaka, 2006). Disulfite converts all ungerminated cytosines to uracil. Since the ungerminated ACGT position was changed to the AUGT position, the ACGT-cleavage restriction enzyme solution could not cut the φ separation region in the miRNA of the mirPS cells (Fig. 7B). The detailed dethiolation map shown by the disulfite DNA sequencing further demonstrates that the OeW gene promoter region loses more than 90% of the thiolation sites in the mirPS cells, similar to those in the human ESWA09 The condition that occurs in the H9 cells (Fig. 7C), which indicates that a pan-genome transformation event does occur to reactivate the sputum to express the 3/4 gene. Example 8 shows the above CpG demethylation experiment. In the experiment to determine the dethiolation positions in the promoter of the gene, we first used disulfite (CpGenome DNA modification tool,

Chemicon, CA) processes the isolated genomic DNA, which converts all unmethylated cytosines to uracil, and then uses the polymerase chain reaction to separate the Oc/K upstream promoter region (long template PCR extension tool,

Roche, IN). Thereafter, the PCR product is collected and eliminated by an equal mixture of one of a plurality of ACGT cleavage restriction enzymes (5 U per restriction enzyme), such as: / / (AACGTT), melon (CACGTC), Pm / / (CACGTG), such as a5/(TACGTA) and example/"(ACGT). Because the unmethylated ACGT in this region is changed to AUGT with disulfite changed 57 201009074, it cannot be used. The ACGT cleavage restriction enzymes were cleaved, so the results of Figure 7B indicate that more than four thiolated ACGT positions in the control group hHFC, PC3 and Colo cells were changed to the demethylation position in the corresponding mirps cells. , the mir-302-mediated demethylation of the Oc/W gene promoter contributes to the reactivation of the gene expression in the mirPS cells derived from the mirPS in the sinus cancer The loss of migration of human ES cells does not migrate. The cells derived from the rapidly metastatic cancer cell line φ are often observed in these mirPS cells (eg, mirPS-PC3 cells). Because ES cells tend to sleep in one place. Forming embryoid bodies in situ (eg, eg), which explains why metastatic humans Adenocarcinoma PC3 cells lose their ability to migrate after ectopic mir-302 transfection. In other examples, 'mir-302 can silence certain cell migration-related genes to avoid normal cell migration and cancer cell invasion'. Cell migration-related genes such as: microtubule-associated protein gene 1Β(Μ4Ρ75), actin-like protein C7X&4), Jingkeli 2(ΑΜ2), temple powder-like protein β precursors 8 4 (/corpse/>), 〇 Myosin light p〇lypeptide kinase (MFXiQ). As shown in Figure 7D and Example 12, metastatic pC3 cells migrated rapidly over time, whereas mirPS-PC3 cells remained stationary. No morphological changes were observed in the other control groups. Therefore, the gene transfer mif-302S of the present invention is sufficient to convert human cancer cells into a more ES-like cell opening state and cell division rate, suggesting a A very beneficial use in cancer treatment. This result suggests a potential therapeutic application for the transfer of these mir_3〇2 into cancer/tumor cells, which can not only treat these malignant cancers/swells The transformation of tumor cells into useful ES stem cells can also reduce the chance of cancer metastasis. More favorable, since these mirPS cell lines are produced by the patient's own cells, and thus they are immunologically compatible with patients, it can be. These mirPS cells are utilized to develop a novel transplantation therapy to repair such cancer/tumor damaged tissues without the risk of immune rejection. Identification of overall ES marker expression using gene withdrawal array analysis φ The pan-genome gene map provides insight into the genetic changes associated with this mir-302-mediated transformation event. After confirming the common performance of standard ES cell markers and gene-transferred mir-302s, we further [before and after the ectopic mir-302 performance in these cells and in these mirps and other human ES cells (eg: Human genomic microarray analysis was performed between WA01-H1 and WA09-H9) to screen for changes in ubiquitin gene phenotypes. The detailed operation procedure is shown in Embodiment 10. Affymetrix gene microarrays (GeneChip U133A & B and U133 plus 2.0 arrays) were used to assess changes in phenotypes of more than 47 human genes. We first used the same mirPS samples to replicate these microarray tests and selected two hundred of the most variable genes (white spots) from one of these tests for further comparison. As shown in Figure 8A (mirPS-Colo) and Figure 9A (mirPS-hHFC), the overall variation of the replication test is less than double (leftmost), which means that the background variation is extremely limited. Based on the decentralized pattern of all microarray-recognized genes, we then calculated the correlation coefficient (CC) between the two sets of comparative transcript databases. Provide another CC rate to display in the 59 201009074

The percentage of similarity in the ubiquitin gene phenotype, which has a threshold of only a one-fold change. Under the definition of such a stringent cC rate, we have found that the phenotypes of these genes of mirPS cells are very similar to those of human ES WA01-H1 (>89%) and WA09-H9 (>86%) cells, however Only a low 47%-53% CC rate was shown between these mi rP S cells and their somatic/cancer cell sources. This strong genetic correlation between human ES and mirpS cells suggests that mir_3〇2s may need to alter thousands of cellular gene expression's including the process of transforming integrated cells/cancer cells into a class of ES mirPS cells. For example, as shown in Figure 8B, sustained and simultaneous observations in these mirps and human ES cell results revealed increased performance of multiple ES genes and a number of carcinogenic, developmental gene-related arrests associated with the cell cycle of mir_3〇2. Referring to Figure 9B, for the gene expression patterns of these mirPS cells, it is also noted that SSEA-1 is moderately expressed in the mirps cells, whereas Klf4 is absent. Figure 8B shows a list of some of the major differentiation-expressing genes between Colo and mirPS-Colo cells. In Figure 8B, we note that the cell cycle checkpoint genes (ie, CDK2, cyclin cydinDl and D2) and the DNA thiol promoter (ie: MECP2 and MECPl-p66) have all been identified as strong targets for mir-302s. . In other words, these pluripotent stem cells are rich in mir-302 microRNA and 0ct4, but only have limited CDK2, cyclin Dl, MECPl-p66 and MECP2. The same results were also observed in Figures 8C and 9B. It is known that the cyclinE-related CDK2 line is required for the S phase of the cell cycle, and inhibition of CDK2 results in a G1 phase check. 201009074 Checkpoint stagnation, fresh and G1 arrest. : Can be ignored in response to leg damage. According to this principle, the CDIC2 π r cell cycle in mirPS cells can reach the __ transfected cancer cell. This cancer mitosis rate, as shown in Figure 5A. Therefore, it is good;! 结果 The result of cycle conversion can be very great for cancer treatment.

Second, the results of inhibiting the activity of MECP2 and MECP1_p66 and the results of -_, etc., resulted in the conversion of cancer cells into benign mkps cells. It is conceivable that the mirPS and the two cells obtained from the patient can progressively help repair the tissue damage of the tumor/cancer. In summary, all of these findings show that the -3G2 gene transfer method of the present invention can be used to transform the genetic map of human somatic/cancer cells into a highly paramarked phenotype that resembles the phenotype of human Es cells.

The pluripotency definition of MirPS cells is the most important characteristic of E S cells. Through manipulation with different factors and/or hormones, human Es cells can differentiate into a three-layered embryonic layer (the ectoderm, the germ layer, and the endoderm), which is the integrity of all adult tissues. Under any treatment of the forest, the embryos of the embryos (5) were xenografted into the uterus or abdominal cavity of SCID-belge mice with insufficient immune function in female pseudopregnant to form a tissue-like cyst of the tumor-like tumor (圊10). These cysts were not observed in other tissue locations. However, unlike the abnormalities, these groups and sacs will scream around them to form an excellent and clear boundary. Moreover, the growth of the squamous sac in the sputum is about 2.5 weeks after transplantation. H seems to have a self-regulating mechanism. 61 201009074 The mainland limits the random growth of 荨 mirPS cells. This self-regulating mechanism also avoids the formation of tumors from these mirPS cells, which provides a means of designing and developing tumor-free pluripotent stem cells in clinical trials and treatments. -

MirPS fine Jfe differentiation is divided into early nicknames | By definition, a pluripotent stem cell can differentiate into various cell types like those derived from embryonic ectoderm, mesoderm and/or endoderm. For example, using various growth factors and/or in vitro treatment of hormones, we can induce ES mirPS cells to differentiate into several cell types of body and/or germ cell lines, including: neuronal progenitors (Fig. 5B) , spermatogonia-like cells (Fig. 11A-E), fibroblasts (Fig. 11F_j), and chondrocytes (Fig. 11K-0). Immunohistochemistry (imhoh) detection has been used to identify markers of these special tissue lineages, which show neuron-specific Tujl and ABCA2, germ cell line-specific Daz a and EE2, fibroblasts Specific atlastini and the first type of procollagen (COL1A1), and chondrocyte-specific tropoelastin and the second type of protoplast ◎ proprotein (COL2A1). In other words, the pluripotent stem cells produced by the present invention can be differentiated into a type of germ cell line cells, spermatogonia-like cells, normal somatic cells, fibroblasts, chondrocytes, and a combination thereof. No signs of tumor formation were observed in these differentiated mirPS cells. In fact, according to the predictions of the "TARGETSCAN" and "PICTAR-VERT" programs on the Sanger website, it is known that many of the oncogenes mir-302s are labeled as "In addition, 'mir-3〇2s can suppress cyclin-related 62 201009074 kinase 2 ( CDK2), cyclin D1 and D2 to avoid rapid growth of tumor cells (Lin et al, 2008b). These findings indicate the tumor-free pluripotency of these mirPS cells. It is envisaged that a variety of molecular treatments can be used to induce more tissue cell types from such mirPS cells. Experiments in vitro in a culture condition free of mammalian cells showed that we have successfully directed mirPS cells to differentiate into three relatively homogeneous somatic cell types (Fig. 11A-0 and Example 11). First, the mirPS cells were treated in a non-mammalian cell culture jdi for six hours by a natural androgenic hormone-dihydrotestosterone (DHT 50 ng/ml), and then processed. The cells (1〇5) were transplanted in vivo into the uterus of a six-week-old female immunodeficiency SCID-beige mouse, and one cyst of the spermatogonia-like cells was produced at the transplant site one week later (Fig. 11A-E). Second, the mirPS cells were treated with the transforming growth factor P1 (TGF41 1〇〇ng/ml) for 12 hours, and then followed by the same transplantation procedure, the mirp cells differentiated into fibroblasts and only one week. Collagen is secreted inside (Fig. 11F-J). Finally, 'the mirPS cells were treated with bone morphogenetic protein 4 (BMP4 100 ng/ml) for η hours' and then the cells were allografted to a six-week-old immune function deficiency SCID-beige small In the liver of the mouse, the mirPS cells differentiated into chondrocytes surrounded by the spleen (Fig. 11K-0). This immune-deficient nude mouse is used to provide an in vivo environment for mimicking transplant therapy. These findings provide strong evidence. We have successfully used the mir-302 gene transfer method of the present invention to generate a novel ES-like pluripotent stem cell line which can be cultured in vitro without a mammalian cell. Guided into a variety of tissue cells in vivo 63 201009074 type. Therefore, the present invention not only converts differentiated somatic cells/cancer cells into a class of ES states, but also maintains ES-like regeneration and pluripotency in a culture condition without mammalian cells. Thus, the present invention provides a powerful new tool and strategy for generating ES-like mir-302-induced pluripotent stem cells (mirPS), particularly from a migratory mir-302. Native cultured mirPS cells of human somatic cells and cancer cells. More preferably, the mirPS cell lines are obtained from human normal hair follicles because they are easier to obtain. Since the intron-derived miRNA biosynthesis pathway is well regulated by multiple intracellular monitoring systems, including: components of mRNA transcription, RNA splicing, exosome processing, and NMD mechanisms', the intron mir-302 of the present invention Performance can be considered safer than the original Pol-III (U6/Hl) driven siRNA/shRNA expression system. In fact, the inventors have observed that the 表现1·ΠΙ-driven expression system tends to overexpress mir-302 and cause the cell cycle to stagnate and die in the G1 phase. The invention further provides a controllable means for converting mammalian somatic cells/Q cancer cells into ES-like mirPS cells and guiding them to form under the combination of a drug-inducible (reM9«/Q^) vector. Useful for normal tissue cells. Since mir-302 has been found to be a potent tumor suppressor gene, the mkps cells produced by the present invention have no risk of tumor formation. The invention has at least five beneficial breakthroughs. The first--- mir_3〇2 expression of the transgenic gene can replace all four large transcription factor genes used in these prior iPS methods to generate more homologous Es-multiple 64 201009074 competent stem cells ( It is only derived from a small number of somatic cells of the patient), which improves the stem cell purity and compatibility of the patient's immune system. Second, because the total length of the mir_3〇2 expressing transgenic genes is relatively small (about one thousand test bases), the method is comparable to the maximum gene transfer transfer rate of 2% in the previous iPS methods. The gene transfer line is extremely high (the success rate is over 91%). Third, the production and culture of mirPS cells is carried out entirely in a condition free of mammalian cells, which avoids the risk of antigen contamination by mammalian cells. Fourth, no oncogenes are used, which avoids the risk of cell variability and tumor formation. Finally, the present invention uses electroporation instead of retroviral infection to deliver the single mir-302-expressing transgene, which avoids the risk of random retrovirus insertion into the host cell genome, which typically causes insertional mutations (inserti〇) Nal mutagenesis). In summary, these advantages address the three main problems of these previous ips methods, which avoid the risks of retroviral infection, oncogene mutations, and uncertain tumorigenesis. θ A. is defined to facilitate the understanding of the present invention. Some terms are defined below: ^^(Nucleotide): - a single molecule of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which comprises: a sugar moiety (pentose) Pent〇se), a phosphate and a nitrogen-containing heterocyclic base (8) are (heterocyclic base). The base is linked to the sugar moiety via giyc〇sidic carbon (the terminal carbon of the pentose sugar), and the combination of the base and the sugar is a nucleoside. a nucleoside nucleotide nucleotide comprising at least one phosphate group bonded to the terminal end of the pentose and the five 65 201009074 end position: comprising two oxygen deoxygenated groups A molecule of nucleic acid or ribonucleic acid, preferably more than three, and usually more than ten. The exact length depends on a number of factors, which in turn depend on the optimal function or use of the 'oligonucleotide. The raised nucleotide can be produced in any manner, including: chemical synthesis, DNA replication, reverse transcription, or a combination thereof Nucleic Acid: a polymer of nucieotide, which can be single or double share. Nucleotide p (pUrine) or pyrimidine nucleotides are structurally a, T, G, C or U different enough to replace normal nucleotides in a nucleic acid molecule. (Nucleic Acid c welcomes m&giiioa) 核酸 a nucleic acid composition refers to a polynucleotide (eg, DNA) (dna)

Or nuclear_acid_in)' can be in the form of a single or double-strand molecular structure. Gene: - A nucleic acid whose nucleotide sequence is encoded for an RNA and/or a polypeptide (protein). One gene can be RNA4Dna. Kikuzu (4) also, bp): thymine (T) and adenine (A), or guanine (G) and cytosol (cyt0Sine ' C) in a double-stranded DNA molecule The partnership. In 66 201009074 RNA, uricil (U) replaces thymine (T). Generally, the joint is connected by hydrogen bonding. _ Precursor messenger RNA (pre-mRNA): a gene primo ribonucleotide transcript that uses a second type of RNA polymerase in eukaryotic cells via an intracellular mechanism ( Pol-II) structure is produced, and this mechanism is called transcription. A pre-mRNA sequence comprises: a 5'-end untranslated region, a 3'-end untranslated region, an exon, and an intron. . Intron: A part or parts of a gene transcriptional molecule sequence that encodes a non-protein reading frame, such as: an in-frame intron, a five-terminal non-translated region (5'- UTR) and three-terminal untranslated area (3,-UTR). jlExonl: A portion or portion of a gene transcriptional molecule sequence that encodes a protein reading frame. Messenger RNA (mRNA): A combination of pre-mRNA exons that form after the intron cleavage structure removes introns and acts as a protein-encoding RNA for protein synthesis. _Complementary deoxyribonucleic acid (cDNA): I - a single strand of DNA complementary to the mRNA sequence, and which does not contain any intron sequences. Sense: A nucleic acid molecule whose sequence and composition are identical to the homologous mRNA. The synonymous nucleic acid structure is represented by the symbol "+", "s" or rsense. 67 201009074 Antisense^: A nucleic acid molecule complementary to an individual mRNA molecule. The "-" symbol indicates the structure of the antisense nucleic acid, or "a" or "antisense" is added before the DNA or RNA. For example: "aDNA" or "aRKTA": at the 5th position of the contiguous nucleotide lacking a terminal end of a bitter acid 'where a five-terminal hydroxyl group of a nuclear acid is linked by a phosphodiester link The three-terminal hydroxyl group of the next nucleotide. There may be other groups at the end such as one or more acid-blocking residues. Three-terminal Xll-endi: one end of the nucleotide lacking a nucleotide at the 3-terminal position' Wherein, the five-terminal hydroxyl group of one nucleotide is linked to the three-terminal hydroxyl group of the next nucleotide by a monophosphate diester linkage. There may be other groups at this end, usually a hydroxide. XI welcomes one nucleic acid molecule by one nucleic acid polymerase. Depending on the polymerase, a template can be single-stranded, double-stranded or partially double-stranded. The synthesized copy is combined with the template, or a double or partial double At least one strand of the stock template is complementary. RNA and DNA are in five to three ends Directional synthesis. The two strands of a nucleic acid duplex are always aligned such that the five ends of the two strands are on opposite ends of the duplex (and if necessary, the three ends of the two strands are also) Nucleic Acid Template: A double-stranded DNA molecule, a double-stranded RNA molecule, a hybrid molecule (eg, a DNA-RNA or RNA-DNA hybrid), or a single strand of DNA or RNA molecule. 68 201009074 Binary (Conserve: If a nucleotide sequence is non-randomly heterozygous with the exact complement of a preselected (reference) sequence, the sequence of the two is consistent. I~^~(Complemetarv sL complementarity Complementation) : ^ Refers to the polynucleotides associated with the base-pairing rule (ie, a sequence of nucleotides such as: sequence "AGT" and sequence "Τ-CA" and "TCU") Complementary. Complementary can be between two strands of DNA, between one strand of DNA and one strand of RNA, or between two strands of RNA. Complementation can be "partial" or "complete" or "whole". Partial complementation occurs when some nucleic acid bases are paired according to the principles of these base pairs (p Artial c〇mplementarity or complementation. Complete or integral complementation occurs when the bases are perfectly matched between the nucleic acid strands. The degree of complementarity between nucleic acid strands is the heterozygous efficiency between nucleic acid strands And intensity has a significant impact. This is especially important for amplification reactions and is also important for detection methods based on binding between nucleic acids. Percent ❹ comPlementarity or complementation refers to the ratio of the number of mismatched bases to the total number of bases in one strand of the nucleic acid. Therefore, the complementarity rate means that half of the test bases are mismatched, while the other half of the test bases are matched. Even if the two strands of nucleic acid differ from the number of bases, the two strands of nucleic acid can complement each other. In this case, the complement occurs between a portion of the longer strands, the bases of the longer strands being paired with the base of the shorter strands. M, #(homologous homology^: means a polynucleotide sequence which is similar to a gene or mRNA sequence. For example, a nucleic acid sequence can be 69 201009074 can be partially or completely identical to a specific gene with all nucleotides and similar Nucleotide shows a 0 complementary checkout (comDlemetarv or mRNA sequence homology. A percentage table of the ratio of homologous acid numbers): Normally paired nuclear complementary nucleosides when DNA or RNA is opened into a double-strand configuration HfComDlemetarv Nucleotide Sequence): a single strand of molecule sufficient to specifically hybridize to iHvbirdize and Hvh DNA or RNA with the desired argon bond on two other strands The nucleotide sequences of the nucleotide sequences are complementary to be heterozygous in the same. Ridification: The formation of a duplex between the nuclear chronological sequences, which is sufficiently j-body. One of the primers (or the splicing model complex (or hybrid) is sufficient: the primer function required for DNA synthesis. Competitive inhibition (ie: non-random). Post-transcriptional gene silence fPosttr; The complexation formed by base pairing is "hybrid" with the target (template), thus implicitly providing a DNA polymerase to initiate a specific interaction between the two complementary polynucleotides (inscriptional Gene Silencine): In mRNA degradation or translation (knockout) or attenuation (knockdo\DNA transgenic genes or small suppression 1 ribonucleic acid interference (one of the gene knockout m under the RNA system) effect, which is usually either foreign / viral k RNA Inference, RNAi): - After squaring in nucleated cells, the transcriptional gene is silently triggered, which can be triggered by small RNA molecules, 201009074 eg microRNA (miRNA), small hairpin RNA (shRNA) and Small interfering ribonucleic acid (siRNA). Such small RNA molecules are generally useful as gene silencers that interfere with the expression of genes within a cell' comprising complete or partial complementation of such small RNa. ^^AMiA{Non-coding RNA): An RNA transcription molecule that cannot be used to synthesize peptides or proteins via an intracellular translation mechanism. A ribonucleic acid (AlieroRNA, miRJSiAl: a single-stranded RNAMiRNA capable of binding to a target gene transcriptional molecule that is partially complementary to the miRNA miRNA is typically about π to 27 oligonucleotides in length and can be compliant with the miRNA The degree of complementarity between mRNAs directly degrades the mRNA targets in their cells, or inhibits the protein translation of their underlying mRNAs. Natural miRNAs can be found in almost all eukaryotic cells, as one of the defenses against viral infections, It also allows for the expression of the gene during the development of plants and animals. ^ Pre-miRNA: a hairpin-type single-strand τ containing a stem-arm that interacts with intracellular RNasellll endonuclease (stem-arm) And a stem-loop region to produce one or more microRNAs (miRNAs) that silence the target gene complementary to the miniribonucleic acid sequences. A pre-miRNA stem structure can be formed - A perfect (100%) or partial (mismatched) hybrid duplex with its dry loop structure attached to one end of the dry arm duplex to form a circular or a hairpin-type annular configuration. 201009074 Small interfering ribose Small interfering RNA (siRMA): short double-stranded ribonucleic acid, which is a ribonucleotide duplex of about 18 to 25 perfect base pairs, and degrades a target gene transcription molecule with almost perfect complementation. Clip-type or short hairpin RNA (small hairDin hairpinRNA > shRNA): a single-stranded ribonucleic acid comprising a pair of partially or fully matched stem-arm nucleotide sequences Acid segmentation forms a type of hairpin structure. Many natural miRNAs are derived from hairpin-type RNA precursors, namely: precursor micronucleic acid (pre-miRNA). ® Dai. (Vector). Moving or stagnating in the environment - a recombinant nucleic acid composition such as: recombinant DNA (rDNA). In general, another nucleic acid molecule is operably linked here. The vector can be automatically replicated in a cell, wherein the vector And the attached fragments are also replicated. One type of preferred vector is an episome, a nucleic acid molecule that can be extrachromosomally replicated. Preferred vectors are those which automatically replicate and express nucleic acids. Ability to boot for one or more A vector encoding a polypeptide and/or a non-coding RNA encoding a gene is referred to herein as a "expression vector (expressi〇nvect〇r)." A particularly important vector allows for the selection of mRNAs produced by using a reverse transcriptase. cDNA A nucleotide sequence in a DNA molecule that encodes an amino acid residue sequence and includes upstream and downstream Α expression control elements. - Nucleic acid, which is recognized by a polymerase molecule 72 201009074, bonding) synthesis. For the purposes of the present invention, it is possible to synthesize the known polyhedral bonding sites, the protons, and any of the sequences available. And a peptide-protein molecule having a structure that encodes - bondable - a ligand that is pre-selected. Ο B. Composition—recombination minus composition 'turns on performance—separated Qin 3〇2 factor' and then induces m(tetra)2-mediated kemer in mammalian cell towels, the recombinant nucleic acid composition comprising: ” a) - recombination, a lean gene, wherein the transgenic gene is homologous to a member of the _3〇2 family, and a recombination non-linear; and b) - a competent candidate, the (iv) face can be used to transmit and express recombination in the mammalian 'animal cell The above-described recombinant transgenic gene further comprises: a) a plurality of exons wherein the exons are linked to form a transcriptional molecule having a desired function; and b) at least one An intron, wherein the intron comprises a recombinant mir_3〇2 homolog, and the exons can be excised via intracellular RNA splicing and processing mechanisms. 73 201009074 Introns of the above-mentioned re-spinning Steps include: a) for the splice junction - the five-terminal receptor frit position; b) "a gene silencing effector meson homologous to the members of the mir-302 family; c) for splice identification a branch point motif; d) one of the polypyrimidines used for splice interaction ; E) are bonded to one member of three-terminal splice acceptor splice site; and f) a plurality of sub-links, for which a 5-terminal end direction of the connection of each of these three components described above. Preferably, the gene silencing effector encodes a nucleic acid composition homologous to 5, _uaagugcuuc CAUGUUU-3' (SEQ. ID. N0.3). The five-terminal receptor splice site comprises a nucleotide sequence of any of 5'-GTAAGAGK-3, (SEQ.ID.N0.4) or GU(A/G)AGU motifs or homologous thereto (eg :5'-GTAAGAGGAT-3' (SEQ.ID.NO.37), 5'-GTAAGAGT-3', 5'-GTAGAGT-3, and 5,-GTAAGT-3,), and the three-terminal receptor splicing The line contains a GWKSCYRCAG CSEQ.ID.NCU;) or a nucleotide sequence of either the CT(A/G)A(C/T)NG motif or homologous thereto (eg, 5, _GATATCCTGC AG-3, (SEQ. ID. NO. 42), 5'-GGCTGCAG-3' and 5'-CCACAG-3'). In addition, a branch point sequence is located between the five-terminal splice site and the three-terminal splice site, and comprises a motif homologous to 5,-TACTWAY-3, (SEQ.ID.N0.6), such as : 5'-TACTAAC-3' and 5'-TACTTAT-3'. In addition, a multi-bite segment 74 201009074 is located near the branch point and the three-terminal splice position, which contains 5'-(TY)m(C/-)(T)nS(C/-)_3' (SEQ. ID. NO. 7) or a high τ or C content sequence homologous to any of 5'-(TC)nNCTAG(G/-)-3' (SEQ.ID.N0.8). The symbols "m" and "η" indicate a plurality of repetitions, y; more preferably, the number of 'm' is equal to 1 to 3 and the number of η is equal to 7 to 12. The symbol "-" means that one of the nuclei can be skipped in the sequence. According to the guidelines for the symbols and formats for nucleotide and/or amino acid sequence data in 37 CFR 1 822, the symbol W refers to an adenine (Α) or thymine ' T) / a pyrimidine ( Uraci b u), symbol κ refers to a bird 嗓吟 (guanine ' pyridine (Ty urination bite (u) ' symbol s refers to a cell or thymus money (T) / urine money (U). Chen Shangqi has The splice body is identified as 5 m Ηέΐ rtiivmin ______! ι · — cytosine ' C or guanine (G), symbol γ means cytosine (c) or thymine (T) / uracil (u) , symbol R refers to an adenine (A) or guanine (G) ' and symbol N refers to adenine (a), cytosine (c), guanine (G) sub-thymidine (thymine / Deoxythymidine, Τ) nucleotides can be replaced by uridine (U). ^ C. Method A method for transforming mammalian cells into pluripotent stem cells

Developmental and cell differentiation-related genes, several of which are identified by mir-302 and H) - recombinant nucleic acid composition 75 201009074 'which can be transmitted, transcribed and processed to be homologous to mir-302 in the cell matrix Encoding rnA; b) treating the cell matrix with the recombinant nucleic acid composition under conditions in which the function of the mir_3〇2_ target gene in the cell matrix is inhibited. More preferably, the recombinant nucleic acid composition can express a recombinant transgenic gene which can induce a Γβ-〇« or Γβ-Ο# vector encoding a recombinant mir-302 family gene group (pre-mir-302s) ; SEQ.ID.NO.29- SEQ.ID.NO.36 chain hybrid) or an artificially reset ❹ mir-302 shRNA homolog (SEQ.ID.NO.27 and SEQ.ID. a hybrid of NO.28). The cell matrix can be expressed in vitro or ex vivo (ex or in vivo 〇νζ·νο) to express pre-mir-3 02s/mir-3 02 shRNA and its target gene. The following examples are intended to illustrate certain preferred embodiments and aspects of the invention and are not to be considered as limiting the scope of the invention. In the experimental documents disclosed below, the abbreviations used are as follows: Μ (mole' molar); mM (mole, millimoiar); (micromolar, micromolar); mol (mole, moles); pmol (pimo Ear, picomolar); gm (grams, grams); mg (mg, milligrams); pg (micrograms 'micrograms); ng (nike 'nangrams); L (liters, liters); ml (ml, milliliters); μ1 ( Microliter, micr〇liters); °C (degrees Celsius, 76 201009074 degrees Centigrade); cDNA (copy or complementary DNA; DNA (deoxyribonucleic acid); ssDNA (single strand of DNA, Single stranded DNA) ; dsDNA (double-stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); PBS (salt buffer) , phosphate buffered saline); NaCl (sodium chloride); HEPES (N-2-ethylpyrazine-N-2-ethene acid, ® N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid ); HBS (HEPES buffered saline, HEPES buffered saline); SDS (twelve burned sulfuric acid) 'Sodium dodecylsulfate); Tris-HCl (triple-ylamino Yue Yue burn - hydrogen chlorides, tris-hydroxymethylaminomethane-hydrochloride); and ATCC (American Culture Collection strain, American Type Culture Collection, Rockville, MD).实施 Example 1 Containing a family of recombinant RGFP genes Listed below are synthetic oligonucleotides used to generate a homologous/intron/intron containing synonymous, antisense or hairpin-type five-mesons: synonymous Nl (Nl-sense ), 5,-GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3' (SEQ.ID.NO.14); antisense N1 (N1-antisense), 5,-CGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGGATCC TCTTAC-3, 77 201009074

(SEQ.ID.NO. 15); synonymous N2, 5,-GTAAGAGGAT CCGATCGCTT GAAGAAGATG GTGCGCTCCT GA_3, (SEQ.ID.NO. 16); antisense N2, 5'-CGCGTCAGGA GCGCACCATC TTCTTCAAGC GATCGGATCC

TCTTAC-3, (SEQ.ID.NO.17); synonymous N3,5,-GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA

GTTAACTTGA AGAAGATGGT GCGCTCCTGA_3' (SEQ.ID.NO. 18); antisense N3, 5'-CGCGTCAGGA GCGCACCATC TTCTTCAAGT TAACTTGAAG

AAGATGGTGC GCTCCTGCGA TCGGATCCTC TTAC-3, (SEQ.ID.NO.19); Synonymous N4, 5'-CGCGTTACTA ACTGGTACCT GTTCTTTTTT TTTTTGATAT

CCTGCAG-3, (SEQ. ID. NO. 20); antisense N4, 5, -GTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3, (SEQ.ID.NO.21). All sequences listed from SEQ. ID. NO. 14 - SEQ ID NO. 11 were phosphated at their five ends. Furthermore, the two RGFP exons were generated by digesting Drfl// restriction enzyme at the 208th nucleotide (nt) position of a red fluorescent AGFP gene (SEQ. ID. NO. 22). The five-terminal and exon are further blunt-ended with t4 DNA polymerase. The 69th amino acid (a, a.) insertion of the HcRedl pigment protein isolated from a novel red-chromophoric pigment protein gene by a purple anemone rnipo 'BD Biosciences, CA) An additional aspartame 78 201009074 acid (aspartate) to generate 'infrared fluorescence emissions that cause less aggregation and nearly twice the intensity of the wavelength of 570-nm. - synonymous Nl-sense to antisense N1-antisense, synonymous N2_sense to antisense N2-antisense, synonymous N3-sense to antisense N3-antisense, and synonymous N4-sense to antisense N4-antisense Practice: Heat each complementary mixture of synonymous and antisense (1:1) sequences to 94 ° C for two minutes, then place in 10 times of PCR buffer at 70 ° C for 10 minutes

❹ Clock (eg: 50 mM Tris-HCl, pH 9.2, 25. (: ; 16 mM (NHASO4 · ' 1.75 mM MgCb). Immediately thereafter, within 1 hour, by using N1+N4, N2+N4 or N3 +N4 (l:l) hybrid mixture from 5 〇. 〇 gradually cooled to 10 ° C, respectively, to perform sequence ligation of Nl, N2 or N3 hybrids with N4 hybrid, then I DNA ligase And a buffer (Roche, IN) was added to the mixture and incubated for 12 hours at 12: (this can form these introns. Thereafter, the two exons are added to the reaction. Medium (1:1:1), and adjust the τ4 DNA junction© enzyme and buffer accordingly to carry out the ligation reaction for another 12 hours. For the selection of the correct recombinant SpRNAi insertion gene, a pair of specific primers 5' -CTCGAGC ATG GTGAGCGGCC TGCTGAA-3,

(SEQ.ID.NO.23) and 5,-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3' (SEQ.ID.NO.24) Amplification of 1 ng of the junction sequence by PCR, which was performed at 94 ° C for one minute. At 52_57. (: One minute, then at 68. (: Two minutes, a total of 25 to 3 cycles ^ will be obtained 79 201009074

The PCR product was chromatographed on a 2% acacia gum, and then a nucleotide extraction sequence (Qiagen 'CA) was used to extract and purify a nucleotide sequence of 9 〇〇 to 11 〇〇 base pair (bp). The composition of the ～ and GFP genes was further confirmed by sequencing. Preferably, in the absence of an intron insertion, the synonymous strand of the genus / ^ · intron sequence 5, _GTAAGTGGTC CGATCGTCGC GACGCGTCAT TACTAACTAT

CAATATCTTA ATCCTGTCCC TTTTTTTTCC ACAGTAGGAC CTTCGTGCA-3' (SEQ.ID.NO.25), and

φ and the inversion of the intron sequence 5,_tgcacgaagg TCCTACTGTG GAAAAAAAAG GGACAGGATT

A AG ATATTG A TAGTTAGTAA TGACGCGTCG CGACGATCGG ACCACTTAC-3' (SEQ. ID. NO. 26). In other embodiments, the recombinant transgene can be hybridized by SEQ. ID. N0.25 and SEQ. ID. NO. 26 with a restriction fragment of Dm//cut and GFP exon. It is made directly and processed according to the same operating procedure as shown above. In this manner, a transgenic gene for the artificially reset mir-302 shRNA meson (encoding SEQ. ID. N0.9) was formed. Since the recombination and AW- and GFP transgenes have a scorpion/and an Xk/restriction at their five and three ends, respectively, they can be easily colonized to have adhesion to the X/w/ A carrier in the adhesive end of the colony. The vector must be a competent organism or a sub-organism selected from the group consisting of a DNA transgene, a plastid, a jumping gene, a transposon, and a viral vector 201009074. In addition, since the insertion position in the intron is flanked by a corpse vw/ and a Mw/limit at its five and three ends, we can remove and replace the insertion with a different insertion sequence. With a meson, the different insert has an adhesion to the PvwJ and Mw/selection sites. Preferably, the insertion sequence is a hairpin-type gene silencing effector having high complementarity to a target gene selected from the group consisting of a fluorescent protein (GFP) gene, a fluorescent enzyme gene, and a lac-Z lactose expression. A group of genes, viral genes, bacterial genes, plant genes, animal genes, and human genes. The complementarity and/or homology ratio between the gene silencing effector and its target gene is in the range of about 30%-100%, and more preferably in the range of 35°/〇 for a hairpin type shRNA meson- 49%, and for both synonymous RNA and antisense RNA mesons range from 90% to 100%. Example 2 The genes were cloned into a scorpion scorpion scorpion mir-302 homologue inserted into the plant and jVAVRGFP due to the recombinant transgene in its five-terminal and three-terminal Each has a specific alpha/limit, which can be easily colonized into a carrier having a relatively adhesive end that adheres to the ΧΑο/ and the restriction. As shown in Figure 3A, we combined the transgenic genes into a linearized ~6,900 卩/?7^-〇?247^ plastid in a 1:1 weight ratio (w/w). 'The mixture was cooled from 65 ° C to 15.0 during one of 50 minutes and then τ4 ligase and buffer were added to the mixture accordingly to join 12 small 81 201009074 at 12 °C. This formed a vector that can induce the brothers AW- and GF corpse. The composition of the vector was confirmed by pCR with specific primers SEQ.ID.N0.23 and SEQ ID N〇24, which were carried out at 94 ° C for one minute and then at 68 ° C for two minutes. 3 loops, further sequencing. In the selection of the lean to one viral vector, the same restriction and ligation procedure was carried out except that the unambiguous linear ' cadaveric UVCX2 retroviral vector (BD Clontech) was used instead. Since the inserting position of the brother's intron is flanked by a pvw/ and a limit at its five and three ends respectively, we can remove and replace the anti with a man-made reset ^^302 shRNA ® meson - Five GFP shRNA mesons, the artificially relocated mir-3 02 shRNA meson has adhesion to the Pvw/ and Mw/selection sites. The reset mir-302 shRNA meson comprises a sequence homologous to 5,-UAAGUGCUUC CAUGUUU-3' (SEQ.ID.N0.3), which comprises: 5'-UAAGUGCUUC CAUGUUUUAG UGU-3, (SEQ.ID. N0.9), 5,-UAAGUGCUUC CAUGUUUUGG UGA-3' (SEQ.ID.NO. 10), 5,-UAAGUGCUUC ^

O CAUGUUUUAG UAG-3' (SEQ.ID.NO.il), 5'-UAAGUGCUUC CAUGUUUCAG UGG-3, (SEQ.ID.N0.12), or 5,-UAAGUGCUUC CAUGUUUGAG UGU-3' (SEQ.ID. N0.13). More preferably, the reset mir-302 shRNA meson comprises 5,-UAAGUGCUUC CAUGUUUUAG UGU-3, (SEQ.ID.N0.9). In other words, the gene silencing effector comprises at least one recombinant RNA sequence which is SEQ.ID.N0.9, SEQ.ID.NO.10, SEQ.ID.NO.n, SEQ.ID.N0.12. SEQ. ID. N0.13, and a set of 82 201009074 group homologs.

The synthetic nucleosides for DNA recombination of the recombinant mir-302 family pre-miRNA or the artificially-reset mir-302 shRNA meson are listed below: Synonymous mir-302a, 5'-GTCCGATCGT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATCTCGAGCT C-3, (SEQ.ID.NO.29); antisense mir-3〇2a,

5,-GAGCTCGAGA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GACGATCGGA C-3, ❹

(SEQ.ID.NO.30); CGCTCCCTTC TTCTGTGACT GTTTTAGTAG (SEQ.ID.NO.31); GACTCCTACT TTCAAAGTCA AAGTTGAAGG (SEQ.ID.NO.32); ACCTTTGCTT TGTGAAACAG

Synonymous mir-302b,5,-ATCTCGAGCT

AACTTTAACA TTGAAAGTAA GAGTCGCTAG TGGAAGTGCT GTGCTTCCAT CGCTA-3’

Antisense mir-302b , 5’-TAGCGCTAGC

AAAACATGGA CAGAAAGCAC GAGCGAGCTC AGCACTTACT TTCCATGTTA GAGAT-3,

Synonymous mir-302c, 5’-CGCTAGCGCT

TAACATGGAG AAGTAAGTGC

GTACCTGCTG TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3, (SEQ.ID.NO.33); antisense

Mir-302c , 5,-ATGTCTAGAC GCCTCCACTG

AAACATGGAA GCACTTACTT CTGTTTCACA 83 201009074 AGCGCTAGCG-3' (SEQ.ID.NO.34);

CAGCAGGTAC

5,-CGTCTAGACA ACTTAGCTAA

CTCCATGTTA

TAACACTCAA

GCCAGGCTAA

AAGCAAAGGT Synonymous mir-302d, ACATGGAAGC GTGCTTCCAT

GTTTGAGTGTTCGACGCGTCAT-3' (SEQ.ID.N0.35); antisense mir-302d, 5,-ATGACGCGTC GAACACTCAA

ACATGGAAGC ACTTAGCCTG GCTTAGCTAA

GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3, (SEQ.ID.NO.36); and synonymous miR-302s,5,-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA

GCCAGGCACA CTAAAACATG GAAGCACTTA

TCGACGCGTC AT-3, (SEQ.ID.NO.27); antisense mir-302s, 5,-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3, (SEQ.ID.NO.28) (Sigma-Genosys, MO). All of these synthetic sequences were purified by PAGE colloid extraction prior to conjugation. In other words, the gene silencing effector has SEQ.ID.NO.29, SEQ.ID.NO.30, SEQ.ID.NO.31, SEQ.ID.NO.32, SEQ.ID.NO.33 A junctional link of a hybrid of SEQ. ID. NO. 34, SEQ. ID. NO. 35, SEQ. ID. NO. 36, and a combination thereof is formed. The recombinant mir-302 family pre-miRNA gene group is formed by four groups of mir-302a-d hybrids, including: synonymous mir_3〇2a and antisense mir-3〇2a, synonymous mir-302b and anti-义 mir_3〇2b, synonym 84 201009074 mir-302c and antisense mir-302c, and synonymous mir-302d and antisense mir-302d. These mir-302a, mir-302b, mir-302c, and mir-302d t-complexes allow PvuIlXhoI, XhoIlNhel, NhellXbal, JSl; Ώ)β//Μ/ζ// restriction enzymes to be eliminated, and After 35 μl of autoclaved ddH20 was collected by a gel extraction filter column (Qiagen, CA), the mixed hybrids were immediately joined to form a A gene group (Roche, 20 U) of the mir-302 family pre-miRNA meson of T4 DNA ligase, and further inserted into these cadaver vw//Mw/linearization and GFP expression vectors. In other embodiments, the artificially reprogrammed mir-302 shRNA is made by hybridizing two synthetic sequences of SEQ.ID.NO.27 and SEQ.ID.NO.28, and then cleaved with Pv restriction enzyme To insert these pvw//M/w/linearization expression vectors. According to the procedure set forth in Figures 2A and 2B, the inducible SpRNAi-RGFP expression containing the recombinant mir-302 family pre-miRNA (i.e., pTet-On-tTS-mir302s) is transmitted to the hHFC Intracellular, however, the vector containing the mir-302 shRNA was introduced into Colo 829 cells. The 5^ see 4/- and GF cadaveric expression vectors can be propagated into a medium containing 500 pg/ml of ampicillin in DH5a LB medium (Sigma Chemical, St Louis, MO). Such propagated expression vectors are isolated and inactivated using a mini-prep or maxi-prep plastid extraction tool (Qiagen, CA). In terms of retroviral vectors, we can also use a packaging cell line, GP2-293 (Clontech, CA), to create an infectious but not self-replicating 85 201009074 (replication-incompetent). The transfected GP2_293 cell line was grown in 1x DMEM medium at 37 ° C and 5% CO 2 . The supplement of the medium was 10% carbon adsorbed fetal bovine serum (FBS) containing 4 mM L - Facial amine, 1 mM sodium pyruvate, 100 pg/ml streptomycin sulfate, and 50 pg/ml neomycin (Sigma Chemical, MO). According to the procedure of a retto-XqRT-PCR titration tool (Clontech, CA), the virus titer was measured by the multiplicity of infection (MOI) 30 before transfection. Example 3 Cell Culture and miRNA of mir-302s Human cancer PC3 and Colo 829 cell lines were obtained from the American Culture Center (ATCC, Rockville, MD), while hHFC and hpESC cell lines were collagenase, respectively. / Trypsin (4:1) is prepared by decomposing 2 to 10 hair follicle roots or 2 mm3 skin explanted from the hair or arm of the inventors. The cells were cultured in RPMI 1640 medium at 37 ° C and 5% CO 2 . The supplement of the medium was 1 〇. /. Fetal bovine serum (FBS), 4 mM L-bromoamide, 1 mM sodium pyruvate and 1 〇〇 pg/ml statin (Sigma Chemical ' MO). The cells were exposed to trypsin-EDTA solution for 1 minute and rinsed once with RPMI, and the isolated cells were re-plated in fresh medium (growth medium) at a ι:ι dilution. Make a 70% to 80% plate full of culture. Regarding the transfer of mir-302s by electroporation, the vector (10-30 pg) was mixed with the host cells (200-2000) in a hypotonic pH buffer 86 201009074 (400 μΐ; Eppendorf). And the electroporation method was carried out for 100 microseconds at 4 〇〇 45 volts to deliver the vector into the host cell genes. After 72 hours, the positive gene-transferred mirps was isolated and collected. The cells were screened by anti-RGFP monoclonal antibody using a FACS flow cytometer (Fig. 3C). The success rate of this novel mir-302s gene transfer method was measured to exceed 91%. In other examples, in the delivery of retroviral vectors, we first cultured P plants to transfer GP2-293 cells (Clontech, CA). ) having a recombinant mir-302 family pre-miRNA meson

SpRNAi-RGFP was inserted into pLNCJQ-var transcriptional virion. The medium of GP2-293 cells (10 ml per medium) (0.25 μιηη) was filtered and incubated at 3 ° ° (: and 5% C 〇 2 for 36 hours) at 37 ° <: and 5% C 〇 2 This was transferred directly to the cell cultures under test for 12 hours. Thereafter, fresh mirPS cell culture medium was added instead of the virus medium and replaced every three days. Since these media contain extremely high design reverse transcription The titer of the viral vector, almost all the cells tested (99.4%-99·8°/〇) were infected with the vector gene and began to express the introns within 24 hours. And RGFP. The positive gene-transferred mirPS cells were isolated and collected 24 hours after infection, and the cells were sorted by anti-RGFP monoclonal antibody (clontech, ca) using a FACS flow cytometer. Example 4 Northern blotting Spelling all RNAs (20 s) on 1 ° / ❶ formaldehyde - acacia and transferred to 87 201009074 nylon membrane (nylon membrane, Schleicher & Schuell, Keene, NH). Synthesis of LNA-DNA probe (sigma_Genosys , MO) can be complemented between these and OTP five-terminal exons or The designed 75-bp junction sequence of the pre_miRNA/shRNA meson, or a complementary gene transcriptional molecule, which was synthesized using the Prime-It II tool (Stratagene, La Jolla, CA). [32P]_dATP appears as a random primer extension ([32p]_dATP > 3000 Ci/mM, Amersham Internationa Arlington Heights, IL) with a 1〇bp-cutoff Micro Bio-Spin column (Bio -Rad, Hercules, CA) 纯化 Purification. Hybrids in 50% freshly deionized formamide (pH 7.0), 5 times Denhardt's solution, 0.5% SDS, 4 times SSPE and 250 mg/mL The denatured salmon sperm DNA fragment (18 hours, 42 ° C) was mixed in. The membrane was washed twice in 2 times SSC, 0.1% SDS (15 minutes, 250C) before autoradiography. And washed once in 0.2 times SSC, 0.1% SDS (45 minutes '37. 。. These results are shown in Figures 4B and 6B. _ Example 5 gDS-PAGE and Western point smashing method for the target protein Immunoblotting (Figures 8C and 9B), after removing the medium Ice-cold PBS was rinsed to about ~70% of the plate to separate the cells, and then a CelLytic-M dissolution/extraction reagent (lysis/extraction reagent, Sigma-Aldrich, MO) was dissolved, which was supplemented with a protease inhibitor, Leupeptin, TLCK, 88 201009074 TAME and PMSF. The cell lines were incubated on a vibrator (10) for 15 minutes at room temperature, then scraped into a microcentrifuge tube and centrifuged at 12,000 xg for 5 minutes to pellet the cell debris. . The cell lysate containing the protein was collected and stored at 7 〇〇C for use. Protein quantification was measured on an E-max micro-disc reader (Molecular Devices, Sunnyvale, CA) using the SOFTmax kit software. Under the conditions of reduction (+50 mM DTT) and non-reducing (no DTT), 30 ton of cell lysate was added to the SDS-PAGE sample buffer and loaded into the polypropylene raft. The amine gel was boiled for 3 minutes before; the molecular weight was determined by comparison with standard protein (Bio-Rad, Hercules 'CA). SDS-polyacrylamide gel electrophoresis was carried out according to these standard operating procedures. The protein decomposed by PAGE was electrolyzed on a nitrocellulose membrane and incubated for 2 hours at room temperature in an Odyssey blocking reagent (Li-Cor Biosciences, Lincoln 'NB). Then, we applied a native antibody to the reagent and incubated the mixture at 4 °C. Native antibody use includes: Oct3/4 (1:500, Santa Cruz), SSEA-3 (1:500,

Santa Cruz), SSEA-4 (1:500, Santa Cruz), Sox2 (1:500 ’

Santa Cruz), Nanog (1:500, Santa Cruz), Klf4 (1:200, Santa Cruz), β-actin (1:2000, Chemicon, Temecula, CA), and RGFP (1:1000, Clontech). After one night, the membrane was rinsed three times with TBS-T and then exposed to goat anti-mouse IgG for 1 hour at room temperature. The goat anti-mouse IgG and Alexa Fluor 680 reactivity Dyes (1:2,000; Invitrogen-Molecular Probes) were combined to form a serogroup 89 201009074 antibody. After three additional TBS-T rinses, the Li-CorOdyssey Infrared Imager (^ Infrared Imager) and Odyssey Software Version 10 were used to manage the fluorescent scanning and image analysis of the immunospot ink method. Example 6 Intron RNA in zebrafish mediates a cause of silence

During transfection, the zebrafish larvae of the Tg (flciz > 7-GAL4: UAS-gfp) variety were reared in a fish tank containing 10 ml of 0.2-fold serum-free RPMI 1640 medium. A transfection premix was prepared by carefully dissolving 60 μM of FuGene liposome transfection reagent (Roche Biochemicals, Indianapolis, IN) in 1 μL of serum-free RPMI 1640 medium. Next, as shown in Example 1-2, the carrier (20 μ§) with an anti_five GFPpre_miRNA meson was mixed with the premixed solution, and then placed on ice for 30 minutes and then directly applied to the aquarium. The Tg0CGAL4: UAS-gfp) on the fish larvae. All two doses (60 pg total) were given at 12 hour intervals. Samples were collected 6 hours after the first transfection. Figure 1B shows the result. Example 7 After the desired experiment, the cell trypsin was hydrolyzed, formed into granules, and resuspended in 丨ml of pBS towel (containing pre-cooled 7 (tetra) sterol under _2 〇c ) 'm fixed cells for 1 hour. The brain is finely granulated and washed once with 1 ml of PBS. The cells were granulated and granulated and resuspended in 1 ml of PBS solution (containing 1 mg/ml propidium iodide, 0.5 mg/ml RNase) at 37〇c 201009074. In the middle of 30 minutes. Then, about 15, cells were analyzed on a BD FACSCalibur flow cytometer (San Jose, CA). The cell dimers were excluded by plotting the pulse width versus pulse area and circled the mono-cells. Use the package software Flowjo to analyze with the "Watson Pragmatic" algorithm

Information collected. As shown in Figure 5A, the first (left) and first (right) peaks of the flow cytometer chart represent the amount of cell populations that are dormant in the G0/G1 and mitotic stages of the entire population of cells tested. Example 8 Demethylated Haitang Road The genomic DNA line from about two million cells was isolated by a DNA isolation kit (Roche) and divided into two small doses. One of the small doses of DNA (2 pg) # a CCGG cleavage restriction enzyme was used to resolve and then evaluated by 1% acacia gel electrophoresis to determine the demethylation of the pan-gen (Figure 7Α). Before and after the disulfite modification, another small dose (2 μΕ) was used to PCR-select the entire 9,400 base pair (bp) five-terminal regulatory region of the promoter (NT-1592 nucleotide 21992184) -22001688). The disulfite modification was carried out using a CpGen〇me DNA modification tool (Chemicon, CA). The DNA was treated with dithionite to convert all ungerminated cytoplasm into urinary, while the thiolated cytosine remained as cytosine. For example, change the unmethylated ACGT bit (instead of the conjugated ACGT) to the augt bit. 91 201009074 PCR primers for the targeted five-terminal promoter region were designed and tested before and after sulfite modification, including: two forward primers 5'-GAGGAGTTGA GGGTACTGTG-3' (SEQ.ID.NO.44) through

Dithionite-modified DNA) and 5'-GAGGAGCT (3A GGGCACTGTG-3' (SEQ.ID.NO.45) (unmodified dna) and a reverse primer 5'-GTAGAAGTGC CTCTGCCTTC C_;3, (SEQ.ID.NO.46). In terms of PCR selection, whether or not these genomic DNAs (50 ng) have been treated with or without disulfite, first in 1x PCR buffer and The primers (150 pmole) were mixed and heated to 94 ° C for 4 minutes, then immediately cooled on ice. Thereafter, % of the following PCR cycles were performed: 1 minute at 92 ° C and 55 ° C 1 minute, then 5 minutes at 70 ° C using a long template PCR extension tool (Roche). The resulting product was collected by a PCR purification kit (Qiagen) and cleaved with various ACGT restriction enzymes. A homogeneous mixture (5 U each) to dissipate 2 pg of DNA, including: U (AACGTT), (CACGTC), Pm// ❹ (CACGTG) ' SnaBI (TACGTA)^ HpyCH4IV (ACGT) 〇m Thereafter, 3% acacia gel electrophoresis was used to evaluate the eliminated fragments (Fig. 7B). In the case of disulfite DNA sequencing analysis (Fig. 7C), Further, a 467-bp target region was amplified by quantitative PCR (qPCR), which was flanked by the OciW transcription initiation site (NT-007592 nucleotide 21996577-21997043). The primer used was a forward primer 5'-GAGGCTGGAG. TAGAAGGATT GCTTTGG-3, (SEQ.ID.NO.47) and a reverse primer 92 201009074 Sub 5, -CCCTCCTGAC CCATCACCTC CACCACC-3, (SEQ.ID.NO.48). The above-mentioned 9400-bp OciW The five-terminal promoter region (50 ng) was mixed with the qpCR primers (100 pni〇le total) in 1x PCR buffer and heated to 94. (1: 2 minutes, then immediately cooled on ice. Perform 2 PCR cycles as follows: at 94. (: 30 seconds, at 68. (: 1 minute, using a high-fidelity PCR extension kit 'Roche) The amplified DNA product with a correct 467-bp length was further fractionated by 3% gel electrophoresis and purified by a colloidal extraction tool (Qiagen) and then used for DNA sequencing. A detailed map of one of these DNA thiolation sites is obtained by comparing unaltered cell bites in the disulfite-modified DNA sequence and in the DNA sequence not modified by disulfite. The unchanging cells are bitten to produce. Example 9 Elicitation of ribonucleoside (miRNA) withdrawal display analysis Under the full plate, small RNAs were isolated from each cell culture using the mz>VanaTM miRNA isolation tool (Amion). °1% furfural was used_ Acacia gel electrophoresis and spectrometer measurements (Bi〇Rad) were used to evaluate the purity and quantity of these isolated small RNAs, which were immediately sent to LC Sciences (San Diego 'CA) for miRNA microarray analysis. Each microarray wafer is hybridized with a single sample labeled Cy3 or Cy5, or background subtracted and normalized with one of the samples labeled Cy3 and Cy5. In a double sample 93 201009074 test, a p-value calculation was performed and a list of more than 3 times the differentiation performance transcripts was generated. In the Cy3 and Cy5 intensity images (blue background) of Fig. 6A, when the signal intensity is increased from the first level to the 65th, 535th level, the corresponding color changes from blue to green, yellow, and then to red. The message strength is above 23, and is considered a positive call in gene expression. In Cy5/Cy3 than in the image (black background), the color is green when Cy3 is higher than Cy5; the color is yellow when Cy3 is equal to Cy5; and the color is red when Cy5 is higher than Cy3. . Example 10 Whole Gene 艚 Microarray Divided into a Human Genome of Over 54,000 Nucleotide Probes GeneChip U133A & B and Plus 2.0 Array (Affymetrix, Santa Clara, CA) It is used to detect the phenotype of 47,000 human gene transcripts of ubiquitin in mirPS cells, as shown in Figures 8A and 9A. Each sample was tested in the third copy and repeated the same experiment four times. All RNA from each sample tested was isolated using RNeasy spin columns (Qiagen). To prepare probes for microarray hybridization, the entire extracted RNA (2 pg) was converted to a double-stranded cDNA using a Superscript selection system (Invitrogen) with a synthetic 〇ligo(dT)24- T7 promoter primer 5, -GGCCAGTGAA TTGTAATACG ACTCACTATA GGGAGGCGG-(dT)24-3' (SEQ.ID.NO.49). The obtained cDNAs were purified by extraction with phenol/chloroform, precipitated with ethanol, and resuspended at a concentration of 0.5 pg/μΐ in secondary water treated with diethyl pyrocarbonate 94 201009074 (diethylpyrocarbonate 'DEPC) (ddH2〇 )in. In vitro transcription was then performed, including: 1 pg of dsDNAs, 7.5 mM uncalibrated ATP and GTP, 5 mM uncalibrated UTP and CTP, and 2 mM sputum

The peptides were labeled with CTP and UTP (biotin-ll-CTP, biotin-16-UTP, Enzo 'Diagnostics), and 20 U of T7 RNA polymerase. The reaction was carried out at 370 C for 4 hours, and the resulting cRNAs were then purified on an RNeasy centrifuge column (Qiagen). A portion of the CRNA sample was separated on a 1% agar extract to examine the length range and then at 94. (: 40 mM Tris-acetate, 100 mM KOAc/30 mM MgOAc was heated for 35 minutes at pH 8.0, and 10 pg of cRNAs were randomly fractionated into an average length of 50 bases. The mixture was continuously mixed for 16 hours at 40 ° C in AFFY buffer (Affymetrix) to complete the hybrid. After the hybridization, 6 times SSPE-T buffer (1 times 0.25 Μ sodium sulphate / 2 times) 15 mM sodium silicate 'pH 7.6 Λ mM EDTA / 0.005. / nucleus.) The array was rinsed three times and then washed with 200 μl of 6 times SSPE-T for 1 hour at 50 ° C. The arrays were additionally 0.5. Double the SSPE-T rinse twice and use 0.5 times SSPE-T at 50. (: Wash for 15 minutes. Then, with 2 pg/ml of aggretin-phycoerythrin (Invitrogen-Molecular Probes) And 1 mg/ml of deuterated BSA (Sigma) The staining test was performed in 6 times SSPE-T (pH 7.6). The arrays were read at 7.5 μπ with a confocal scanner 'Molecular Dynamics'. In order to identify the change in the background, we use the same sample to replicate the microarray tests and select two hundred genes (圊8) White point) is compared to the step of step 2010 20107474, which is slightly presented on one side of the test. The sample signals are normalized using the total average difference between the perfect matching probe and the mismatched probe. Use Affymetrix Microarray Suite version 5.0, Expression ConsoleTM version 1.1.1 (Affymetrix) and Genesprings (Silicon · Genetics) software to analyze changes in the overall ubiquitin gene expression pattern (green dot in Figure 8A). More than doubled (〇 The change in gene expression rate of ne-fold) is considered as positive differential genes. In the gene cluster test, a plug-in Genetrix (Epicenter Software) is used in conjunction with Affymetrix software to be used in each microarray. The internal housekeeping gene ❿ controls the average normalization of the sample signal. After normalization, when the signal intensity increases from level 1 to level 65, 535, the corresponding color changes from green to black to red. At level 23,000 The strength of the message above (red) is considered a forward call, and a northern ink dot analysis can be detected as positive. Example ii Immunodetection Xie Wei was subjected to xenografting of these mirPS-derived embryoid bodies to "Zhouda female pseudopregnancy immunity except for the treatment of mirPs culture-free cells containing no mammalian cells without any treatment A teratoma-like cyst (10) 1()) can be formed in the spleen or abdominal cavity of a SCID_bdge mouse with insufficient function. This immunocompromised nude mouse is used to provide an integrated internal environment for mimicking transplant therapy. The method of making the rice it It pregnant mouse is: after the intraperitoneal injection of humans, the human growth hormone (human men〇pausal § bribe, plus g) two, '; after / main shot human fluff Membrane gland hormone ch〇ri〇nic 96 201009074 gonadotrophin, hCG) - day. In vitro molecularly guided stem cells differentiated into germline lineages, mirPS cells were maintained in DMEM/F12 (1:1; high glucose) medium in poly-guanine/base membrane cover at 370C and 5% CO 2 ( Polyporithine/laminin-coated for 12 hours, the supplement of the culture solution is 10% carbon adsorption FBS, 4 mM L-age guanamine, 1 mM sodium citrate, 5 ng/ml activin and 50 ng/ml Dihydrotestosterone (DHT). Thereafter, the proteolytically hydrolyzed the cells was washed with 1 PBS and collected in four small doses of frozen Matrige (100 μM each) and a small dose of 1 μM in 1 PBS. After that, we immediately transferred the cells to the six-week-old SCID-beige nude mouse hind limb muscle, peritoneum, uterus, subcutaneous skin (including Matrigel) and tail vein (including PBS). The mice were anesthetized with diethyl ether during the experimental treatment. One week later, spermatogonial cells were found only in the uterine region. In terms of fibroblast differentiation, we performed the same procedure as shown above before allogeneic transplantation, but using a standard DMEM medium containing no pan red for 6 hours, the supplement of the culture solution was 100/〇FBS, 4mML-bran Indoleamine, 1 mM sodium pyruvate, 5 ng/ml Noggin protein and 100 ng/ml transforming growth factor ji (TGF-pl). Fibroblasts were found in the uterus a week later. In terms of chondrocyte differentiation, we performed the same procedure as before, but using standard RPMI 1640 medium for 6 hours. The composition of the medium was 10% FBS, 4 mM L-guanamine, 1 mM sodium pyruvate and 1 ng/ml bone morphogenetic protein 4 (BMP4). Chondrocytes are found only in the liver area. 97 201009074 In the case of immunodetection of specific tissue markers, these tissue samples were fixed overnight in 4% paraformaldehyde at 4 °C. These samples were washed successively with 1x PBS, methanol, isopropanol and tetrahydronaphthalene before being embedded in the stone ants. The embedded samples were then cut at a thickness of 7-10 μηη on a microtome and mounted on a clean TESPA coated slide. The waxes of the slides were then removed with xylene and fixed under coverslips using mounting gel (Rising media; Richard Allan Scientific » Kalamazoo » MI), followed by hematoxylin and sputum Red® (eosin) (H&E, Sigma) stained for morphological observation. Immunohistochemistry (IHC) staining tools were purchased from Imgenex (San Diego, CA). Procedures for antibody dilution and immunostaining are performed according to the manufacturer's recommendations. Native antibodies used include: Tujl (1:500, Abeam Inc., Cambridge, MA), ABCA2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), Dazla (1:100, Abeam), EE2 (1: 100, Santa Cruz), atlastinl (1:200 ' Santa Cruz), COL1A1 (1:500, Santa Cruz), COL2A1 (1:500, Santa Cruz), tropoelastin (1:200, Abeam), and RGFP (1: 500, Clontech). A goat anti-rabbit or horse anti-mouse anti-system calibrated by fluorescence staining was used as a secondary antibody (1:2,000, Invitrogen-Molecular Probes). Positive results were observed under a 100x microscope with full field scan and measured at a magnification of 200x or 400x using a Metamorph image processing program (Nikon 80i and TE2000 microscopic quantitative analysis system) for quantitative analysis. 98 201009074 Example 12 Cell Migration Test In a 96-well plate, we placed a PC3 and a mirPS-PC3 cell in each well and recorded their movement and interaction. The cells were grown in RPMI 1640 medium at 37 ° C and 5% CO 2 . The supplement of the medium was 10% carbon adsorption FBS, 4 mM L-clinamide, 1 mM sodium pyruvate, 5 ng. /ml activin, 5 ng/ml Noggin protein, 3 ng/ml bFGF and 0.5 μM GSK-3 inhibitor XV. The cells were individually separated and collected using a pair of MO-188NE 3D hydraulic fine micromanipulators under a TE2000 inverted microscopic system (Nikon). The micromanipulator has a Cell holder. The entire micromanipulator and microscope system are placed on a shockproof table. Record these photos every 15 seconds (magnification 400x and 600x) for 6 hours. The cell migration is determined by tracking the movement of the cells in the photographs and the morphology of the cells. As shown in Figure 7D, metastatic cancer PC3 cells exhibit a rapid fusiform movement as described by ATCC, wherein the mirPS-PC3 cells remain in the placement position' which exhibits a circular dormant phenotype. Example 13 Statistical Fractions These results are expressed as mean ± standard deviation (mean ± SE). The statistical analysis of the data was calculated by one-way ANOVA. 99 201009074 When the main effects are obvious, the Dunnettis post-hoc test is used to identify groups that are significantly different from the control group. When comparing between the two treatment groups, a two-tailed student test (tw〇_tailed student纟test) was used. For experiments involving more than two treatment groups, the variance analysis (AN0VA) was performed according to a post-hoc multiple range test. The probability value p<〇.〇5 is considered to have statistical significance. All values are determined by a two-tailed test (tw〇_taile(j test).

BRIEF DESCRIPTION OF THE DRAWINGS The following illustrations are for illustrative purposes only and are not limiting of the invention: Figures 1A-B depict the mechanism of in vivo synthesis of intron miRNAs and their relative gene silencing effects. (Fig. 1A) Transcription of an intron miRNA into a portion of the precursor message RNA (p_RKA) which contains a protein-encoded exon and a non-coding intron. The introns are spliced out and some of the secondary structures are further digested to induce a silent gene in the small miRNA 77, and the exons are ligated to form mature mRNA. For the labeling of eggs from f synthesis. (Item 1B) Example of transfecting the intron miRNA gene of the design into the zebrafish zebrafish shows a strong gene silencing effect on the target green EGFp expression (> 80% inhibition, left The fourth column), however, other non-standard mesons are not affected by the shadow 100 201009074, including (from the first column to the fifth column): one of the miRNAs without the miRNA (1), against the ancestor-p24 (2 Or an intron of a pre-miRNA meson that binds to either protein/« ton/?/(3), and an anti-^GFP pre-miRNA meson but a functional 5'-splice site Intron (5). The anti-five GFP pre-miRNA was inserted into the 5'-proximal intron region of the red-shifted fluorescent marker (GFP) gene. Figure) shows the mature ❹ miRNA family generated only by the splicing products of the pre-miRNA-insertion 'instead of the null and GF corpse (-) or the defective, which means intron synthesis in intron miRNAs RNA splicing is required. 2A and 2B depict the structure and strategy of a modified Tei-O« vector containing φ toilet eGFi5 transgenic gene (ie, 户2^〇„々灯_(10)^必) to express the mir-302spre-miRNA gene group Or shRNA. An electroporation-type gene transfer delivery method is used to transfer the mir-302-transferred gene into a somatic/cancer cell. Figure 3A-3C shows human normal hair follicle hHFC cells and cancer. Melanoma Col cells are transformed into ES-like pluripotent stem (mirPS) cells, which are transfected with a pre-designed Γβ-〇« mir-302-expressing transgenic gene (Fig. 3A). «Recombinant shirts in the inducible vector (ie, must) _3〇2_ The expression of the transgenic keise (ie SpRNAi-RGFP). The SpRMAi-RGFP transgenic line is 370 base pairs (bp) The source region is flanking and is used to recombine the target position of the human cell genome of 101 201009074. (Fig. 3B) The construction of the mir-302 pre-miRNA gene group (mir-302s), which is merged into the transgenic gene of the plant. Part of the intron. (Fig. 3C) Selection of positive mir-302-transduced mirPS cells using a FACS flow cytometry The flow cytometer classifies cells for antibodies against RGFP-tagged proteins. The success rate of transgenic gene delivery of the present invention is currently measured to be about 91% to 93%. Figures 4A-4B show the miRNAs in the mirps cell 7^-〇„shirt_3〇2-expressing the integration and integration of the transgenic genes, which result in the mir_3〇2 family members (mir-302s) under the control of various concentrations of deoxygenated hydroxyl (Dox) Induced performance. (Fig. 4A) Quantitative PCR (qPCR; left panel) analysis of genomic DNA isolated from different mirps cell lines, showing that all mjjps cells carry only one or two accompanying copy sequences of the transgene, in which Transduction was detected in hHFC and Colo cells; gene (control group). Fluorescence in situ hybridization ^piu〇rescent plus-fishing/hybridization, FISH; right panel) The experiment further shows that the transgenic gene is inserted into a specific location of the human genome. This restriction gene transfer insert implies that the concentration of the overall mir-302 expression can affect the survival of the mirps cells (Fig. 4B). The northern blot analysis and histogram display corresponding to the Dox concentration that induces mir_3〇2s performance. . Figures 5A-5C show changes in the conversion of human normal hair follicles (hHFC) and cancer melanoma cancer Colo cells to ES-like pluripotent stem (mirps) cells. (Fig. 5A) In these mir-302-transduced cells (ie ··mirps-hHFC and mirPS-Colo cells) 102 201009074

Morphological changes and cell proliferation rates. The shape of ES-like round cells and the very slow cell regeneration rate were found in hydroxy-302-transduced cells with deoxyhydroxyl (Dox) induction. (Fig. 5B) Formation of embryonic bodies (EB) derived from such mirps cells and guided differentiation into neuronal precursor cells with positive Tujl and/or ABCA2 markers. (Fig. 5C) A time course change pattern of EB formed from a single mirPS cell after limiting dilution. These cell cycles are estimated to be approximately 2 〇 24 hours. Figures 6A-6B show the correlation between mir_3〇2s transfection and ES marker expression. (Fig. 6A) Microarray analysis of overall miRNA expression, showing that all mir_3〇2 family members (mir-302s) are predominantly expressed in dmirx-induced mirps cells, rather than in s Xuan et al. In the cells (n=3, p<〇〇1). (Fig. 6B) Northern point ink and western point ink analysis method, the lions Dgx Weizhi these mkps cells showed abundant ES cell markers, including: Qet3/4 _4), %ΕΑ_3, SSEA-4, Sox2 and Nanog However, Klf4 (n=4, p < 〇〇 1), k, which performed less, is very similar to the markers of human ES WAG1_H1 and WAG9_H9 (10). Figures 7A-7D show that the HpaII digestion of the gene in the various mirps cell lines (Fig. 7A) shows that the CpG is low in the ubiquitin size of the harvested cells. _) will cut the ship's acct via the disulfite modified Yang 3/4 promoter 9,4 〇〇 test pair in the regulatory region, which shows the unmethylated lion in all secret cells (or 103 201009074 AUCT) Significant increase in position. (Fig. 7C) Disulfite DNA sequence showing a detailed thiolated map on the side of the start of the Oct3/4 promoter. Black circles and white circles indicate the thiolated and unmethylated cytosine positions, respectively. (Fig. 7D) Loss of migration ability in mirPS-PC3 cells compared to the initial metastatic cancer PC3 cells of mirPS-PC3 cells. Figure 8A-8B shows the analysis of pan-gene expression in colo, mirPS-Colo and human ES WA01-H1 (H1) and WA09-H9 (H9) cells. (囷8A) Comparison of altered gene expression patterns using Human genome GeneChip Ul33A & B© and plus 2.0 arrays (Affymetrix), shown in mirPS-Colo and Hl (89%) and H9 (86) High similarity between °/〇), but less similar to cancer-type Colo (53%) cells. White dots indicate highly variable genes compared to the stable expression gene (green dot). (Fig. 8B) Microarray recognizes functional gene clusters of differentiation-expressing genes' which demonstrates in mpreS cells: significant increase in es cell markers and melanoma oncogenes, developmental messages, and mir-302-target cell proliferation and DNA A significant reduction in methylation genes is found to be similar to that detected in H1 and H9 cells (η = 4, ρ < 〇.〇ι). Figure 8C shows Northern blotting and western blotting analysis confirming the correlation between the expression patterns of mir-302s, human ES cell markers, and predicted mir_3〇2 genes in mirps_c〇1〇 cells, which is related to human Es H1 and The pattern of expression in H9 cells was similar to 'except for the expression of Klf4 (n=3., ρ<〇.〇1). Figure 9Α-9Β shows the ubiquitin gene expression between hHFC, mirPS-hHFC and human ES WA01-H1 (HI) and WA09>H9 (H9) cells 104 201009074 Analysis. (Fig. 9A) Comparison of altered gene expression patterns using the human genome gene chip ui33 plus 2.0 array (Affymetrix) showing high similarity between mirPS-hHFC and Hl (96%) and H9 (91%), but It is less similar to hHFC (47%-56%) somatic cells. (Fig. 9B) Western blot analysis confirming the correlation between these expression patterns of mir-3〇2s, human ES cell markers and predicted mir-302 genes in mirPS-hHFC cells, which is related to human Es HI and These patterns of expression in H9 cells are similar except for the appearance of Klf4 and Klf5. The listed mir-302 genes include: seventeen transcriptional regulators, one tissue protein deacetylase (HDA4), two thiol-based CpG-binding proteins (MECP1-p66 and MECP2), and three cell cycles. Checkpoint proteins (CDK2, cyclin D1 and D2). Figure 10 shows teratomas-like native tissue (n/total = 6/6) derived from mirps grafts in the uterus or peritoneal cavity of female pseudopregnant immune function SCID-beige mice. These differentiated tissues include all three embryonic vegetative layers: ectoderm, mesoderm and endoderm, which are succinctly stained after hematoxylin and eosin (Η & E) staining. Different cell morphology is used to judge. The microscope photographs were taken at 2 〇〇χ magnification using a Nikon ΤΕ2000 system. Figure 11 - 110 shows the guiding pluripotency of mirPS cells. DHT, TGF-βΙ and BMP4 were treated from top to bottom, respectively, and the mirPS cells were induced to differentiate into spermatogonia (Fig. 11A-E) and fibroblasts (Fig. 11F_ and chondrocyte) (Fig. 11K) -0) Tissue cells' their lines are treated ex vivo in mice with insufficient immune function. The immune function is abundant in _environmental simulation transplantation. The photomicrograph shown from left to right indicates: use Heterogeneous differential interference hematoxylin staining (Fig. ΠΑ, F, K), marked with the transgenic gene mir_3〇2 labeled RGFP marked bright field (red) (Fig. 11B, G, L), with 4,6-mercapto- 2-phenylpurine (4,6-diamidino-2-phenylindole)-like immunostaining of the first tissue marker (blue DAPI) (Fig. 11C, H, M), second labeled with luciferin Tissue-tagged immunostaining (green EGFP) (Fig. 11D, I, N), and the combination of all three fluorescent markers (Fig. 11E, J, 0). Displayed in small windows in these RGFP-bright fields Morphology of differentiated mirPS cells at high magnification (600x) 106 201009074 [Sequence Table] <110> Lin, Shi-Lung Lin Xilong Wu, David TS Wu Tangxi<1 20> Use of inducible recombinant ribonucleic acid to generate embryonic stem cells without cancer cell characteristics/Generation of Tumor-Free. Embryonic Stem-Like Pluripotent Cells Using Inducible Recombinant RNA Agents <150> 12/149725 <151> 2008-05 -07 <150> 61/191327 <151> 2008-09-08

<150> 61/193438 <151> 2008-11-28 <150> PCT/US09/30432 <151> 2009-01-08 <150> 12/318806 <151> 2009-01-08 <160> 53 <170> Patentln version 3.5 <210> 1 <211> 12 <212> DNA <213> Artificial sequence <220><223> Chemical synthesis nucleotides <400 〉 1 12 gctaagccag gc <210〉 2 <211> 12 <212> DNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide 107 201009074 <400>2 gcctggctta gc <;210> 3 <211> 17 <212> RNA <213> Artificial sequence <220><223> Chemical synthesis nucleotides <400> 3 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 synthetic nucleotides 201009074 <400> 6 tactway <210> 7 <211> 17 <212> DNA <213> artificial sequence <220><223> chemical synthesis oligo Glycoside <400〉 7 tytycttttt tttttts

<210> 8 <211> 19 <212> DNA <213> Artificial sequence <220><223> Chemical synthesis nucleotides <220><221> misc_feature <222> ): (15) <223> n is a, c, g, or t <400>8 tctctctctc tctcnctag <210> 9 <211> 23 <212> RNA <213> artificial sequence <220><223>Chemical Synthetic Nucleotide<400> 9 uaagugcuuc cauguuuuag ugu <210> 10 201009074 <211> 23 <212> RNA <213> Artificial Sequence <220><223> Chemistry Synthetic nucleotides <400> 10 uaagugcuuc cauguuuugg uga <210>11 <211> 23 <212> RNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 11 uaagugcuuc cauguuuuag uag <210> 12 <211> 23 <212> RNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 12 uaagugcuuc cauguuucag ugg &lt ;210> 13 <211> 23 <212> RNA <213> artificial sequence <220><223> chemical synthesis nucleotides ≪400> 13 uaagugcuuc cauguuugag ugu <210> 14 201009074 <211> 42 <212>DN A <213> Artificial sequence <220><223> Chemical synthesis oligonucleotide <400> 14 Gtaagaggat ccgatcgcag gagcgcacca tcttcttcaa ga 42 <210> 15 <211> 46 <212> DNA <213> Artificial sequence

<220><223>Chemical Synthesis Oligonucleotide <400> 15 46 42 cgcgtcttga agaagatggt gcgctcctgc gatcggatcc tcttac <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <220><223>Chemical Synthetic Nucleotide<400> 16 gtaagaggat ccgatcgctt gaagaagatg gtgcgctcct ga <210> 17 <211> 46 <212> DNA <213> Artificial Sequence <220><223> Chemical synthetic nucleotides <400> 17 cgcgtcagga gcgcaccatc ttcttcaagc gatcggatcc tcttac 46 <210> 18 111 201009074 <211> 70 <212> DNA <213> artificial sequence <220><223> chemical synthesis Nucleotide <400 18 gtaagaggat ccgatcgcag gagcgcacca tcttcttcaa gttaacttga agaagatggt gcgctcctga <210> 19 <211> 74 <212> DNA <213> artificial sequence <220><223><400> 19 cgcgtcagga gcgcaccatc ttcttcaagt taacttgaag aagatggtgc gctcctgcga tcggatcctc ttac <210> 20 <211> 47 <212> DNA <213> artificial sequence <220><223> Chemical Synthetic Nucleotide <400 20 cgcgttacta actggtacct cttctttttt tttttgatat cctgcag <210> 21 <211> 45 <212> DNA <213> Artificial Sequence <220><223> Chemical Synthesis Oligonucleotide <400> 21 201009074 45 gtcctgcagg atatcaaaaa aaaaagaaga ggtaccagtt agtaa <210> 22 <211> 689 <212> DNA <213> artificial sequence <220><223> (ifeimicto crispa) derived HcRedl chromoprotein gene in the 69th amino acid inserted aspartate (aspartate (Asp)) to generate a mutant red fluorescent protein gene <400> 22 atggtgagcg gcctgctgaa ggagagtatg 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 > Fan manufacturing sequence < 220 ><223> Chemical Synthetic Nucleotide <400> 23 113 201009074 ctcgagcatg gtgagcggcc tgctgaa 27 <210 24 <211> 27 <212> DNA <213> Artificial Sequence <220><223>Chemical Synthetic Nucleotide<400> 24 tctagaagtt ggccttctcg ggcaggt 27 <210> 25 <211> 89 <212> DNA <213> Artificial Sequence <220><223> Chemistry Synthetic nucleotides <400> 25 gtaagtggtc cgatcgtcgc gacgcgtcat tactaactat caatatctta atcctgtccc ttttttttcc acagtaggac cttcgtgca

60 89 <210> 26 <211> 89 <212> DNA <213> artificial sequence <220><223> chemical synthetic nucleotide <400 26 tgcacgaagg tcctactgtg gaaaaaaaag ggacaggatt aagatattga tagttagtaa

60 tgacgcgtcg cgacgatcgg accacttac <210> 27 <211> 82 <212> DNA <213> artificial sequence 114 89 201009074 <220> <223> chemical synthesis nucleotides <400> 27 gtccgatcgt cataagtgct tccatgtttt Agtgtgctaa gccaggcaca ctaaaacatg 60 gaagcactta tcgacgcgtc at 82 <210> 28 <211> 82 <212>DNA <213> artificial sequence <220>

<223> Chemical Synthetic Nucleotide <400> 28 60 82 60 91 atgacgcgtc gataagtgct tccatgtttt agtgtgcctg gcttagcaca ctaaaacatg gaagcactta tgacgatcgg ac <210> 29 <211> 91 <212> DNA <213> Artificial Sequence <;220< 223 > chemical synthesis nucleotides <400> 29 gtccgatcgt cccaccactt aaacgtggat gtacttgctt tgaaactaaa gaagtaagtg cttccatgtt ttggtgatgg atctcgagct c <210> 30 <211> 91 <212> DNA <213> artificial sequence<220><223>Chemical Synthetic Nucleotide<400> 30 gagctcgaga tccatcacca aaacatggaa gc This cttactt ctttagtttc aaagcaagta 60 115 201009074 catccacgtt taagtggtgg gacgatcgga c <210> 31 <211> 95 <212> DNA <213> Artificial sequence <220><223>Chemical synthetic nucleotide <400> 31 atctcgagct cgctcccttc aactttaaca tggaagtgct ttctgtgact ttgaaagtaa gtgcttccat gttttagtag gagtcgctag cgcta <210> 32 <211> 95 <212> DNA <213> Artificial sequence <220><223> chemical synthesis oligonucleotide<400> 32 tagcgctagc gactcctact aaaacatgga agcacttact ttcaaagtca cagaaagcac ttccatgtta aagttgaagg gagcgagctc gagat <210> 33 <211> 90 <212> DNA <213> artificial sequence <220 <223> chemical synthesis nucleotides <400>; 33 cgctagcgct acctttgctt taacatggag gtacctgctg tgtgaaacag aagtaagtgc ttccatgttt cagtggaggc gtctagacat

<210> 34 <211> 90 <212> DNA 60201009074 <213> artificial sequence <220 <223> chemical synthesis oligonucleotide <400> 34 atgtctagac gcctccactg aaacatggaa gcacttactt ctgtttcaca cagcaggtac ctccatgtta aagcaaaggt agcgctagcg 90

<210> 35 <211> 82 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 35 cgtctagaca taacactcaa acatggaagc acttagctaa gccaggctaa gtgcttccat gtttgagtgt tcgacgcgtc at 60 82 ❹ <210> 36 <211> 82 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 36 atgacgcgtc gaacactcaa acatggaagc acttagcctg gcttagctaa gtgcttccat gtttgagtgt tatgtctaga Eg 60 82 <210> 37 <211> 10 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 37 117 201009074 gtaagaggat <210> 38 <211> 6 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 38 guragu <210> 39 <211> 8 <212>; DNA <213> artificial sequence <220><223> chemical synthetic nucleotide <400> 39 gtaagagt <210> 40 <211> 7 <212> DNA <213> artificial sequence<213>;220><223>Chemistry Colony nucleotides <400> 40 gtagagt <210> 41 <211> 6 <212> DNA <213> artificial sequence <220><223> chemical synthesis nucleotides <400> 41 201009074 gtaagt <210> 42 <211> 12 <212> DNA <213> artificial sequence <220><223> chemical synthesis oligonucleotide <400> 42 gatatcctgc ag

<210 43 <211>11 <212>DNA<213> Artificial sequence <220><223>Chemical synthesis oligonucleotide <400> 43 wuccaagggg g <210> 44 <211> 20 <212> DNA <213> artificial sequence <220><223>primer<400> 44 gaggagttga gggtactgtg <210> 45 <211> 20 <212>DNA <213> artificial sequence<212> 220> <223>primer <400> 45 201009074 gaggagctga gggcactgtg <210> 46 <211> 21 <212> DNA <213> artificial sequence <220><223>primer<400> 46 gtagaagtgc ctctgccttc c <210> 47 <211> 27 <212> DNA <213> artificial sequence <220><223> primer <400> 47 gaggctggag tagaaggatt gctttgg <210> 48 <211&gt 27 <212> DNA <213> artificial sequence <220><223> primer <400> 48 ccctcctgac ccatcacctc caccacc <210> 49 <211> 63 <212>DNA <213> Sequence <220><223>primer<400> 49 60 201009074 63 ggccagtgaa ttgtaatacg Actcactata gggaggcggt tttttttttt tttttttttt ttt <210> 50 ' <211> 69

<212> RNA - <213> artificial sequence <220><223> intron <400> 50 60 69 ccaccacuua aacguggaug uacuugcuuu gaaacuaaag aaguaagugc uuccauguuu ❹ uggugaugg <210> 51 <211> 73 <212> RNA <213> artificial sequence <220><223> intron <400> 51 60 73 gcucccuuca acuuuaacau ggaagugcuu ucugugacuu ugaaaguaag ugcuuccaug uuuuaguagg agu ❿ <210> 52 <211> 68 <212> RNA <213&gt Artificial sequence <220><223> intron <400> 52 60 ccuuugcuuu aacauggagg uaccugcugu gugaaacaga aguaagugcu uccauguuuc aguggagg <210> 53 121 68 201009074 <211> 62 <212> DNA <213> artificial sequence<;220><223> intron <400> 53 taacactcaa acatggaagc acttagctaa gccaggctaa gtgcttccat gtttgagtgt 60 tc 62

122

Claims (1)

201009074 VII. Patent Application Range: 1. A method for converting at least one cell into at least one tumor-free pluripotent stem cell, comprising the steps of: 0) constructing a recombinant nucleic acid composition capable of being delivered, Transcribed and processed into at least one gene silencing effector that interferes with a plurality of cellular genes designated by mir-302; (b) treating a cellular matrix with the recombinant nucleic acid composition. 2. The method of claim 1, wherein the cell line is selected from the group consisting of a mammalian cell, a human cell, a normal somatic cell, a diseased somatic cell, a tumor cell, a cancer cell, a human hair follicle cell, and a cell line. Human skin wide cells, and their combined cells. In the method, the 5H cell matrix exhibits a plurality of cellular genes designated by mir-302. The step of treating the cell matrix in mir-:., /, is carried out under conditions in which the cellular genes are inhibited. 5. The method of claim 1 is heavy, wherein the recombinant nucleic acid is composed The inclusions include at least a heavy, and intron, sequence such as a homolog of horse and Qin 3〇2. 6. If requested! The method ώ mm ^ U recombinant nucleic acid composition comprises a vector, a shoulder autosome, a viral vector, an inverted vector, and a combination thereof. 123 201009074 7. The method of claim 1, wherein the recombinant nucleic acid composition comprises a drug A gene expression promoter that can be induced. 8. The method of claim 7, wherein the drug-inducible gene expression promoter is controlled by a tetracycline derivative or equivalent. 9. The method according to claim 8, wherein the tetralin derivative derivative or the like is selected from the group consisting of G418, tetracycline, deoxygenated, neomycin, ampicillin, ketomycin, derivatives thereof, And their combinations. 0_ The method of claim 1, wherein the recombinant composition comprises a ~ 或 or a gene expression system. The method of claim 1, wherein the recombinant nucleic acid composition comprises: a five-terminal donor splice site, an intron insertion site, a branch point motif, a multi-bite segment, and a three-terminal receptor Splice bit. The method of claim 11, wherein the recombinant nucleic acid composition is formed by a program selected from the group consisting of chemical synthesis, nucleocyanate recombination, genetic engineering, and combinations thereof. ^ 13_ The method of claim 12 Wherein the five-terminal donor splice site comprises or is homologous to a sequence of SEQJD*N0·4. 14. The method of claim 12, wherein the five-terminal donor splice line is homologous to 5'-GTAAG-3 The method of claim 12, wherein the branch point motif comprises or is homologous to a SEQ.ID.N0.6 sequence. The method of claim 12, wherein the branch point motif Including • 5'-TACTAAC-3' or homologous thereto. 17. The method of claim 12, wherein the multi-aged segment comprises a singularity, an ankle 7 sequence or a SEQ.ID.N0.8 sequence, or the same The method of claim 12, wherein the tri-terminal receptor splice site comprises or is homologous to a SEQ.ID.NO.5 sequence. 19. The method of claim 12, wherein the tri-terminal receptor The splice position is homologous to 5'-CTGCAG-3'. 20. The method of claim 12, wherein the intron insertion bit comprises the same as mir_3〇2 The method of claim 1, wherein the recombinant nucleic acid sequence further comprises a complex exon, which is selected from the group consisting of a glory protein marker gene, a glory enzyme gene, lac_Z lactose expression gene, embryonic stem cell marker gene, viral gene, bacterial gene, cell marker gene, skip gene, transposon, and combinations thereof. 22. The method of claim ,, wherein the gene silencing effector comprises a SEQ. A sequence homologous to the sequence of ID.NO.1 or a sequence of SEQ.ID.N0.2. 125 201009074 23. The method of claim 1, wherein the gene silencing effector is a sequence of SEQ.ID.N0.3 Homologous or complementary, or both. 24. The method of claim 1, wherein the gene silencing effector is a recombinant hairpin RNA comprising a sequence homologous to a SEQ.ID.N0.9 sequence A sequence as claimed in claim 2, wherein the gene silencing effector is a recombinant nucleic acid sequence which is a hybrid of a sequence of SEQ.ID.NO.27 and a sequence of SEQ.ID.NO.28 Formed as a method of requesting The silent effector includes a recombinant RNA 'which includes an SEq id n〇9 sequence, an SEq ID N〇1〇 sequence, a SEQ.ID.NO.ll sequence, a SEQJD.NO. 12 sequence, and a SEQJD. The sequence of the NO. 13 sequence, and combinations thereof, is homologous. 27. The method of claim 1, wherein the gene silencing effector is formed by a joining junction of one of the following hybrids: a SEQ.ID.NO.29 sequence, a SEQ.ID.NO.30 sequence, a SEQ.ID.N0.31 sequence, a SEQ.ID.NO.32 sequence, a SEQ.ID.NO.33 sequence, a SEQ.ID.NO.34 sequence, a SEQ.ID.NO.35 sequence, a sequence SEQ ID NO: The method of claim 1, wherein the recombinant nucleic acid composition is selected from the group consisting of a tetracycline reaction element, a virus or a second type RNA polymerase promoter ( Pol-II), or both, a K〇zak consistent translation initiation site, a polyadenylation message, a plurality of restriction/selection sites, and combinations thereof. 126 201009074 29. The method of claim 1 wherein The recombinant nucleic acid composition is selected from an origin of replication, an SV4〇 precocious promoter for expressing at least one antibiotic resistance gene in a competent prokaryotic cell, for use in a mammalian cell. An alternative source of SV4〇 replication, and combinations thereof. % As in the method of claim i, the heavy acid composition - Gene transfer method is introduced to the cell towel, and the method of recording is stored from liposome transfection, chemical sense, gene recombination, disease subtraction, transposon insertion, skip gene insertion, microinjection, electroporation , gene gun penetration, and combinations thereof. The sub-system is treated with an intracellular mechanism exosome, and the nonsense mediates a decrease. 31. For example, the method of δ ̄ green, wherein the mechanism is selected from rNA splicing. , solution, and combinations thereof. ❹ 32. The method of claim 1, wherein the cell marker is 0et3/4. wherein the b-stem stem cell exhibits an embryonic stem. 33. The method of claim 1 is cultured under conditions of cells. Wherein the b-stem stem cell line is in a cell-free cell of the method of claim 1. The cell of the cell of claim 1 is characterized by the method of claim 1, wherein the cell is differentiated into a cell type. The cells are differentiated into a spermatophore 127 201009074 The dry blister is differentiated into a normal body 36. The method of claim 1, wherein the multicellular cell 37. 38. Mingtian runs into a fiber Group of cells differentiate into a cartilage The method of one request, wherein the pluripotent stem cells mother weave. The method of item 1 of the request, wherein the pluripotent stem cells. The method of claim 1, wherein the pluripotent stem cell line is selectively isolated using mir-302 microribozyme nucleic acid and Oct3/4 as a label. 4. A recombinant nucleic acid composition for inducing a mir trap mediated gene silencing effect comprising: at least one intron encoding a pluripotent stem cell capable of transforming at least one cell into a tumor-free bacterium A gene silencing effector in which the intron is spliced out in the cell to induce a mil>_3G2• mediated gene silencing effect. 41. The composition of claim 4, wherein the neonucleic acid composition is formed in a process selected from the group consisting of chemical synthesis, nucleotide recombination, genetic engineering, and combinations thereof. 42. The composition of claim 4, wherein the intron comprises: (a) an intron insertion site encoding a gene silencing effector homologous to mir-302; (b) - five-terminal supply Somatic splice junction and a tri-terminal receptor splice site; 128 201009074 (C) a branch point motif; and (d)-polypyrimidine segment. 43. The method of claim 42 + Λ, the sandwich nucleus_containing I, comprises a nucleic acid sequence comprising a dry loop structure homologous to the -SEQ IDαΝ sequence or a SEQ.ID.NCU sequence. The composition of the apposition term 42, wherein the intron insertion site comprises a nucleic acid sequence homologous or complementary to a SEQ. ID. Na3 sequence, or both are 45. The composition of claim 42 Wherein the intron insertion site comprises a hairpin-type precursor mini-ribonucleic acid (pre-miRNA) sequence comprising a sequence selected from the group consisting of a SEQ.ID.N0.9 sequence, a sequence of SEQ.ID.NO.10, SEQ.ID.NO.11 sequence, a SEQ.ID.NO.12 sequence and a SEQ.ID.NO.13 sequence. 46. The composition of claim 42, wherein the branch point motif comprises a nucleotide of adenosine (A) in a nucleic acid sequence comprising or homologous to a sequence of SEQ.ID.N0.6 . 47. The composition of claim 42, wherein the branch point motif comprises a nucleoside (A) nucleotide that is at least one oligonucleotide comprising a homologous to 5'-TACTAAC-3' Sequence within a nucleic acid sequence. 129. The composition of claim 42, wherein the polypyrimidine segment comprises a nucleic acid sequence having a high τ or c content, which comprises a sequence of SEQ.ID.N0.7 or a sequence of SEQ.ID.N0.8 |J, or homologous to it. 49. The composition of claim 42, wherein the five-terminal donor splice site comprises a nucleic acid sequence comprising a sequence of SEQ. ID. N0.4 or homologous thereto. 50. The composition of claim 42, wherein the five-terminal donor splice site comprises a nucleic acid sequence comprising 5,-GTAAG-3, or homologous thereto. 51. The composition of claim 42, wherein the triad acceptor splice comprises a nucleic acid sequence comprising or homologous to a SEQ. ID. N0.5 sequence. 52. The composition of claim 42, wherein the triad acceptor splice comprises a nucleic acid sequence' comprising 5,-CTGCAG-3, or homologous thereto. 53. The composition of claim 4, wherein the gene silencing effector comprises a nucleic acid sequence' which is homologous or complementary to a sequence of SEQ. ID. N0.3, or both. 54. The composition of claim 4, wherein the gene silencing effector comprises a nucleic acid sequence comprising a SEQ.ID.NO.10 sequence, a SEQ.ID.NO.ll sequence, and a SEQ.ID.NO .12 sequence, and/or a SEQ.ID.MX13 sequence. 55. The composition of claim 4, wherein the gene silencing effector comprises a recombinant nucleic acid sequence formed by a junction of one of the following hybrids: a 130 201009074 SEQ. ID. NO. 29 sequence, a SEQ .ID.NO.30 sequence, _ SEQ.ID.NO.31 sequence, a SEQ.ID.NO.32 sequence, - SEQ.ro.NO.33 sequence, a SEQ.ID.NO.34 sequence, _ • SEQ.ID.NO.35 sequence, a SEQ.ID.NO.36 sequence, and its group. 56. The composition of claim 40, wherein the gene silencing effector comprises a nucleic acid sequence comprising a sequence of SEQ. ID. N0.9. 57. The composition of claim 40, wherein the gene silencing effector comprises a heavy ® set nucleic acid sequence which is a SEQ.ID.NO.27 sequence or a SEQ.ID.NO.28 sequence hybrid form. 58. At least one embryo-like pluripotent stem cell is produced by the method of claim 1. The embryonic pluripotent stem cell of claim 58, wherein the whole cell or a part of the cell of the embryonic pluripotent stem cell is applied by a donor cell nuclear transfer technique (SCNT). 60. The embryonic pluripotent stem cell of claim 58, wherein the embryonic multi-parallel stem cell cell-cell extract comprises at least one organ of the embryonic pluripotent stem cell. Or cytoplasm. 131