TWI461531B - Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant rna agents - Google Patents

Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant rna agents Download PDF

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TWI461531B
TWI461531B TW098114454A TW98114454A TWI461531B TW I461531 B TWI461531 B TW I461531B TW 098114454 A TW098114454 A TW 098114454A TW 98114454 A TW98114454 A TW 98114454A TW I461531 B TWI461531 B TW I461531B
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TW201009074A (en
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Shi Lung Lin
David Ts Wu
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Shi Lung Lin
David Ts Wu
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使用可誘導之重組核醣核酸生成不具癌細胞特性之類胚胎幹細胞Use of inducible recombinant ribonucleic acid to produce embryonic stem cells that do not have cancer cell characteristics

本發明一般而言係關於用來發育、生成及篩選不含腫瘤之類胚胎幹(ES)多能性細胞的一種手段及方法,其使用在感興趣之該等細胞中之一重組腫瘤抑制因子微型核醣核酸(miRNA)或小髮夾型RNA(shRNA)試劑的基因轉殖表現。更明確言之,本發明係關於用來生成一非自然發生之重組內含子及其組成物的一種基因轉殖方法及由可誘導性核酸組成,其可經剪接及處理而在哺乳動物細胞中形成小型核醣核酸(RNA)基因靜默效應子(如miRNA先驅物(pre-miRNA)及/或shRNA)內,然後誘發在發育及細胞分化相關之基因及致癌基因中的某些特定基因靜默效應,導致將該等細胞轉化成類ES多能性狀態中。較佳地,該等小型RNA基因靜默效應子包括類腫瘤抑制子之miRNA,像是:mir-302a、mir-302b、mir-302c、mir-302d,及其人造重設之shRNA同源衍生物與其組合。換句話說,如此生成之該等多能性類幹細胞可在無哺乳細胞(feeder-free)之細胞培養條件下培養。感興趣之該等細胞包括體外(in vitro )、離體(ex vivo )及/或體內(in vivo )分離之體細胞或癌症細胞。The present invention relates generally to a method and method for developing, producing, and screening embryonic stem (ES) pluripotent cells that do not contain tumors, using recombinant tumor suppressor factors in one of the cells of interest. Gene transfer performance of microRNA (miRNA) or small hairpin RNA (shRNA) reagents. 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 (RNA) 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 , causing the cells to be transformed into an ES-like pluripotent state. Preferably, the small RNA gene silencing effector comprises a tumor suppressor-like miRNA, such as: mir-302a, mir-302b, mir-302c, mir-302d, and artificially reprogrammed shRNA homologous derivatives thereof Combine with it. In other words, the pluripotent stem cells thus generated can be cultured in a cell culture condition free of feeder-free. Such cells of interest include somatic or cancer cells isolated in vitro , ex vivo , and/or in vivo .

近來在人類幹細胞之研究已顯示在移植治療中之一高度期望的潛力。然而,用來選殖人類幹細胞之該等來源受限,且極難控制其純度及品質。在1998年,James Thomson等人(如: 美國專利第5,843,780號、第6,200,806號、第7,029,913號,及第7,220,584號)自人類胚胎之晚囊胚(late blastocysts)分離第一株人類胚胎幹(ES)細胞株(Thomson等人 (1998)Science 282 :1145-1147)。H1及H9細胞係得自該等分離之人類ES細胞的兩株典型細胞株。兩年後,Gearhart等人(如:美國專利第6,090,622號、第6,245,566號,及第6,331,406號)也發展出一種自後囊胚(post-blastocyst)人類胚胎分離類ES原生生殖細胞之方法。因為該等ES細胞分離方法的方式必須破壞原先胚胎,引發了許多倫理及人道關注,其質疑將該等ES細胞用於臨床治療上的正當性。Recent studies in human stem cells have shown a highly desirable potential in one of transplant treatments. However, such sources for the selection of human stem cells are limited and it is extremely difficult to control their purity and quality. In 1998, James Thomson et al. (eg, U.S. Patent Nos. 5,843,780, 6,200,806, 7,029,913, and 7,220,584) isolated the first human embryonic stem (ES) from late blastocysts of human embryos (ES). Cell lines (Thomson et al ., (1998) Science 282 : 1145-1147). The H1 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) also developed a method for isolating ES primary germ cells from post-blastocyst human embryos. Because of the manner in which these ES cell isolation methods must destroy the original embryo, many ethical and human concerns have arisen, questioning the legitimacy of using these ES cells for clinical treatment.

在最近幾年,治療安全及使用知情同意上的問題也受到注意。例如,因為ES細胞生長需要由周圍「哺乳細胞」纖維母細胞所釋放的一些不確定因子,所有人類ES細胞較佳地係在一層小鼠或人類纖維母細胞上生長(Reubinoff等人之美國專利第6,875,607號)。然而,該等纖維母細胞哺乳細胞呈現不同之表面抗原,其通常污染ES細胞抗原之純度並可引起病人的免疫排斥。儘管已發展了一些無哺乳細胞之培養條件,該等無哺乳細胞方法中沒有一個能將該未分化之ES細胞狀態維持一段長時間。不幸地,在目前任何可得之培養條件下,人類ES細胞株不能達到100%的群體純度。甚至是在最佳之含哺乳細胞培養條件下,一些(約5%-10%或更多)ES細胞總是傾向分化成其它組織細胞類型並失去其幹細胞特性。得自該等ES細胞之 最常觀察到的細胞類型之一係畸胎瘤(teratoma)。畸胎瘤係通常得自人類生殖系細胞的一種腫瘤,其包含類似胚胎內胚層(endoderm)、中胚層(mesoderm)及外胚層(ectoderm)組織之多重類腫瘤細胞類型。因此,如何避免哺乳細胞之細胞污染、增加幹細胞純度並減少腫瘤形成之風險係本幹細胞研究的三個主要課題。In recent years, attention has also been paid to the issues of treatment safety and the use of informed consent. For example, because ES cell growth requires some uncertainty from the release of surrounding "mammalian cells" fibroblasts, all human ES cells are preferably grown on a layer of mouse or human fibroblasts (US Patent Reubinoff et al.) No. 6,875,607). However, the fibroblasts of the fibroblasts exhibit different surface antigens, which generally contaminate the purity of the ES cell antigen and can cause immune rejection in the patient. Although some culture conditions without mammalian cells have been developed, none of these non-mammal cell methods maintain this undifferentiated ES cell state for a long 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% to 10% or more) ES cells tend to differentiate into other tissue cell types and lose their stem cell properties. From these ES cells One of the most commonly observed cell types is teratoma. Teratoma is a tumor commonly obtained from human germline cells that contains multiple types of tumor cell types like embryonic endoderm, mesoderm and ectoderm. Therefore, how to avoid cell contamination of mammalian cells, increase the purity of stem cells and reduce the risk of tumor formation are the three main subjects of stem cell research.

2006年時,Takahashi與Yamanaka引介誘導性多能性幹細胞(induced pluripotent stem cells,iPS cells)(Cell 126 :663-676)。藉由使用四個轉錄因子基因[Oct3/4 (Oct4 ),Sox2c-MycKlf4 ]之反轉錄病毒傳送進入小鼠纖維母細胞內,Takahashi與Yamanaka成功在體外將該等體纖維母細胞轉化並轉形成類ES之iPS細胞株。此方法之成功率在該全部所測試之細胞群中估計少於0.002%-2%。在2007年,該等iPS細胞之基因及行為特性經觀察係類似該等小鼠ES細胞的特性(Okita等人,(2007)Nature 448 :313-317;Wernig等人,(2007)Nature 448 :318-324)。同時,Yu等人使用類似的方法,但以另一組部分不同轉錄因子,發展出得自人類纖維母細胞之新穎iPS細胞株,該等轉錄因子包括:Oct4、Sox2、Nanog及LIN28(Yu等人,(2007)Science 318 :1917-1920)。然而,Yu的方法遠較Takahashi的方法效率低。該等iPS細胞應用之優點顯示出若配合體細胞核轉移(somatic cell nuclear transfer,SCNT)技術,不僅解決了原先ES細胞方法的倫理問題,也提供了對病人友 善的潛在療法(Meissner等人,(2006)Nature 439 :212-215)。此iPS型SCNT治療已測試用來處理基因轉殖小鼠模型中的鐮刀細胞貧血症(Hanna等人,(2007)Science 318 :1920-1923)。然而,仍有兩個未解決的問題;其一係反轉錄病毒之基因轉殖使用,其二係致癌基因之使用(如:c-MycKlf4 )。反轉錄病毒感染係可將四個大型轉錄因子基因轉殖地傳送進標的細胞內的唯一有效手段;然而,多個反轉錄病毒載體隨機插入該標的細胞基因體也可能影響其它非標的基因。這造成疑問,因為由於不確定之反轉錄病毒插入通常會引起細胞變異,特別是當一或多個轉殖基因係致癌基因時。如何避免由該等轉錄因子誘發iPS細胞形成腫瘤目前為止係難解的。In 2006, Takahashi and Yamanaka introduced induced pluripotent stem cells (iPS cells) ( Cell 126 : 663-676). By using four transcription factor genes [ Oct3/4 ( Oct4 ), Sox2 , c-Myc and Klf4 ] retroviruses into mouse fibroblasts, Takahashi and Yamanaka successfully performed these fibroblasts in vitro. Transformed and transformed into an ES-like iPS cell line. The success rate of this method is estimated to be less than 0.002% to 2% in all of the tested cell populations. In 2007, the genetic and behavioral properties of these iPS cells were observed to be similar to those of mouse ES cells (Okita et al., (2007) Nature 448 :313-317; Wernig et al. (2007) Nature 448 : 318-324). At the same time, Yu et al. used a similar method, but developed a novel iPS cell line derived from human fibroblasts with a different set of different transcription factors, including: Oct4, Sox2, Nanog, and LIN28 (Yu et al. (2007) Science 318 : 1917-1920). However, the method of Yu is far less efficient than the method of Takahashi. The advantages of these iPS cell applications show that the use of somatic cell nuclear transfer (SCNT) technology not only addresses the ethical issues of the original ES cell approach, but also provides potential patient-friendly therapies (Meissner et al., ( 2006) Nature 439 : 212-215). This iPS type SCNT treatment has been tested to treat sickle cell anemia in a gene transfer mouse model (Hanna et al., (2007) Science 318 : 1920-1923). However, there are still two unresolved problems; one is the use of retrovirus gene transgenes, and the second is the use of oncogenes (eg c-Myc and Klf4 ). Retroviral infection is the only effective means of transgenic four large transcription factor genes into the target cell; however, random insertion of multiple retroviral vectors into the target cell genome 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.

iPS細胞之另一缺點係其非均質性。為生成一iPS細胞,至少三或更多個不同之轉錄因子基因必須經由反轉錄病毒感染來插入單一細胞基因體中。然而,由於不同之反轉錄病毒轉殖基因呈現不同比率之傳送效率及插入變異,多個反轉錄病毒插入通常造成宿主細胞基因體中各式各樣之轉殖基因組合。僅有具適當比率及數目之該等四個轉錄因子基因的細胞可變成具有良好多能性的iPS細胞。此係為何在反轉錄病毒插入後該等iPS細胞僅表現整個細胞群的0.002%-2%,而佔該等細胞超過98%之其它細胞係以複雜之不確定的轉殖細胞組合而轉形。為了收集具正確轉殖基因組合之純iPS細胞,需要一系列繁瑣的細胞篩選程序(Shinya Yamanaka之美國專利第7,250,255號)。 雖然iPS細胞形成機制尚未清楚,但是此技術不需要多個轉錄調節子來協調在胚胎基因之再活化及發育訊息之去活化間的轉換。Oct4-Sox2-c-Myc-Klf4Oct4-Sox2-Nanog-LIN28 基因的組合效應似乎直接活化某些胚胎基因;然而,該等效應如何造成取消體細胞分化所必要之整體發育訊息尚未確定。除轉殖基因Oct4外,iPS方法所使用之所有其它轉殖基因實際上均包含在某些組織細胞發育的起始階段中。藉由將該等發育訊息放在一起,該等發育訊息之協調或擾亂以某種方式終止了細胞分化,然後將該細胞轉形回類ES狀態。這非自然機制,且可包含不確定之風險,像是細胞變異及腫瘤形成。Another disadvantage of iPS cells is their heterogeneity. To generate an iPS cell, at least three or more different transcription factor genes must be inserted into a single cell genome via retroviral infection. However, since different retroviral transgenic genes exhibit different ratios of delivery efficiency and insertional variation, multiple retroviral insertions typically result in a diverse combination of transgenic genes in the host cell genome. Only cells with the appropriate ratio and number of these four transcription factor genes can become iPS cells with good pluripotency. Why does this iPS cell represent only 0.002% to 2% of the entire cell population after retroviral insertion, while other cell lines that account for more than 98% of these cells are transformed with complex and indeterminate combinations of transgenic cells. . In order to collect pure iPS cells with the correct combination of transgenic genes, a series of cumbersome cell screening procedures are required (Shinya Yamanaka, U.S. Patent No. 7,250,255). Although the mechanism of iPS cell formation is not known, this technique does not require multiple transcriptional regulators to coordinate the transition between reactivation of embryonic genes and deactivation of developmental messages. The combined effect of the Oct4-Sox2-c-Myc-Klf4 or Oct4-Sox2-Nanog-LIN28 genes appears to directly activate certain embryonic genes; however, the overall developmental message necessary for such effects to abolish somatic differentiation has not been established. With the exception of the transgenic gene Oct4, all other transgenic genes used in the iPS method are actually included in the initial stages of development of certain tissue cells. By putting these developmental messages together, the coordination or disruption of such developmental messages somehow terminates cell differentiation and then transforms the cells back into an ES-like state. This is an unnatural mechanism and can involve uncertainties such as cell variability and tumor formation.

體細胞核轉移(SCNT)之實驗已顯示體細胞核及卵母細胞之細胞質的雜合可形成多能性類幹細胞,其指出在該卵母細胞之細胞質中的某些母系元素而非該細胞核中之轉錄因子在核轉化中佔有重要角色(Simonsson and Gurdon,(2004)Nat Cell Biol. 6:984-990)。在桑葚胚時期前之自然受精卵及早期接合子中,母系元素負責正常幹細胞再生及多能性的調節及維持,其全然無腫瘤形成之風險。這是為何在32-64細胞(桑葚胚)時期前之胚胎細胞係全部相同且全能的。母系元素在卵子生成期間產生並存在於最初胚胎形成時期所需之成熟卵母細胞中。缺乏Dicer(岱塞爾,微型核醣核酸生體合成作用時所需之一致性核醣核酸酶)之小鼠卵母細胞制止減數分裂I之分裂期,其指出微型核醣核酸係卵母細胞中的主要母系元素之一 (Murchison等人,(2007)Genes Dev. 21 :682-693)。在鼠科動物卵母細胞中,核糖核酸佔據母系元素之一大部分,其相當於全部基因體轉錄分子之約45%(Stitzel等人,(2007)Science 316 :407-408)。在母系-胚系轉變期間,該等母系RNA很快地降解,且該胚系基因之轉錄很早就在該2-4細胞時期時啟動,並產生用於胚胎發育的訊息(O’Farrell等人,(2004)Curr.Biol. 14 :R35-45)。可以想見,許多該等母系RNA係該等胚系基因之抑制子,以在胚胎發育的最初時期同步化發育訊息並維持全能性/多能性之ES細胞再生。因此,母系RNA可能係ES細胞維持及再生所必要的該等主要母系元素之一。Experiments with somatic cell nuclear transfer (SCNT) have shown that cytoplasmic heterogeneity of somatic nuclei and oocytes can form pluripotent stem cells, which indicate certain maternal elements in the cytoplasm of the oocyte rather than in the nucleus. Transcription factors play an important role in nuclear transformation (Simonsson and Gurdon, (2004) Nat Cell Biol. 6: 984-990). In the natural fertilized eggs and early zygote before the morula stage, the 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 before the 32-64 cell (mulberry embryo) period are all identical and versatile. Maternal elements are produced during egg production and are present in the mature oocytes required during the initial embryogenesis. A mouse oocyte lacking Dicer (consistent ribonuclease required for microribonuclear biosynthesis) stops the meiotic division of meiosis I, which is indicated in the microribonucleotide oocyte One of the main maternal elements (Murchison et al., (2007) Genes Dev. 21 : 682-693). In murine oocytes, ribonucleic acid occupies a majority of maternal elements, which is equivalent to about 45% of all genomic transcriptional molecules (Stitzel et al., (2007) Science 316 : 407-408). During maternal-germline transformation, these maternal RNAs degrade rapidly, and transcription of this germline gene is initiated very early in the 2-4 cell phase and produces messages for embryonic development (O'Farrell et al. (2004) Curr. Biol. 14 : R35-45). It is envisaged that many of these maternal RNAs 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, maternal RNA may be one of these major maternal elements necessary for ES cell maintenance and regeneration.

總而言之,ES細胞維持及再生之自然方式係仰賴某些母系RNA(作為抑制子)而非用於iPS技術之該等四個轉錄因子(作為活化子)。為了生成模擬ES細胞維持及再生之自然機制的類ES多能性細胞,吾人極度需要一種新策略來識別及評估該等母系RNA的功能。可將該等已識別之母系RNA傳送進人類成人幹細胞或體細胞,以維持該等幹細胞特性或將該等體細胞轉化成一類ES狀態中,或兩者皆可。因此,吾人需要用來生成類ES多能性細胞之有效、簡單且安全之方法及試劑組成物,較佳地係使用母系RNA。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 maternal RNAs. 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, we need an efficient, simple, and safe method and reagent composition for generating ES-like pluripotent cells, preferably using maternal RNA.

本發明提供一種方法,供至少一個哺乳動物細胞轉化為至少一個多能性類幹細胞。該方法包含以下步驟:提供至少一細胞 基質,其表現複數個mir-302的標的細胞基因;提供至少一個重組核酸組成物,其可經傳送、轉錄及處理成在該細胞基質中與mir-302同源的至少一基因靜默效應子;及在mir-302所標的該等細胞基因受抑制的情況下,以該重組核酸組成物處理該細胞基質。換句話說,本發明提供一種用來發育、生成及篩選類胚胎幹(ES)多能性細胞之方法,其使用類小髮夾型之重組微型核醣核酸(miRNA)試劑的異位表現,這些miRNA如mir-302a、mir-302b、mir-302c、mir-302d,及其人造重設之miRNA先驅物(pre-miRNA),及/或小髮夾型RNA(shRNA)同源衍生物與其組成物。非自然發生/人造/人工mir-302試劑之設計包括小髮夾型RNA(shRNA)及/或小干擾RNA(siRNA)同源衍生物或基因群的失配(mismatched)及完美配對(perfectly matched)結構(constructs),這全部都可改善標的專一性並減少基因轉殖傳送及基因靜默所需之mir-302數量(拷貝數(copy number))。The invention provides a method for the conversion of at least one mammalian cell into at least one pluripotent stem cell. The method comprises the steps of: providing at least one cell a matrix that exhibits a plurality of target cell genes of mir-302; provides at least one recombinant nucleic acid composition that can be transmitted, transcribed, and processed into at least one gene silencing effector homologous to mir-302 in the cell matrix; And in the case where the cellular genes identified by mir-302 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) pluripotent cells using ectopic expression of a small hairpin-type recombinant microribonucleic acid (miRNA) reagent. miRNAs such as mir-302a, mir-302b, mir-302c, mir-302d, and their artificially-reset miRNA precursors (pre-miRNA), and/or small hairpin RNA (shRNA) homologous derivatives and their constituents Things. The design of the non-naturally occurring/artificial/artificial mir-302 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 improve the specificity of the target and reduce the number of mir-302 (copy number) required for gene transfer delivery and gene silencing.

天然微型核醣核酸(miRNA)通常係約長度18-27之核苷酸(nucleotides,nt),並可依其相互之互補程度而直接降解其標的訊息RNA(messenger RNA,mRNA)或抑制該標的mRNA之轉譯。mir-302家族(mir-302s)係一群高同源性之基因間miRNA,其分享超過89%之同源性且在幾乎所有的哺乳動物間係一致的。Mir-302家族包含四個成員,其經共同轉錄作為一非編碼(non-coding)之RNA基因群,包括:mir-302b、mir-302c、mir-302a、mir-302d及mir-367,其以五端至三端之方向連接(Suh 等人,(2004)Dev.Biol. 270 :488-498)。雖然mir-367及mir-302家族係共同表現,由於其對抗不同組之標的基因係截然不同的種子基序(seed motif),其功能實際上係彼此不同的。頃發現在許多哺乳動物之早期接合子及胚胎幹(ES)細胞中mir-302家族之表現量極高(Tang等人,(2007)Genes Dev. 21 :644-648;Suh等人,(2004)Dev.Biol. 270 :488-498)。在生長緩慢之ES細胞中表現量最豐富,並在細胞分化及/或增殖後快速地減少。小鼠的卵母細胞缺乏岱塞爾(Dicer),其係miRNA生體合成作用所需的一種一致性核醣核酸酶,因此該等卵母細胞停留於減數分裂第一階段的分裂期(meiosis I),這表示該等miRNA在卵子生成中扮演決定性的角色(Murchison等人,(2007)Genes Dev. 21 :682-693)。此外miRNA具有小型抑制RNA的特性,其可抑制具高互補程度(complementarity)之標的基因的轉譯(Bartel,D.P.(2004)Cell 116 :281-297),mir-302家族可能係負責避免ES細胞在早期胚胎形成期間任何可能之早熟分化的主要母系抑制子。該等發現暗示mir-302家族在正常ES細胞維持及再生中扮演重要的角色。Natural microRNAs (miRNAs) are usually about 18-27 nucleotides (nucleotides, nt), and can directly degrade their target messenger RNA (mRNA) or inhibit the target mRNA according to their mutual complementarity. Translation. The mir-302 family (mir-302s) is a group of highly homologous intergenic miRNAs that share more than 89% homology and are consistent across almost all mammals. The Mir-302 family contains four members that are co-transcribed as a non-coding RNA gene group, including: mir-302b, mir-302c, mir-302a, mir-302d, and mir-367. It is connected in a five-terminal to three-terminal direction (Suh et al., (2004) Dev. Biol. 270 : 488-498). Although the mir-367 and mir-302 families are co-expressed, their functions are actually different from each other due to their distinct seed motifs against different sets of target genes. It has been found that the mir-302 family is highly expressed in early mammalian and embryonic stem (ES) cells in many mammals (Tang et al., (2007) Genes Dev. 21 :644-648; Suh et al., (2004). Dev. Biol. 270 : 488-498). It is most abundant in ES cells that grow slowly and rapidly decreases after cell differentiation and/or proliferation. Mouse oocytes lack Dicer, which is a consensus ribonuclease required for miRNA biosynthesis, so these oocytes stay in the first stage of meiosis. I), which means that these miRNAs play a decisive role in egg production (Murchison et al. (2007) Genes Dev. 21 :682-693). In addition, miRNAs have small RNA-inhibiting properties that inhibit the translation of target genes with high complementarity (Bartel, DP (2004) Cell 116 :281-297), and the mir-302 family may be responsible for avoiding ES cells. Major maternal suppressor of any possible early maturity differentiation during early embryogenesis. These findings suggest that the mir-302 family plays an important role in the maintenance and regeneration of normal ES cells.

所有mir-302成員在其五端前十七個(17)核苷酸中分享一完全相同(100%)的序列(包括完全種子基序),以及在其第23個核苷酸成熟miRNA序列中具有整體83%-96%的同源性。該種子基序位在成熟miRNA序列之五端前八個核苷酸中,其決定在該miRNA及其標的基因間的鍵合專一性及效率。根據連結至 Sanger miRBase::Sequences網站(http://microrna.sanger.ac.uk/)資料庫之「TARGETSCAN」(http://www.targetscan.org/vert_42/)及「PICTAR-VERT」(http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi?)程式的預測,其經導向幾乎對抗相同的細胞基因,包括在人類及小鼠中超過445株的一致性基因。此外,mir-302也與mir-93、mir-367、mir-371、mir-372、mir-373,及mir-520家族成員分享某些重疊之標的基因。大多數之該等標的基因係發育訊息及轉錄因子,其與早期胚胎形成期間之譜系專一性細胞分化的初始及/或促進作用有關(Lin等人,(2008b)RNA 14 :2115-2124)。許多該等標的基因也係熟知之致癌基因。因此,mir-302家族之功能更可能抑制發育訊息及分化相關轉錄因子的整體生成,而非如先前之iPS方法係在某些胚胎訊息傳遞路徑上建立轉錄刺激。此外,由於許多該等標的發育訊息及分化相關之轉錄因子係致癌基因,mir-302家族也可如一腫瘤抑制子般作用,以防止正常ES細胞再生偏差而形成腫瘤。換句話說,本發明提供一種方法,其用以生成不含腫瘤之類ES多能性細胞。例如,類胰島素生長因子(insulin-like growth factors,IGF)係用於專對神經元細胞譜系之分化的潛在發育訊息,該等訊息係經由Ras/Raf/有絲分裂劑活化蛋白質激酶(mitogen-activated protein kinase,MAPK)路徑或經由磷脂醯肌醇3-激酶(phosphatidylinosital 3-kinase;PI3K)/Akt之訊息傳導路徑。相同之訊息傳遞路徑也 與許多腫瘤/癌症轉形相關,像是:腦瘤、乳癌、肺癌、前列腺癌,及皮膚黑色素癌。本發明者發現IGF受體(IGFR)-Ras/PI3K訊息傳遞路徑中超過十八個成員係mir-302家族的強力標的,這表示在哺乳動物卵母細胞及ES細胞中有一極嚴密的封鎖以防止神經元細胞分化。在許多其它各種細胞譜系上也觀察到類似的mir-302家族抑制效應。根據上述證據,本發明者咸信mir-302家族係正常ES細胞之維持與再生的主要調節者,其可能將已分化之體細胞轉化為均質的類ES狀態。All mir-302 members share an identical (100%) sequence (including the complete seed motif) and the 23rd nucleotide mature miRNA sequence in the first seventeen (17) nucleotides of its five ends. It has an overall homology of 83%-96%. 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 "TARGETSCAN" (http://www.targetscan.org/vert_42/) and "PICTAR-VERT" linked to the Sanger miRBase::Sequences website (http://microrna.sanger.ac.uk/) The http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi?) program predicts that it is 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 members of the mir-93, mir-367, mir-371, mir-372, mir-373, and mir-520 families. 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) RNA 14 : 2115-2124). Many of these target genes are also well known to be oncogenes. Thus, the function of the mir-302 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 delivery 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 tumors from degenerating in normal ES cell proliferation. 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 for the differentiation of neuronal cell lineages, which activate mitogen-activated proteins via Ras/Raf/mitogens. The kinase, MAPK) pathway or signaling pathway via phospholipidlinosital 3-kinase (PI3K)/Akt. The same message transmission pathway 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 signaling pathway has a strong target of more than eighteen member lines of the mir-302 family, indicating a very tight blockade in mammalian oocytes and ES cells. Prevent neuronal cell differentiation. A similar mir-302 family inhibitory effect was also observed on many other various cell lineages. Based on the above evidence, the inventors of the present invention, the mir-302 family, are major regulators of maintenance and regeneration of normal ES cells, which may transform differentiated somatic cells into a homogeneous ES-like state.

為測試mir-302家族之功能,本發明者已根據天然內含子miRNA生體合成機制(圖1A)發展出第二型RNA聚合酶驅動(Pol-II-driven)之miRNA表現系統,並順利地使用此系統體外地及體內地產生天然miRNA家族以及人造shRNA(圖1B)。廣義而言,該內含子係一基因之非編碼序列,其包括同一讀框內含子(in-frame intron)、五端非轉譯區(5’-UTR)及三端非轉譯區(3’-UTR)。吾人先前之研究已證實有效成熟之miRNA可得自哺乳動物基因之該等內含子區域,稱之為內含子微型核醣核酸(intronicmiRNA)(Lin等人,(2003)Biochem Biophys Res Commun. 310 :754-760;Lin等人,(2005)Gene 356 :32-38)(Lin等人,(2003)Biochem Biophys Res Commun. 310 :754-760;Lin等人,(2005)Gene 356 :32-38)。因為約有50%之哺乳動物miRNA係存在蛋白質編碼基因之該等內含子內,故內含子miRNA表現係哺乳動物中常見之現象(Rodriguez等人,(2004)Genome Res. 14 :1902-1910)。如圖1A所示,內含子miRNA生體合成係仰賴新生之第二型RNA聚合酶介導(nascent Pol-II-mediated)之pre-mRNA轉錄及內含子剪接/切除之間的結合交互作用,其係發生在靠近基因體核染質周圍纖絲的某些細胞核區域內(Ghosh等人,(2000)RNA 6 :1325-1334;Lin等人,(2008a)Frontiers in Bioscience 13 :2216-2230)。該等miRNA係在其宿主基因(pre-mRNAs)之先驅轉錄分子內以第二型RNA聚合酶(Pol-II)轉錄,並以剪接體及其它核醣核酸酶III(RNaseIII)等核酸內切酶剪接以形成成熟之miRNA(Lin等人,2003;Danin-Kreiselman等人,(2003)Mol Cell 11 :1279-1289);然而,Drosha 可能不需要此種程序(Ruby等人,(2007)Nature 448 :83-86)。結果,內含子miRNA生體合成係以多個細胞內監測系統嚴密調控,包括:第二型RNA聚合酶(Pol-II)轉錄、RNA剪接、外體處理及無義介導RNA降解(nonsense-mediated RNA decay,NMD)。換句話說,該類miRNA基因靜默效應子係由一細胞內機制釋放,該機制係選自RNA剪接、外體處理、無義介導RNA降解、及其組合。由於此高度細胞內監測,可避免在其它shRNA/siRNA表現系統中所發現的RNA過飽和問題,因而造成在標的基因上一更有效、標的專一性且更安全的基因靜默效應(Lin等人,2008a)。To test the function of the mir-302 family, the inventors have developed a second-type RNA polymerase-driven miRNA expression system based on the natural intron miRNA biosynthesis mechanism (Fig. 1A). This system was used to generate native miRNA families as well as artificial shRNAs in vitro and in vivo (Fig. 1B). Broadly speaking, the intron is a non-coding sequence of a gene comprising the same in-frame intron, a five-terminal non-translated region (5'-UTR), and a three-terminal non-translated region (3) '-UTR). Previous studies by ours have demonstrated that effective mature miRNAs can be derived from these intron regions of mammalian genes, termed intron microRNAs (Lin et al., (2003) Biochem Biophys Res Commun. 310 : 754-760; Lin et al. (2005) Gene 356 : 32-38) (Lin et al., (2003) Biochem Biophys Res Commun. 310 : 754-760; Lin et al., (2005) Gene 356 : 32- 38). Since about 50% of mammalian miRNAs are present in these introns of protein-coding genes, intron miRNAs are a common phenomenon in mammals (Rodriguez et al., (2004) Genome Res. 14 :1902- 1910). As shown in Figure 1A, intron miRNA biosynthesis relies on the binding interaction between pre-mRNA transcription and intron splicing/resection of nascent Pol-II-mediated nascent Pol-II-mediated Role, which occurs in certain nuclear regions close to the fibrils around the nuclear chromosomal (Ghosh et al., (2000) RNA 6 : 1325-1334; Lin et al., (2008a) Frontiers in Bioscience 13 : 2216- 2230). These miRNAs are transcribed as a second-type RNA polymerase (Pol-II) in the precursor transcription molecule of their host genes (pre-mRNAs), and splicing and other endonucleases such as RNase III Splicing to form mature miRNAs (Lin et al, 2003; Danin-Kreiselman et al, (2003) Mol Cell 11 : 1279-1289); however, Drosha may not require such a procedure (Ruby et al., (2007) Nature 448 :83-86). As a result, intron miRNA biosynthesis is tightly regulated by multiple intracellular monitoring systems, including: type 2 RNA polymerase (Pol-II) transcription, RNA splicing, exosome treatment, and nonsense-mediated RNA degradation (nonsense- Medicated RNA decay, NMD). In other words, the miRNA gene silencing effector is released by an intracellular mechanism 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). ).

藉由模擬該天然內含子miRNA路徑(圖1A),本發明者發明一種新穎內含子miRNA表現系統來轉錄紅色色偏螢光蛋白質 (red-shifted fluorescent protein,RGFP )之一重組轉殖基因,稱之為SpRNAi-RGFP ,其包含可產生內含子miRNA及/或類shRNA基因靜默效應子的一種人造/人工剪接勝任內含子(splicing-competent intron,SpRNAi )。該SpRNAi 在該SpRNAi-RGFP 基因之pre-mRNA內以Pol-II共同轉錄,並藉由RNA剪接來切開。接著,進一步將所剪接之SpRNAi 處理成成熟之基因靜默效應子,像是:天然miRNA及人造shRNA,因而在標的基因上引發特定之後轉錄基因靜默(posttranscriptional gene silencing,PTGS)效應。同時,在內含子剪接後,將該SpRNAi-RGFP 基因轉錄分子之外顯子鏈接在一起以形成用來轉譯之RGFP標記蛋白質的一成熟mRNA,該RGFP標記蛋白質可用來識別miRNA/shRNA表現。在其它實施例中,一些功能性蛋白質外顯子可用來代替RGFP以提供額外之基因功能,像是:用於體細胞轉化之胚胎幹(ES)基因標記。換句話說,該基因靜默效應子可經由後轉錄基因靜默、轉譯抑制、RNA干擾,及/或無義介導降解來誘發一細胞內基因靜默效應。由於目前在脊椎動物中所發現超過1000個天然miRNA種類尚未清楚其功能,且仍不斷地識別出更多新的miRNA,本發明之內含子miRNA表現系統可作為用來在體內及體外地測試該等miRNA功能的有力工具。By simulating the natural intron miRNA pathway (Fig. 1A), the inventors invented a novel intron miRNA expression system to transcribe a recombinant transgenic gene of red-shifted fluorescent protein ( RGFP ). , referred to as SpRNAi-RGFP , which comprises a splicing-competent intron ( SRNAI ) that produces an intron miRNA and/or a shRNA-like gene silencing effector. The SpRNAi is co-transcribed with Pol-II in the pre-mRNA of the SpRNAi-RGFP gene, and is cleaved by RNA splicing. Subsequently, the spliced SpRNAi is further processed into a mature gene silencing effector, such as a natural miRNA and an artificial shRNA, thereby initiating a specific posttranscriptional gene silencing (PTGS) effect on the target gene. At the same time, after intron splicing, the exon of the SpRNAi-RGFP 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 function, such as: embryonic stem (ES) gene signature for somatic cell transformation. In other words, the 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 identified, the intron miRNA expression system of the present invention can be used as a test for in vivo and in vitro. A powerful tool for these miRNA functions.

SpRNAi 內含子包括數個一致之核苷酸組成部分,其包含:一五端剪接位(5’-splice site;SEQ.ID.NO.4)、一分支點基序 (branch-point motif,BrP;SEQ.ID.NO.6)、一多嘧啶段(poly-pyrimidine tract,PPT;SEQ.ID.NO.7及SEQ.ID.NO.8),及一三端剪接位(3’-splice site;SEQ.ID.NO.5)。此外,一小髮夾型miRNA或shRNA先驅物係插在該五端剪接位及該分支點基序之間。此部分之內含子通常在RNA剪接及處理期間形成一套馬索(lariat)結構。此外,該SpRNAi 之三端包含一多重轉譯停止密碼子區域(T codon),以增加內含子RNA剪接及NMD處理的正確性。當此T codon在一細胞質mRNA中出現時,其發出活化該NMD系統的訊息以降解該細胞中所累積的任何未建構之RNA。然而,會保留高度建構之shRNA及先驅miRNA(pre-miRNA)以供Dicer 進一步地酶切來分別形成成熟之siRNA及miRNA。對於基因轉殖表現而言,吾人在RGFP 基因(SEQ.ID.NO.22)之DraII 限制位(第208個核苷酸)中人工地合併SpRNAi 。此形成了一重組之SpRNAi-RGFP 轉殖基因。酶切具DraII切位之RGFP 可在每一端產生具有三個內凹核苷酸的AG-GN核苷酸斷口,其在SpRNAi 插入後將分別形成五端剪接位及三端剪接位。因為此內含子插入中斷了RGFP蛋白質的完整性,其可藉由內含子剪接來恢復,故吾人能經由在該等轉染細胞中出現之紅色RGFP來判定該內含子miRNA/shRNA之釋放及該RGFP mRNA之成熟。該RGFP 基因也包含多個外顯剪接促進子(exonic splicing enhancer,ESE)以增加RNA剪接之正確性及效率。The SpRNAi intron comprises a plurality of identical nucleotide components comprising: a five-terminal splice site (5'-splice site; SEQ. ID. NO. 4), a branch-point motif (branch-point motif) , BrP; SEQ.ID.NO.6), poly-pyrimidine tract (PPT; SEQ.ID.NO.7 and SEQ.ID.NO.8), and a three-terminal splice position (3' -splice site; SEQ.ID.NO.5). In addition, a small hairpin miRNA or shRNA precursor is inserted between the five-terminal splice site and the branch point motif. The introns of this portion typically form a set of lariat structures during RNA splicing and processing. In addition, the three ends of the SpRNAi contain a multiple translation stop codon region (T codon) to increase the correctness of intron RNA splicing and NMD processing. 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, highly constructed shRNAs and precursor miRNAs (pre-miRNAs) will be retained for Dicer to further digest to form mature siRNAs and miRNAs, respectively. For gene transfer performance, we artificially incorporated SpRNAi in the DraII restriction (208th nucleotide) of the RGFP gene (SEQ.ID.NO.22). This resulted in a recombinant SpRNAi-RGFP transgene. Enzyme-cleavage of RGFP with DraII cleavage produces an AG-GN nucleotide break with three concave nucleotides at each end, which will form a five-terminal splice site and a three-terminal splice site, respectively, after SpRNAi insertion. Since this intron insertion interrupts the integrity of the RGFP protein, which can be restored by intron splicing, we can determine the intron miRNA/shRNA via the red RGFP present in the transfected cells. Release and maturation of the RGFP mRNA. The RGFP gene also contains multiple exonic splicing enhancers (ESEs) to increase the correctness and efficiency of RNA splicing.

在較佳之具體實施例中,本發明係一可誘發miRNA/shRNA表現系統(圖2A及2B),其改善體內及體外之miRNA/shRNA表現程度的控制。此種改善不僅適用於較安全之電穿孔法(electroporation)/顯微注射法(micro-injection)來進行轉殖基因傳送,以代替易發生腫瘤的反轉錄病毒感染(retroviral infection),同時也避免了在該等轉染細胞中RNA過度累積的可能性。根據此種改善,本發明者已成功地生成各種mir-302轉導多能性幹(mir-302-transduced pluripotent stem,mirPS)細胞株,其係源自正常表皮皮膚細胞(mirPS-hpESC)、正常頭髮毛囊細胞(mirPS-hHFC)之人類原代培養(primary culture)與癌症乳腺癌MCF7(mirPS-MCF7)、前列腺癌PC3(mirPS-PC3)及皮膚黑色素癌Colo 829(mirPS-Colo)細胞。如圖2A及2B所示,本發明者首先將人造mir-302家族pre-miRNA/shRNA結構(圖3B)合併至SpRNAi-RGFP 轉殖基因之內含子插入位(即:MluI-PvuI 限制/選殖位),然後將該SpRNAi-RGFP 轉殖基因插進去氧羥(Dox)-可誘發pTet-On-tTS 載體之多重選殖位(即:XhoI-ClaI 限制位),因而形成pTet-On-tTS-mir302s 轉殖基因載體(圖3A)。換句話說,該重組核酸組成物(如SpRNAiSpRNAi-RGFP 及諸如此類)包含藥物可誘發之基因表現載體。此外,該SpRNAi-RGFP 也可合併進基因表現載體中,其係選自質體、病毒載體、反轉位子、及其組合。該SpRNAi-RGFP 轉殖基因係以370鹼基對(base-pair,bp)同源區域為側翼,該同源區域係用來重組插入該 宿主細胞基因體的標的位。在基因轉殖傳送方面,pTet-On-tTS-mir302s 載體(10-30μg)與該等宿主細胞(200-2000)在一低滲性PH緩衝液(400μl;有蓋試管(Eppendorf))中混合,且在400-450伏特下實行電穿孔法100微秒(μsec)以將該轉殖基因傳送進該等宿主細胞基因體內。在72小時後,使用FACS流式細胞計數儀及anti-RGFP及anti-Oct3/4單株抗體兩者以供分離並收集基因轉殖mirPS成功之細胞(圖3C)。此新穎mir-302家族基因轉殖方法之成功率超過91%,其遠高於原先iPS方法所發現之0.002%-2%的成功率。該人造mir-302家族之所有序列係根據Sanger miRBase::Sequences程式之序列資料庫而化學合成。pTet-On-tTS 載體編碼CMV驅動tTS 抑制子基因(CMV-driven tTS inhibitor gene)以去活化該轉殖基因之TRE-CMV 啟動子。當出現去氧羥(Dox)時,tTS之抑制功能則經Dox中和,因此表現出SpRNAi-RGFP 轉殖基因及其編碼mir-302家族。In a preferred embodiment, the invention is a miRNA/shRNA expression system (Figs. 2A and 2B) that improves control of the extent of miRNA/shRNA expression in vivo and in vitro. This improvement applies not only to safer electroporation/micro-injection for transgenic gene delivery, but also to retrograde tumor-prone retroviral infections. The possibility of excessive accumulation of RNA in these transfected cells. Based on such improvements, the present inventors have succeeded in generating various mir-302-transduced pluripotent stem (mirPS) cell lines derived from normal epidermal skin cells (mirPS-hpESC), Human primary culture of hair follicle cells (mirPS-hHFC) and cancer breast cancer MCF7 (mirPS-MCF7), prostate cancer PC3 (mirPS-PC3), and cutaneous melanoma Colo 829 (mirPS-Colo) cells. As shown in Figures 2A and 2B, the inventors first incorporated the artificial mir-302 family pre-miRNA/shRNA structure (Fig. 3B) into the intron insertion site of the SpRNAi-RGFP transgene (ie: MluI-PvuI restriction/ Insertion of the SpRNAi-RGFP transgene into deoxyhydroxyl (Dox)-induced pluripotency of the pTet-On-tTS vector (ie: XhoI-ClaI restriction), thus forming pTet-On -tTS-mir302s transfer gene vector (Fig. 3A). In other words, the recombinant nucleic acid composition (such as SpRNAi , SpRNAi-RGFP, and the like) comprises a drug -evokable gene expression vector. In addition, the SpRNAi-RGFP can also be incorporated into a gene expression vector selected from the group consisting of a plastid, a viral vector, an inverted position, and combinations thereof. The SpRNAi-RGFP transgenic line is flanked by a 370 base pair bp homologous region that is used to recombine the target position of the host cell genome. In terms of gene transfer delivery, the pTet-On-tTS-mir302s vector (10-30 μg) was mixed with the host cells (200-2000) in a hypotonic pH buffer (400 μl; Eppendorf). An electroporation method of 100 microseconds (μsec) was performed at 400-450 volts to deliver the transgene into the host cell genes. After 72 hours, both FACS 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-302 family gene transfer method is over 91%, which is much higher than the success rate of 0.002%-2% found in the original iPS method. All sequences of this artificial mir-302 family were chemically synthesized according to the sequence library of the Sanger miRBase::Sequences program. pTet-On-tTS vector encoding CMV driven tTS repressor gene (CMV-driven tTS inhibitor gene) to remove the activated TRE-CMV promoter of the gene transfected colonization. When deoxyhydroxyl (Dox) is present, the inhibitory function of tTS is neutralized by Dox, thus exhibiting the SpRNAi-RGFP transgene and its coding mir-302 family.

在另一較佳具體實施例中,本發明提供一種基因工程方法,其用來建構包含至少一個所需之介子的一人工/人造SpRNAi 內含子,該介子係用來產生mir-302家族或類mir-302之miRNA、shRNA及/或反義RNA基因靜默效應子。該SpRNAi 係藉由合成寡核苷酸元素的鏈結而形成,以供RNA剪接,合成寡核苷酸元素像是:五端剪接位、BrP、PPT、三端剪接位,及一些鏈接寡核苷酸。寡核苷酸合成器可化學合成並鏈結該等元素。換 句話說,該內含子(如:SpRNAi )係以一化學合成方法來合成。在其它實施例中,該等要素之鏈結可藉由酵素限制及接合來達成。換句話說,該內含子(如:SpRNAi )亦可藉由核苷酸重組方法來形成。所得之內含子(如:SpRNAi )可直接用來轉染至感興趣之該等細胞內或是進一步地合併在宿主基因中以與該基因轉錄分子(pre-mRNA)共同表現。因此,目前發明之重組核苷酸組成物進一步地包含重組內含子,其編碼至少一個像是mir-302的RNA基因靜默效應子。一般而言,該等內含子插入方法包括酵素限制/選殖、同源DNA重組、轉位子合併、跳躍基因整合、反轉錄病毒感染,及其組合。該宿主基因係選自螢光蛋白質(GFP)標記基因、胚胎幹(ES)標記基因、螢光酵素、lac-Z乳糖表現基因、病毒基因、轉位子、跳躍基因、人工重組基因及天然細胞基因。換句話說,該重組核酸組成物進一步包括複數個外顯子,其係選自螢光蛋白質標記基因、螢光酵素基因、lac-Z乳糖表現基因、胚胎幹細胞標記基因、病毒基因、細菌基因、細胞標記基因、跳躍基因、轉位子、及其組合。在不受限制下,本發明較佳使用用來指出mir-302家族表現的修飾紅螢光蛋白質(RGFP)基因。In another preferred embodiment, the invention provides a genetic engineering method for constructing an artificial/artificial SpRNAi intron comprising at least one desired meson for producing a mir-302 family or Silence effector of miRNA, shRNA and/or antisense RNA gene of mir-302. The SpRNAi is formed by synthesizing a strand of an oligonucleotide element for RNA splicing, and the synthetic oligonucleotide elements are: a five-terminal splice site, a BrP, a PPT, a three-terminal splice site, and some linked oligonucleotides. Glycosylate. An oligonucleotide synthesizer can chemically synthesize and link these elements. In other words, the intron (eg, SpRNAi ) is synthesized by a chemical synthesis method. In other embodiments, the linkage of the elements can be achieved by enzyme restriction and engagement. In other words, the intron (such as: SpRNAi) may be formed by recombinant methods nucleotides. The resulting intron (eg, SpRNAi ) can be used directly to transfect into such cells of interest or further incorporated into the host gene for expression with the gene transcriptional molecule (pre-mRNA). Thus, the recombinant nucleotide composition of the present invention further comprises a recombinant intron encoding at least one RNA gene silencing effector such as mir-302. In general, such intron insertion methods include enzyme restriction/selection, homologous DNA recombination, transposon merging, skip gene integration, retroviral infection, and combinations thereof. The host gene is selected from the group consisting of a fluorescent protein (GFP) marker gene, an embryonic stem (ES) marker gene, a luciferase, a lac-Z lactose expression gene, a viral gene, a transposon, a skip gene, a recombinant gene, and a natural cell gene. . In other words, the recombinant nucleic acid composition 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, Cell marker genes, skip genes, transposons, 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.

在一態樣中,該SpRNAi 內含子包含一五端剪接位,其與5’-GTAAGAGK-3’(SEQ.ID.NO.4)或GU(A/G)AGU基序(SEQ.ID.NO.38)(即:5’-GTAAGAGGAT-3’(SEQ.ID.NO.37)、5’-GTAAGAGT-3’(SEQ.ID.NO.39)、 5’-GTAGAGT-3’(SEQ.ID.NO.40)以及5’-GTAAGT-3’(SEQ.ID.NO.41))任一者同源,而其三端係三端受體剪接位(3’-acceptor splice site),該三端受體剪接位與GWKSCYRCAG(SEQ.ID.NO.5)或CT(A/G)A(C/T)NG基序(即:5’-GATATCCTGC AG-3’(SEQ.ID.NO.42)、5’-GGCTGCAG-3’及5’-CCACAG-3’))任一者同源。換句話說,該重組核酸組成物之內含子包含五端供體剪接位(5’-donor splice site)、內含子插入位、分支點基序、多嘧啶段,及三端受體剪接位。此外,分支點序列位在該等五端及三端剪接位之間,其包含與5’-TACTWAY-3’(SEQ.ID.NO.6)基序同源之基序,像是:5’-TACTAAC-3’及5’-TACTTAT-3’。換句話說,該分支點基序包括該SEQ.ID.NO.6序列或與該SEQ.ID.NO.6序列同源。該分支點序列之腺核苷「A」核苷酸在幾乎所有之剪接體內含子中,藉由細胞(2’-5’)寡腺苷合成酶及剪接體來形成一部分之(2’-5’)鏈接套馬索內含子RNA。此外,多嘧啶段係位在接近該分支點及三端剪接位之間,其包含與5’-(TY)m(C/-)(T)nS(C/-)-3’(SEQ.ID.NO.7)或5’-(TC)nNCTAG(G/-)-3’(SEQ.ID.NO.8)基序任一者同源之高T或C含量序列。符號「m」及「n」指示多個重覆,其1;更佳地,m的數量等於1~3且n的數量等於7~12。符號「-」係指在該序列中可被略過之核苷酸。也有一些鏈接核苷酸序列係用來連接所有該等合成內含子元素。根據37 CFR 1.822中關於用 於核苷酸及/或氨基酸序列資料之符號及格式的準則,符號W係指腺嘌呤(A)或胸嘧啶(T)/尿嘧啶(U),符號K係指鳥嘌呤(G)或胸嘧啶(T)/尿嘧啶(U),符號S係指胞嘧啶(C)或鳥嘌呤(G),符號Y係指胞嘧啶(C)或胸嘧啶(T)/尿嘧啶(U),符號R係指腺嘌呤(A)或鳥嘌呤(G),且符號N係指腺嘌呤(A)、胞嘧啶(C)、鳥嘌呤(G)或胸嘧啶(T)/尿嘧啶(U)。In one aspect, the SpRNAi intron comprises a five-terminal splice site with a 5'-GTAAGAGK-3' (SEQ. ID. NO. 4) or GU (A/G) AGU motif (SEQ. ID) .NO.38) (ie: 5'-GTAAGAGGAT-3' (SEQ.ID.NO.37), 5'-GTAAGAGT-3' (SEQ.ID.NO.39), 5'-GTAGAGT-3' ( SEQ.ID.NO.40) and 5'-GTAAGT-3' (SEQ.ID.NO.41)) are homologous, and their tri-terminal tri-terminal receptor splice sites (3'-acceptor splice site) The tripartite receptor splice site is associated with the GWKSCYRCAG (SEQ.ID.NO.5) or CT(A/G)A(C/T)NG motif (ie: 5'-GATATCCTGC AG-3' (SEQ. ID.NO.42), 5'-GGCTGCAG-3' and 5'-CCACAG-3')) are homologous. In other words, the intron of the recombinant nucleic acid composition comprises a 5'-donor splice site, an intron insertion site, a branch point motif, a polypyrimidine segment, and a triad receptor splicing Bit. 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.NO.6) motif, such as: 5 '-TACTAAC-3' and 5'-TACTTAT-3'. In other words, the branch point motif comprises or is homologous to the SEQ.ID.NO.6 sequence. The adenosine "A" nucleotide of this branch point sequence is partially formed by the cell (2'-5') oligoadenylate synthetase and the splice form in almost all of the spliced introns (2'- 5') Link the set of horseshoe intron RNA. In addition, the polypyrimidine moiety is located close to the branch point and the three-terminal splice site, and comprises 5'-(TY)m(C/-)(T)nS(C/-)-3' (SEQ. A high T or C content sequence homologous to any of the 5'-(TC)nNCTAG(G/-)-3' (SEQ.ID.NO.8) motifs. The symbols "m" and "n" indicate multiple repetitions, 1; More preferably, the number of m is 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 the sequence. There are also some linked nucleotide sequences that are used to link all of these synthetic intron elements. 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 adenine (A) or thymine (T) / uracil (U), symbol K refers to Guanine (G) or thymidine (T) / uracil (U), symbol S refers to cytosine (C) or guanine (G), symbol Y refers to cytosine (C) or thymidine (T) / Uracil (U), the symbol R refers to adenine (A) or guanine (G), and the symbol N refers to adenine (A), cytosine (C), guanine (G) or thymidine (T) /Uracil (U).

在另一態樣中,表現各種內含子基因靜默效應子之多個轉殖基因及/或載體可用來達成多個標的基因上的基因靜默。在其它實施例中,多個基因靜默效應子可由內含子介子產生。換句話說,該內含子插入位包含至少一個與mir-302同源的基因靜默效應子。例如:已報導在斑馬魚中之anti-EGFP 含pre-miRNA內含子的異位表現產生兩個不同長度之miRNA,稱之為miR-EGFP(282/300)及miR-EGFP(280-302),這指出該SpRNAi 之介子可產生多個基因靜默效應子(Lin等人,2005)。在某些例子中,內含子基因靜默效應子可與標的基因轉錄分子(即:mRNA)雜合以形成用來引發二級RNA干擾效應(RNAi)的雙股siRNA。因為該等基因靜默效應子不斷地由該基因轉殖載體製造,其將紓解有關體內快速RNA降解之疑慮。此策略之優點係經由該載體型基因轉殖轉染或病毒感染的穩定傳送,其提供可靠之長期基因靜默效力。此外,本發明可在特定RNA啟動子之控制下生產RNAi相關之基因靜默效應子,包括:miRNA、shRNA及siRNA,該特定RNA啟動子係選自第 二型RNA聚合酶(Pol-II)、病毒聚合酶、第三型RNA聚合酶(Pol-III),及四環黴素反應元素控制RNA聚合酶(tetracycline responsive element-controlled RNA polymerase,TRE)啟動子。該等病毒啟動子係類Pol-II RNA啟動子,其係自巨細胞病毒(cytomegalovirus,CMV)、反轉錄病毒長終端區(retrovirus long-terminal region,LTR)、B型肝炎病毒(hepatitis B virus,HBV)、腺病毒(adenovirus,AMV)及腺相關病毒(adeno-associated virus,AAV)中分離而得。例如:慢病毒LTR啟動子足以提供超過每一細胞5×105 個拷貝的pre-mRNA轉錄分子。也可在該病毒聚合酶啟動子之前插入藥敏感抑制子(即:tTS )以控制該等基因靜默效應子之轉錄率。該抑制子可用化學藥劑或抗生素來抑制,該等化學藥劑或抗生素係選自氨基醣類抗生素(G418)、四環黴素(tetracycline)、去氧羥(doxycyclin)、新黴素(neomycin)、安比西林(ampicillin)、康黴素(kanamycin)及其衍生物等等之群。換句話說,目前發明之重組核酸組成物的表現可用一類藥物抗生素衍生物來調節,像是:四環黴素衍生物例如:去氧羥係四環黴素衍生物其中之一。In another aspect, multiple transgenic genes and/or vectors that exhibit various intron gene silencing effectors can be used to achieve gene silencing on multiple target genes. In other embodiments, multiple gene silencing effectors can be produced by intron mesons. In other words, the intron insertion site comprises at least one gene silencing effector homologous to mir-302. For example, it has been reported that the ectopic expression of anti- EGFP- containing pre-miRNA introns in zebrafish produces two different lengths of miRNA, called miR-EGFP (282/300) and miR-EGFP (280-302). ), which it indicates that the SpRNAi may generate a plurality of muting meson effector gene (Lin et al., 2005). In some instances, an intron gene silencing effector can be hybridized to a target gene transcription molecule (ie, mRNA) to form a double stranded siRNA that is used to elicit a secondary RNA interference effect (RNAi). Because these gene silencing effects are continually produced by the gene transfer vector, they will alleviate concerns about rapid RNA degradation in vivo. The advantage of this strategy is the stable delivery of transfection or viral infection via this vector type gene, which provides reliable long-term gene silencing efficacy. In addition, the present invention can produce RNAi-related gene silencing effectors under the control of a specific RNA promoter, including: miRNA, shRNA and siRNA, the specific RNA promoter is selected from the second type RNA polymerase (Pol-II), Viral polymerase, type III RNA polymerase (Pol-III), and tetracycline responsive element-controlled RNA polymerase (TRE) promoter. The promoters of the virus are Pol-II RNA promoters, which are derived from cytomegalovirus (CMV), retrovirus long-terminal region (LTR), hepatitis B virus (hepatitis B virus). , HBV), adenovirus (AMV) and adeno-associated virus (AAV) were isolated. For example: a lentiviral LTR promoter is sufficient to provide more than 5 × 10 5 per cell copies of transcript pre-mRNA molecule. Drug sensitive repressors (i.e., tTS ) can also be inserted prior to the viral polymerase promoter to control the transcription rate of the gene silencing effectors. The inhibitor may be inhibited by a chemical agent or an antibiotic selected from the group consisting of amino sugar antibiotics (G418), tetracycline, doxycyclin, neomycin, A group of ampicillin, kanamycin, its derivatives, and the like. In other words, the performance of the recombinant nucleic acid composition of the present invention can be regulated by a class of pharmaceutical antibiotic derivatives, such as one of tetracycline derivatives such as deoxyhydroxytetracycline derivatives.

依據本發明,所需內含子RNA介子係以細胞內機制來切除並釋放,然後在特定基因標的上引發所欲之基因靜默效應,該特定基因標的與該RNA介子具有高互補性;而該宿主基因轉錄分子之該等外顯子係鏈接在一起以形成用來產生具所欲蛋白功能的成熟mRNA,這些報導或標記蛋白質可選自紅/綠螢 光蛋白質(RGFP/EGFP)、胚胎基因標記、螢光酵素、β半乳醣苷酶(lac-Z)及其衍生物。該報導/標記蛋白之表現可用於識別在該等轉染細胞中該等內含子基因靜默效應子的數量程度及位置,以及有助於確認所得基因靜默效應。在其它實施例中,以該外顯子鏈接形成的成熟mRNA可用在習知之基因治療以代替受損或遺失之基因功能,或增加特定基因表現。換句話說,該等基因靜默效應子(與mir-302同源)也可包括反義RNA、核醣酵素(ribozyme)、短臨時RNA(short temporary RNA,stRNA)、細微非編碼RNA(tiny non-coding RNA,tncRNA)、Piwi交互作用RNA(Piwi-interacting RNA,piRNA)、雙股RNA(double-stranded RNA,dsRNA)、siRNA、shRNA、miRNA,及其前驅物(即:pri-/pre-miRNA)。該等內含子基因靜默效應子之使用係用來將不想要之標的基因靜默的有力工具,該等標的基因係選自外源基因、致病轉殖基因、病毒基因、突變基因、致癌基因、疾病相關非編碼RNA基因與許多其它類型之蛋白質編碼以及非編碼細胞基因。According to the present invention, the desired intron RNA meson is excised and released by an intracellular mechanism, and then elicits a desired gene silencing effect on a specific gene target, the specific gene target having high complementarity with the RNA meson; The exon lines of the host gene transcriptional molecule are linked together to form a mature mRNA for producing a desired protein function, and the reporter or marker protein may be selected from red/green Photoprotein (RGFP/EGFP), embryonic gene marker, luciferase, beta-galactosidase (lac-Z) and its derivatives. The expression of the reporter/marker protein can be used to identify the extent and location of the intron gene silencing effectors in the transfected cells, as well as to confirm the resulting gene silencing effect. In other embodiments, mature mRNAs formed with the exon linkages can be used in conventional gene therapy to replace impaired or lost gene function, or to increase specific gene expression. In other words, 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, tncRNA), 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, mutant genes, oncogenes. Disease-associated non-coding RNA genes are encoded with many other types of proteins as well as non-coding cellular genes.

因為一些天然pre-miRNA的幹環(stem-loop)結構太大且/或太複雜,無法配合該SpRNAi-RGFP 轉殖基因,本發明者通常使用人造重設tRNAmet 環(即5’-(A/U)UCCAAGGGGG-3’)(SEQ.ID.NO.43)來代替該等天然pre-miRNA環。該tRNAmet 環顯示出能如同天然miRNA般經由相同之Ran-GTP及Exportin-5傳送機制有效地協助將人造重設之miRNA自細胞核輸出至細 胞質(Lin等人,(2005)Gene 356 :32-38)。有利的是,本發明目前使用一對人造改善pre-mir-302環,包括:5’-GCTAAGCCAG GC-3’(SEQ.ID.NO.1)及5’-GCCTGGCTTA GC-3’(SEQ.ID.NO.2),其提供與天然pre-miRNA相同的細胞核輸出效率,但不會干擾tRNA輸出。並且,此改善加強了mir-302a-mir-302a*及mir-302c-mir-302c*雙鏈體(duplex)之形成,其可增加mir-302家族的整體功能及穩定度。該等新穎pre-miRNA環之設計係以mir-302b/mir-302a之tRNAmet 環與短幹環(short stem-loop)的組合來修正,其係高度地表現在胚胎幹細胞中而非表現在其它分化組織細胞中。因此,在mir-302家族中使用該等重組/人造/人工pre-miRNA/shRNA環將不會干擾我們身體中之天然miRNA路徑,因而較不具細胞毒性且更加安全。Because the stem-loop structure of some natural pre-miRNAs is too large and/or too complex to fit the SpRNAi-RGFP transgene, the inventors typically use artificially reconfigured tRNA met loops (ie 5'-( A/U) UCCAAGGGGG-3') (SEQ. ID. NO. 43) is substituted for these natural pre-miRNA loops. This tRNA met loop has been shown to efficiently assist in the export of artificially reprogrammed miRNAs from the nucleus to the cytoplasm via the same Ran-GTP and Exportin-5 delivery mechanisms as natural miRNAs (Lin et al., (2005) Gene 356 :32- 38). Advantageously, the present invention currently employs a pair of artificially improved pre-mir-302 loops, including: 5'-GCTAAGCCAG GC-3' (SEQ. ID. NO. 1) and 5'-GCCTGGCTTA GC-3' (SEQ. ID.NO.2), which 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 function and stability of the mir-302 family. The design of these novel pre-miRNA loops is modified by the combination of the tRNA met loop of mir-302b/mir-302a and the short stem-loop, which is highly expressed in embryonic stem cells and not in others. Differentiated into tissue cells. Therefore, the use of these recombinant/artificial/artificial pre-miRNA/shRNA loops in the mir-302 family will not interfere with the natural miRNA pathways in our body and is therefore less cytotoxic and safer.

mir-302 pre-miRNA家族之基因群(cluster)係藉由合成mir-302同源衍生物之雜合及鏈結/接合來形成,其在五端至三端方向上包含四個部分:mir-302a、mir-302b、mir-302c及mir-302d pre-miRNA(圖3B)。所有該等人造重設之mir-302 miRNA/shRNA分子在其前十七個核苷酸中具有完全相同之五端(如:5’-UAAGUGCUUC CAUGUUU-3’(SEQ.ID.NO.3))。以下列出用於mir-302 pre-miRNA基因群之DNA重組的合成寡核苷酸:包括:同義mir-302a(mir-302a-sense),5’-GTCCGATCGT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATCTCGAGCT C-3’(SEQ.ID.NO.29);反義mir-302a(mir-302a-antisense),5’-GAGCTCGAGA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GACGATCGGA C-3’(SEQ.ID.NO.30);同義mir-302b,5’-ATCTCGAGCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTAG CGCTA-3’(SEQ.ID.NO.31);反義mir-302b,5’-TAGCGCTAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGCTC GAGAT-3’(SEQ.ID.NO.32);同義mir-302c,5’-CGCTAGCGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3’(SEQ.ID.NO.33);反義mir-302c,5’-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCGCTAGCG-3’(SEQ.ID.NO.34);同義mir-302d,5’-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGACGCGTC AT-3’(SEQ.ID.NO.35);及反義mir-302d,5’-ATGACGCGTC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3’(SEQ.ID.NO.36)(Sigma-Genosys,St.Louis,MO)。在其它實施例中,吾人可使用人造重設之shRNA,其係以合成同義mir-302家族,5’-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA GCCAGGCACA CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3’(SEQ.ID.NO.27)與反義mir-302家族,5’-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3’(SEQ.ID.NO.28)之雜合來形成,以代替簡單內含子插入的mir-302pre-miRNA基因群。換句話說,該等較佳的基因靜默效應子之一係以SEQ.ID.NO.27與SEQ.ID.NO.28.雜合形成的一重組核酸序列。該mir-302 shRNA向所有天然之mir-302成員分享超過91%的同源性,並標靶人類中相同之細胞基因。The mir-302 pre-miRNA family of genes 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 -302a, mir-302b, mir-302c and mir-302d pre-miRNA (Fig. 3B). All such artificially-reset mir-302 miRNA/shRNA molecules have identical five-terminal ends in their first seventeen nucleotides (eg, 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID.NO.3) ). The synthetic oligonucleotides for DNA recombination of the mir-302 pre-miRNA gene group are listed below: including: synonymous mir-302a (mir-302a-sense), 5'-GTCCGATCGT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATCTCGAGCT C-3' (SEQ.ID.NO.29); antisense mir-302a (mir-302a-antisense), 5'-GAGCTCGAGA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GACGATCGGA C-3' (SEQ .ID.NO.30); Synonymous mir-302b, 5'-ATCTCGAGCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTAG CGCTA-3' (SEQ.ID.NO.31); antisense mir-302b, 5'-TAGCGCTAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGCTC GAGAT-3' (SEQ.ID.NO.32); Synonymous mir-302c, 5'-CGCTAGCGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3' (SEQ.ID.NO.33) ; antisense mir-302c, 5'-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCGCTAGCG-3' (SEQ.ID.NO.34); synonymous mir-302d, 5'-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA 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 an artificially-reset shRNA that synthesizes the synonymous mir-302 family, 5'-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA GCCAGGCACA CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3' (SEQ.ID.NO.27) Antisense mir-302 family, 5'-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3' (SEQ.ID.NO.28) hybrid formation to replace the simple intron insertion of mir-302pre-miRNA Gene group. 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 SEQ.ID.NO.28. The mir-302 shRNA shares more than 91% homology to all natural mir-302 members and targets the same cellular genes in humans.

關於該mir-302 pre-miRNA/shRNA之內含子插入,由於該重組SpRNAi-RGFP 轉殖基因之插入位在其五端及三端分別以PvuIMluI 限制/選殖位作為切位,該先驅介子可藉由各種pre-miRNA/shRNA介子簡單地移除及取代(如:mir-302 pre-miRNA/shRNA),其具有配合該等PvuIMluI 限制位的相配粘著端(cohesive end)。藉由針對各種基因轉錄分子來改變該等內含子的介子,本發明之內含子mir-302家族表現系統可用作體外及體內地誘發標的基因靜默的有力工具。對於長度確認及轉殖基因純化而言,該mir-302插入SpRNAi-RGFP 結構(10ng)係以聚合酶連鎖反應(polymerase chain reaction,PCR)與一對寡核苷酸引子(即:5’-CTCGAGCATG GTGAGCGGCC TGCTGAA-3’(SEQ.ID.NO.23)及5’-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3’(SEQ.ID.NO.24))來複製,其在94℃、52-57℃及之後在68℃各執行一分鐘,共執行25-30個循環。所得之PCR產物(~900-1100bp)在2%洋菜膠上層析,然後以膠體萃取工具(gel extraction kit)來萃取及純化(Qiagen,CA)。在確認該DNA序列後,該插入mir-302之SpRNAi-RGFP 的純化轉殖基因經進一步地插入一pTet-On-tTS 載體之限制/選殖位(即:XhoI-ClaI 位),以形成用於細胞內表現的pTet-On-tTS-mir302s 轉殖基因表現載體(圖3A)。Regarding the intron insertion of the mir-302 pre-miRNA/shRNA, since the insertion site of the recombinant SpRNAi-RGFP transgene is nicked at its five-terminal and three-terminal ends with PvuI and MluI restriction/selection positions, respectively The precursor meson can be simply removed and replaced by various pre-miRNA/shRNA mesons (eg, mir-302 pre-miRNA/shRNA) with a cohesive end that matches the PvuI and MluI restriction sites. . The intron mir-302 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 these introns for various gene transcriptional molecules. For length confirmation and purification of the transgenic gene, the mir-302 was inserted into the SpRNAi-RGFP structure (10 ng) by polymerase chain reaction (PCR) with a pair of oligonucleotide primers (ie: 5'- CTCGAGCATG GTGAGCGGCC TGCTGAA-3' (SEQ.ID.NO.23) and 5'-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3' (SEQ.ID.NO.24)) were replicated at 94 ° C, 52-57 ° C and after Each of the 68 ° C is performed for one minute, and a total of 25-30 cycles are performed. The resulting PCR product (~900-1100 bp) was chromatographed on 2% acacia and extracted and purified using a gel extraction kit (Qiagen, CA). After confirming the DNA sequence, the purified transgenic gene of SpRNAi-RGFP inserted into mir-302 is further inserted into a restriction/selection position of a pTet-On-tTS vector (ie, XhoI-ClaI position) for formation. The pTet-On-tTS-mir302s transgenic gene expression vector expressed in cells (Fig. 3A).

可使用選自以下之一種方法來達成將該pTet-On-tTS-mir302s 轉殖基因載體傳送進哺乳動物細胞內:微脂體/化學轉染法(liposomal/chemical transfection)、電穿孔法(electroporation)、基因槍穿透法(gene gun penetration)、轉位子/反轉位子插入法(transposon/retrotransposon insertion)、跳躍基因整合(jumping gene integration)、顯微注射(micro-injection),及反轉錄病毒/慢 病毒感染(retroviral/lentiviral infection)。為避免隨機轉殖基因插入及細胞變異的風險,本發明者較佳地使用與同源重組配合之電穿孔法以將該pTet-On-tTS-mir302s 轉殖基因載體傳送進感興趣之宿主細胞。例如:該SpRNAi-RGFP 轉殖基因係以一370鹼基對(bp)同源區域為側翼,以用來重組插入靠近LOC727977所在區域之三端鄰近處的人類染色體6中,在此區域中並無已知之基因編碼。頃偵測到在此單一位置中精確地插入SpRNAi-RGFP (圖4A)。因此在Dox存在時,該SpRNAi-RGFP 轉殖基因及其編碼mir-302家族之表現全係依照該pTet-On-tTS 載體之TRE-CMV 啟動子的活化而定。本發明者已在人類正常及癌症體細胞兩者中測試了此種可誘發mir-302家族表現的新穎方法,該等體細胞包括:正常表皮皮膚細胞、正常頭髮毛囊細胞、癌症乳腺癌MCF7、前列腺癌PC3,及黑色素癌Colo細胞。換句話說,該哺乳動物細胞可轉化為一多能性類幹狀態,該哺乳動物細胞係選自人類細胞、正常體細胞、患病體細胞、腫瘤或癌症細胞、人類頭髮毛囊細胞、人類皮膚細胞及其組合。所得之該等mir-302轉導多能性幹(mir-302-transduced pluripotent stem,mirPS)細胞在相同之基因體位置處全都僅攜帶該SpRNAi-RGFP 轉殖基因之一或二個共存的重覆序列(concomitant copies)(圖4A),這表示該整個mir-302濃度可影響該等mirPS細胞的存活。吾人也已觀察到mir-302家族及其標記RGFP mRNA之表現程度將隨著Dox濃度提高而增加(圖 4B)。整個mir-302濃度必須表現得超過30倍(folds)但低於50倍,以觸發將該體細胞轉化為類ES多能性幹細胞。由於在頭髮毛囊及皮膚細胞中其極低的表現率(僅增加約16倍),所以由Pol-III或CMV啟動子驅動之目前可得的直接(外顯)siRNA/shRNA表現系統無法運作。這可能是因為該天然mir-302基因群之樣板太短且具結構,以致無法以Pol-III或CMV啟動子直接地轉錄。因此,本發明提供一種可誘發機制,其以具體藥物(即:Dox)在體外及體內地控制mir-302家族的表現程度,這可作為除了該等細胞內監測系統外的一第二道防線。在本發明之該等mirPS細胞中不會偵測到因RNA累積或過飽和所造成的細胞毒性。The pTet-On-tTS-mir302s gene vector can be delivered into mammalian cells using a method selected from the group consisting of liposomal/chemical transfection, electroporation. ), gene gun penetration, transposon/retrotransposon insertion, jumping gene integration, micro-injection, and retrovirus / retroviral/lentiviral infection. In order to avoid the risk of randomization of gene insertion and cell variability, the present inventors preferably use electroporation in combination with homologous recombination to deliver the pTet-On-tTS-mir302s transgene vector into a host cell of interest. . For example, the SpRNAi-RGFP transgenic line is flanked by a 370 base pair (bp) homology region for recombination into human chromosome 6 adjacent to the three ends of the region where LOC727977 is located, in this region There are no known genetic codes. Accurate insertion of SpRNAi-RGFP in this single position was detected (Fig. 4A). Thus, in the presence of Dox, the SpRNAi-RGFP transgene and its expression in the mir-302 family are all based on the activation of the TRE-CMV promoter of the pTet-On-tTS vector. The present inventors have tested novel methods for inducing mir-302 family expression in both human normal and cancer somatic cells, including: normal epidermal skin cells, normal hair follicle cells, cancer breast cancer MCF7, Prostate cancer PC3, and melanoma Colo cells. In other words, the mammalian cell can be transformed into a pluripotent dry state selected from human cells, normal somatic cells, diseased somatic cells, tumor or cancer cells, human hair follicle cells, human skin. Cells and combinations thereof. The resulting mir-302-transduced pluripotent stem (mirPS) cells all carry only one or two coexisting weights of the SpRNAi-RGFP transgene in the same locus position. Concomitant copies (Fig. 4A), indicating that the entire mir-302 concentration can affect the survival of the mirPS cells. We have also observed that the degree of expression of the mir-302 family and its labeled RGFP mRNA will increase with increasing Dox concentration (Fig. 4B). The entire mir-302 concentration must be expressed more than 30 fold but less than 50 fold to trigger the conversion of the somatic cells to ES-like pluripotent stem cells. The currently available direct (exogenous) siRNA/shRNA expression system driven by the Pol-III or CMV promoter is inoperable due to its extremely low rate of expression in hair follicles and skin cells (only a 16-fold increase). This may be because the template for the native mir-302 gene group is too short and structural to be directly transcribed by the Pol-III or CMV promoter. Accordingly, the present invention provides an inducible mechanism for controlling the degree of expression of the mir-302 family in vitro and in vivo with a specific drug (ie, Dox), which serves as a second line of defense in addition to such intracellular monitoring systems. . Cytotoxicity due to accumulation or supersaturation of RNA is not detected in the mirPS cells of the present invention.

本發明已採用該可誘發轉殖基因表現系統的新穎設計及策略,稱之為如先前提及之pTet-On-tTS-mir302s ,並將其用於基因轉殖地表現在人類體細胞/癌症細胞中之mir-302家族(mir-302s)的該等成員或同源衍生物,因此可將該等體細胞/癌症細胞轉化成類胚胎幹(ES)的多能性狀態。在較佳具體實施例中,本發明提供一種用於使用藥物可誘發重組核酸組成物之方法,該組成物可被傳送、轉錄及處理而成在人類細胞中之類mir-302 miRNA/shRNA分子/同源衍生物,因此在該等細胞中之mir-302標的之發育及分化相關基因誘發特定之基因靜默效應,本方法包含以下步驟:a)提供:i)細胞基質,其表現mir-302家族標的之複數個發育及分化相關基因,及ii)重組核酸組成 物,其可轉錄編碼複數個非編碼mir-302 miRNAs/shRNAs或其同源衍生物的分離RNA,該複數個非編碼mir-302 miRNAs/shRNAs或其同源衍生物藉由細胞內機制依次被處理成成熟之mir-302 miRNAs/shRNAs或其同源衍生物中,因此能抑制在該細胞基質中之該等標的基因的功能;b)以該重組核酸組成物來處理該細胞基質,其係在使在該細胞基質中之該等標的基因功能受抑制的狀況下處理。更佳的是,該藥物可誘發重組核酸組成物係Tet-On 載體,其包含插入重組mir-302家族基因群(mir-302s;SEQ.ID.NO.29-SEQ.ID.NO.36之雜合)或人造重設mir-302 shRNA同源衍生物(即:SEQ.ID.NO.27及SEQ.ID.NO.28之雜合)任一個於SpRNAi-RGFP 轉殖基因中。該細胞基質可用體外(in vitro)、離體(ex vivo)及/或體內(in vivo)任一種方式表現該mir-302 miRNA/shRNA及其標的基因。之後,將該細胞基質轉化或轉形成類ES狀態,其不僅呈現標準ES細胞標記,如:Oct3/4SSEA3SSEA4Sox2Nanog 、及LIN-28 ,並且包含高度去甲基化基因體,其類似經過幹細胞轉化之接合子基因體。The present invention has employed a novel design and strategy for inducing a transgenic gene expression system, referred to as pTet-On-tTS-mir302s as previously mentioned, and used for gene transfer in human somatic/cancer cells. Such members or homologous derivatives of the mir-302 family (mir-302s) can thus convert such somatic/cancer cells into a pluripotent state of embryonic stem-like (ES). In a preferred embodiment, the present invention provides a method for inducing a recombinant nucleic acid composition using a drug, which can be transmitted, transcribed, and processed into a mir-302 miRNA/shRNA molecule in a human cell. a homologous derivative, such that the developmental and differentiation-related genes of the mir-302 in these cells induce a specific gene silencing effect, the method comprising the steps of: a) providing: i) a cell matrix that exhibits mir-302 a plurality of developmental and differentiation-related genes of the family target, and ii) a recombinant nucleic acid composition transcribed to encode a plurality of isolated RNAs encoding non-coding mir-302 miRNAs/shRNAs or homologous derivatives thereof, the plurality of non-coding mir- 302 miRNAs/shRNAs or homologous derivatives thereof are sequentially processed into mature mir-302 miRNAs/shRNAs or homologous derivatives thereof by intracellular mechanisms, 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 that inhibit the function of the target genes in the cell matrix. More preferably, the pharmaceutical compositions can induce recombinant nucleic acid based Tet-On vector, which comprises inserting a recombinant mir-302 family gene cluster (mir-302s; SEQ.ID.NO.29-SEQ.ID.NO.36 of Heterozygous) or artificially reset mir-302 shRNA homologous derivatives (ie, heterozygous for SEQ.ID.NO.27 and SEQ.ID.NO.28) in either of the SpRNAi-RGFP transgenic genes. The cell substrate can express the mir-302 miRNA/shRNA and its target gene in any of in vitro, ex vivo and/or in vivo manners. Thereafter, the cell matrix is transformed or transformed into an ES-like state that exhibits not only standard ES cell markers such as: Oct3/4 , SSEA3 , SSEA4 , Sox2 , Nanog , and LIN-28 , but also highly demethylated genomes. It is similar to the zygote genome transformed by stem cells.

應用此發明,本發明者已在七個方面成功地達成mir-302誘發多能性幹(mirPS)細胞生成:第一,已生成源自相同細胞類型之兩個同族系的mirPS細胞株;其一係來自人類正常頭髮毛囊細胞(hHFC),而另一者係來自癌症黑色素癌Colo細胞,兩者皆可體外地形成類胚胎體(圖5A-5C)。第二,已使用該等mirPS 轉錄分子之微型核醣核酸(miRNA)微陣列及北方點墨分析法(northern blot analyses)證實mir-302家族之表現提高(圖6A-6B)。第三,已偵測到該標準胚胎幹(ES)細胞標記之表現提高,包括:Oct3/4、SSEA-3、SSEA-4、Sox2及Nanog(圖6B、8B-8C及圖9B)。第四,已觀察到整體之基因體DNA去甲基化,其類似經過該轉化程序後之接合子基因體的狀態(圖7A-7C)。第五,該等mirPS細胞之泛基因體之基因表現型式已顯示出與人類ES WA01(H1)及WA09(H9)細胞之樣式有超過86%的高相似度(圖8A及圖9A)。第六,將該等mirPS細胞衍生之類胚胎體(embryoid bodies,EB)體內移植至免疫功能不足之SCID-beige小鼠中可形成類畸胎瘤組織囊腫,其包含所有三個胚胎生殖層(外胚層、中胚層及決定性的內胚層)(圖10)。然而,不像畸胎瘤,該等組織囊腫與其周圍組織間形成一極佳且清楚之邊界。並且,該等囊腫在小鼠中的成長在移植後約2.5週時中止。這看來似乎是有一種自我調節之機制可體內地限制該等mirPS衍生EB細胞的隨機生長。最後,可將mirPS細胞之分化導引以形成各種身體系及生殖系組織細胞類型,像是:神經元前身(neuronal progenitor)、軟骨細胞(chondrocyte)、纖維母細胞(fibroblast)及類精原細胞原生細胞(spermatogonia-like primordial cell),其係體外地使用各種荷爾蒙及/或生長因子來處理(圖11A-11O)。此外,本發明者已成功地使用電穿孔型基因轉殖傳送來形成該等mir-302所誘發之mirPS細胞株,其避免反轉錄 病毒感染及細胞突變的風險(圖2A及圖2B)。該等發現提供了微型核醣核酸(miRNA)誘發幹細胞生成的強力證據,其中mir-30家族之異位表現不僅能將成人身體細胞及癌症細胞兩者皆轉化為類ES多能性狀態,而且能在無哺乳細胞培養條件下維持該等類ES mirPS細胞的再生及多能性。由於mir-302家族之功能可用91%-93%之高成功率來將正常及癌症組織細胞兩者皆轉化為類ES多能性幹細胞,此新穎發明之該等發現可在幹細胞及癌症治療兩者中提供有利之應用。Using this invention, the inventors have successfully achieved mir-302 induced pluripotent stem (mirPS) cell production in seven aspects: first, mirPS cell lines derived from two homologous lines of the same cell type have been generated; One is from human normal hair follicle cells (hHFC) and the other is from cancer melanoma Colo cells, both of which can form embryoid bodies in vitro (Figures 5A-5C). Second, the mirPS have been used MicroRNAs (miRNA) microarrays of transcriptional molecules and northern blot analyses confirmed increased performance in the mir-302 family (Figures 6A-6B). Third, increased performance of the standard embryonic stem (ES) cell markers has been detected, including: Oct3/4, SSEA-3, SSEA-4, Sox2, and Nanog (Figures 6B, 8B-8C, and Figure 9B). Fourth, overall genomic DNA demethylation has been observed, similar to the state of the zygote genotype following this transformation procedure (Figures 7A-7C). Fifth, the gene expression patterns of the ubiquitosomes of these mirPS cells have been shown to have a high similarity to the patterns of human ES WA01 (H1) and WA09 (H9) cells (Fig. 8A and Fig. 9A). Sixth, transplanting such 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 ( Ectodermal, mesoderm and definitive endoderm) (Fig. 10). 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 discontinued approximately 2.5 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, chondrocyte, fibroblast, and spermatogonial cells. Spermatogonia-like primordial cells, which are treated in vitro using various hormones and/or growth factors (Figures 11A-11O). Furthermore, the present inventors have successfully used electroporation-type gene transfection to form mirPS cell strains induced by these mir-302, which avoid reverse transcription Risk of viral infection and cell mutation (Figure 2A and Figure 2B). These findings provide strong evidence that microRNAs (miRNAs) induce stem cell production, in which the ectopic manifestations of the mir-30 family not only convert both adult body cells and cancer cells into ES-like pluripotent states, but also The regeneration and pluripotency of these ES mirPS cells were maintained in the absence of mammalian cell culture conditions. Since the function of the mir-302 family can convert both normal and cancer tissue cells into ES-like pluripotent stem cells with a high success rate of 91%-93%, the novel invention can be found in both stem cells and cancer treatment. Advantageous applications are provided.

由以上發現可知,本發明者亦已習得mir-302不僅能大幅地抑制細胞週期素相關激酶2(cyclin-dependent kinase 2,CDK2)、細胞週期素(cyclin)D1及D2的表現以減弱細胞增殖及遷移率,而且能抑制MECP2及MECP1-p66的活動以誘發基因體DNA的整體去甲基化(圖8B-8C及圖9B)。眾所周知細胞週期素cyclin E相關之CDK2係進入細胞循環S期所必須的,且CDK2之抑制可導致G1期檢驗點停滯,其中細胞週期素cyclin D1在回應DNA損害時可超越G1期停滯。根據此原則,以mir-302來抑制CDK2及細胞週期素cyclin D1兩者暗示一項事實:mirPS細胞具有一極慢的分裂率。如圖5A及圖5C中所示,mirPS細胞之平均細胞循環係約20-24小時,其遠慢於其身體/癌症對等物的平均細胞循環(每細胞循環約4-6小時)。腫瘤/癌症細胞無法在如此低之細胞增殖率下生存。藉由控制該細胞循環率,mir-302也可影響細胞之存活。因此,此癌症-幹細胞循環轉換 的結果對癌症治療有極大的好處。此外,對MECP2及MECP1-p66活動的抑制與圖7A-7C的結果一致,其顯示將惡性癌症細胞後天轉化成良性mirPS細胞。可以想見,從病人處所得之該等mirPS細胞可進一步幫助修補癌症之組織傷害。藉由抑制基因體印痕(genomic imprinting)及細胞命運決定(cell fate determination)所必要的細胞基因,mir-302家族不僅能將分化體細胞/癌症細胞轉化為類ES多能性狀態,同時也能在無哺乳細胞培養條件下保持此類ES狀態。此外,由於CDK2,細胞週期素cyclin D1及D2之抑制亦可減弱腫瘤/癌症細胞之該等細胞增殖及遷移率,mir-302家族扮演了對抗腫瘤細胞生長及形成之強力腫瘤抑制子的角色(Lin等人,2008b)。mir-302家族之此腫瘤抑制子特徵可協助生成用於臨床移植、幹細胞及癌症治療上的無腫瘤誘發多能性幹細胞。根據吾人目前之發現,mir-302誘發mirPS細胞轉化之機制較該等轉錄因子誘發iPS細胞之機制更加安全、清楚及可理解。From the above findings, the present inventors have also learned that mir-302 can not only significantly inhibit the expression of cyclin-dependent kinase 2 (CDK2), cyclin D1 and D2 to attenuate cell proliferation. And mobility, and can inhibit the activity of MECP2 and MECP1-p66 to induce global demethylation of genomic DNA (Fig. 8B-8C and Fig. 9B). It is well known that the cyclin E-related CDK2 line is required for entry into the S phase of the cell cycle, and inhibition of CDK2 can lead to arrest of the G1 phase checkpoint, in which cyclin D1 can exceed G1 arrest in response to DNA damage. According to this principle, inhibition of both CDK2 and cyclin D1 by mir-302 suggests 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 the average cell cycle of their body/cancer counterparts (about 4-6 hours per cell cycle). Tumor/cancer cells cannot survive at such low cell proliferation rates. By controlling the rate of cell turnover, mir-302 can also affect cell survival. Therefore, this cancer-stem cell cycle conversion The results have great benefits for cancer treatment. Furthermore, inhibition of MECP2 and MECP1-p66 activity was consistent with the results of Figures 7A-7C, which showed that malignant cancer cells were acquired into benign mirPS cells. It is conceivable that the mirPS cells obtained from the patient can further help repair the tissue damage of the cancer. By inhibiting the cellular genes necessary for genomic imprinting and cell fate determination, the mir-302 family can not only transform differentiated somatic cells/cancer cells into ES-like pluripotent states, but also This ES state is maintained in the absence of mammalian cell culture conditions. In addition, due to CDK2, inhibition of cyclin D1 and D2 also attenuates the cell proliferation and migration of tumor/cancer cells, and 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 feature of the mir-302 family can assist in the generation of tumor-free pluripotent stem cells for clinical transplantation, stem cell and cancer therapy. According to our current findings, the mechanism by which mir-302 induces mirPS cell transformation is safer, clearer, and understandable than the mechanism by which these transcription factors induce iPS cells.

總而言之,本新穎發明mir-302家族表現系統提供一安全且有力的工具以供新穎之類ES多能性幹細胞生成,特別是源自身體毛髮毛囊細胞之原生培養,因為毛髮較容易取得。由於內含子miRNA路徑係以多個細胞內監測系統(像是:mRNA轉錄、RNA剪接、外體處理及NMD)嚴密調控,因此該路徑較習知之siRNA/shRNA路徑更加有效及安全(Lin等人,2008a)。本發明具有至少五個有益之突破。第一,mir-302表現轉殖基因 可代替在該等先前之iPS方法中所用的所有四個大型轉錄因子基因,其用來生成僅源自一些病人體細胞之更具同源性的類ES多能性幹細胞,這可改善病人之免疫系統的幹細胞純度及相容性。第二,因為該mir-302表現轉殖基因之總長度係相對地較小(約千個鹼基),與該等iPS方法中之最大值2%相較,本方法之轉殖基因傳送係極高的(成功率超過91%)。第三,該mirPS細胞之生成及培養完全在無哺乳細胞狀況下進行,避免了哺乳細胞抗原污染的風險。第四,本發明未使用致癌基因,此避免了細胞變異及腫瘤形成的風險。最後,本發明者使用電穿孔法代替反轉錄病毒感染來傳送單一mir-302表現轉殖基因,此避免了隨機反轉錄病毒插入該宿主細胞基因體的風險,其常會引起插入突變(insertional mutagenesis)。事實上,mir-302已顯示係強力之腫瘤抑制子,其甚至可將各種腫瘤/癌症細胞轉化為類ES多能性幹細胞(優先權由Lin等人之美國專利申請案第12/149,725號擁有)。總結來說,該等優點解決了該等iPS方法之三個主要問題,其避免了反轉錄病毒感染、致癌基因突變及不確定之腫瘤發生的風險。In summary, the novel mir-302 family expression system of the present invention provides a safe and powerful tool for the generation of novel ES pluripotent stem cells, particularly native cultures derived from hair follicle cells of the body, as hair is readily available. Since the intron miRNA pathway is tightly regulated by multiple intracellular monitoring systems (eg, mRNA transcription, RNA splicing, exosome processing, and NMD), this pathway is more efficient and safer than the well-known siRNA/shRNA pathway (Lin et al. People, 2008a). The invention has at least five beneficial breakthroughs. First, mir-302 expresses the transgenic gene Can replace all four large transcription factor genes used in these prior iPS methods, which are used to generate more homologous ES-like pluripotent stem cells derived from only a few patient somatic cells, which can improve the patient's Stem cell purity and compatibility of the immune system. Second, because the total length of the transgenic gene of the mir-302 is relatively small (about a thousand bases), the transgenic gene delivery system of the method is compared with the maximum of 2% of the iPS methods. Extremely high (with a success rate of over 91%). Third, the production and culture of the mirPS cells were carried out completely without lactating cells, avoiding the risk of antigen contamination by mammalian cells. Fourth, the present invention does not use oncogenes, which avoids the risk of cell variability and tumor formation. Finally, the inventors used electroporation instead of retroviral infection to deliver a single mir-302-expressing transgene, which avoids the risk of random retrovirus insertion into the host cell genome, which often causes insertional mutagenesis. . In fact, mir-302 has been shown to be a potent tumor suppressor, which can even convert various tumor/cancer cells into ES-like pluripotent stem cells (priority is owned by US Patent Application Serial No. 12/149,725, to Lin et al. ). In summary, these advantages address the three main problems of these iPS methods, which avoid the risks of retroviral infection, oncogene mutations, and uncertain tumors.

本發明除了生成類ES多能性細胞株的一般功能外,本發明之可能應用進一步包括以下方法:維持無哺乳細胞及無腫瘤ES細胞培養狀況、避免癌症細胞分化及轉化、分離純或同源幹細胞群、成人幹細胞株之體外選殖及純化、體外導引幹細胞分化成純身體組織,以及使用所得之類ES多能性幹細胞進行 移植及幹細胞治療的發展。本發明也可用來作為一種工具,其可研究幹細胞功能及機制,或是可提供用來針對特定用途而改變幹細胞之特性的組成物及方法。在其它具體實施例中,本發明之該等類ES多能性幹細胞可由正常及癌症體細胞以及哺乳動物(像是:人類、猴、狗、貓、大鼠及小鼠)之成年幹細胞生成。In addition to the general function of the ES-like pluripotent cell line, the present invention further includes the following methods: maintaining the culture state of non-mammalian cells and tumor-free ES cells, avoiding differentiation and transformation of cancer cells, and isolating 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 use of ES pluripotent stem cells such as those obtained The development of transplantation and stem cell therapy. The invention may also be used as a tool to study stem cell function and mechanism, or to provide compositions and methods for modifying the characteristics of stem cells for a particular use. In other embodiments, such ES pluripotent stem cells of the invention can be produced from normal and cancer somatic cells as well as adult stem cells of mammals such as humans, monkeys, dogs, cats, rats, and mice.

儘管現將參考附圖描述本發明之特定具體實施例,但應了解此等具體實施例僅作為實施例之用,並僅例示性舉出少數之可能特定具體實施例,其可代表本發明之原理的應用。熟習本技術者人士顯然應了解各種改變及修正皆屬隨附申請專利範圍中所進一步定義的本發明之精神、範疇及構想內。While the invention has been described with respect to the specific embodiments of the present invention, it will be understood that The application of the principle. It is obvious to those skilled in the art that the various changes and modifications are within the spirit, scope and concept of the invention as further defined in the appended claims.

本發明提供一種新穎核酸組成物及基因轉殖方法,其使用一可誘發重組類微型核醣核酸(miRNA)小髮夾型核醣核酸(shRNA),將哺乳動物體細胞/癌細胞之基因及行為特徵轉化成一類胚胎幹(ES)多能性狀態。換句話說,本發明提供一種方法,其將至少一個哺乳動物細胞轉化為至少一個多能性類幹細胞。該方法包含以下數個步驟:提供至少一個細胞基質,其表現複數個由mir-302所標的之細胞基因;提供至少一個重組核酸組成物,其可被傳送、轉錄及處理成在該細胞基質中與mir-302同源的至少一個基因靜 默效應子;及在由mir-302所標的之該等細胞基因受抑制的一條件下,以該重組核酸組成物處理該細胞基質。不同於原先shRNA之設計,本發明之shRNA可包含類似於天然mir-302先驅物(pre-mir-302)的一失配(mismatched)幹臂(stem-arm)。另外,本發明之shRNA亦可包含一改良之pre-mir-302幹環(stem-loop),如:5’-GCTAAGCCAG GC-3’(SEQ.ID.NO.1)及5’-GCCTGGCTTA GC-3’(SEQ.ID.NO.2),其可提供與天然pre-miRNA相同的核輸出效率,且不會干擾tRNA輸出。換句話說,該基因靜默效應子包含與SEQ.ID.NO.1序列或SEQ.ID.NO.2序列任一者同源的一序列。在未受限於任何特定理論下,此種轉化係應用於一新發現之mir-302-介導基因靜默機制,其可由能夠表現一mir-302家族基因群(mir-302s)或一mir-302-同源shRNA任一者之一重組轉殖基因的轉染來觸發。The present invention provides a novel nucleic acid composition and gene transfer method using a small micro hairpin-type ribonucleic acid (shRNA) which induces recombinant microRNAs (miRNA) to genetically and behaviorally characterize mammalian somatic cells/cancer cells. Transformed into a class of embryonic stem (ES) pluripotent states. 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 cell matrix that expresses a plurality of cellular genes designated by mir-302; providing at least one recombinant nucleic acid composition that can be delivered, transcribed, and processed into the cell matrix At least one gene static homologous to mir-302 The effector; and the cell matrix is treated with the recombinant nucleic acid composition under conditions in which the cellular genes identified by mir-302 are inhibited. Unlike the design of the original shRNA, the shRNA of the present invention may comprise a mismatched stem-arm similar to the native mir-302 precursor (pre-mir-302). In addition, the shRNA of the present invention may further comprise a modified pre-mir-302 stem-loop, such as: 5'-GCTAAGCCAG GC-3' (SEQ. ID. NO. 1) and 5'-GCCTGGCTTA GC. -3' (SEQ. ID. NO. 2), which provides the same nuclear export efficiency as native pre-miRNA without interfering with tRNA output. In other words, the gene silencing effector comprises a sequence homologous to either the SEQ.ID.NO.1 sequence or the SEQ.ID.NO.2 sequence. Without being bound by any particular theory, this transformation is applied to a newly discovered mir-302-mediated gene silencing mechanism that can express a mir-302 family of genes (mir-302s) or a mir- Transfection of one of the 302-homologous shRNAs is triggered by transfection of the recombinant transgene.

在一較佳具體實施例中,本發明之基因轉殖表現的設計係基於天然內含子miRNA生體合成之路徑(圖1A)。本發明者設計了一種新穎核酸組成物,其表現編碼一紅色色偏螢光蛋白質(RGFP)之一重組轉殖基因,即:SpRNAi-RGFP ,其包含可經由細胞內RNA剪接及處理機制來製造內含子miRNA及/或類shRNA基因靜默效應子的一人造/人工剪接勝任內含子(SpRNAi )(Lin等人,2003,2006a、2006b)。實施例1中描述用來設計及建構該SpRNAi 內含子及SpRNAi-RGFP 轉殖基因的操作程序。該SpRNAi 在該SpRNAi-RGFP 基因之先驅轉錄分子(pre-mRNA)內以 哺乳動物第二型RNA聚合酶(Pol-II)共同轉錄,並以RNA剪接/處理來切開。之後,進一步將所剪接之SpRNAi 處理成成熟之基因靜默效應子,如:天然miRNAs及人造shRNAs,以使得在標的基因上觸發特定的後轉錄基因靜默(posttranscriptional gene silencing,PTGS)效應。在此例中,本發明生成一種重組mir-302s及/或一種與該mir-302s同源之shRNA。同時,在內含子剪接後,將該SpRNAi-RGFP 基因轉錄分子之外顯子鏈接在一起以形成一成熟之RGFP mRNA,用來轉譯成有益於識別所需miRNA/shRNA表現之一紅色螢光標記蛋白質。在其它實施例中,可使用一些功能性蛋白質外顯子來代替RGFP以提供額外之基因功能,如:ES細胞標記基因Oct4Sox2NanogLIN-28SSEA3SSEA4In 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 that expresses a recombinant transgene encoding a red-colored fluorescent protein (RGFP), namely: SpRNAi-RGFP , which comprises a mechanism that can be produced via intracellular RNA splicing and processing mechanisms. An artificial/artificial splicing of an intron miRNA and/or shRNA-like gene silencing effector intron ( SRNAI ) (Lin et al., 2003, 2006a, 2006b). The procedure for designing and constructing the SpRNAi intron and the SpRNAi-RGFP transgene is described in Example 1. The SpRNAi is co-transcribed in the pre-transcriptional molecule (pre-mRNA) of the SpRNAi-RGFP gene by mammalian type 2 RNA polymerase (Pol-II), and is cleaved by RNA splicing/treatment. Thereafter, the spliced SpRNAi is further processed into mature gene silencing effectors, such as natural miRNAs and artificial shRNAs, such that a specific posttranscriptional gene silencing (PTGS) effect is triggered 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 exon of the SpRNAi-RGFP gene transcriptional molecule is linked together to form a mature RGFP mRNA, which is translated into red fluorescence which is useful for identifying the desired miRNA/shRNA expression. Mark the protein. In other embodiments, some functional protein exons may be used in place of RGFP to provide additional gene functions, such as the ES cell marker genes Oct4 , Sox2 , Nanog , LIN-28 , SSEA3, and SSEA4 .

在另一較佳具體實施例中,本發明提供一種用來將哺乳動物體細胞/癌細胞轉化為類ES多能性幹細胞之新穎方法(圖2A及圖2B),其使用一分離之藥物可誘發核酸組成物,該組成物可被傳送、轉錄及處理成在哺乳動物細胞中之類mir-302 miRNA/shRNA分子/同源物;因此在該等細胞中之mir-302所標的之發育及分化相關基因中誘發特定之基因靜默效應,該方法包含以下步驟:a)提供一細胞基質,其表現複數個由mir-302家族所標的之發育及分化相關基因,及b)提供一重組核酸組成物,其能夠轉錄編碼複數個非編碼mir-302 miRNAs/shRNAs或其同源物的一分離RNA,其可藉由細胞內機制依次被處理為成熟之mir-302 miRNAs/shRNAs或其同源物,因此能抑制該等標的基因在該細胞基質中的功能;c)在該細胞基質中之該等標的基因功能受抑制的條件下,以該重組核酸組成物處理該細胞基質。更佳地係,該藥物可誘發重組核酸組成物係一Tet-On 載體,其包含以一重組mir-302家族基因群(mir-302s;SEQ.ID.NO.29-SEQ.ID.NO.36之雜合物)或一人造重設mir-302 shRNA同源物(即:SEQ.ID.NO.27及SEQ.ID.NO.28之雜合物)任一者插入的SpRNAi-RGFP 轉殖基因。換句話說,該重組核酸組成物包含一藥物可誘發之基因表現載體。此外,該重組核酸組成物可包含一基因表現載體,其係選自質體、病毒載體、反轉位子、及其組合組成之群。另外,重組核酸組成物包含一Tet-OnTet-Off 基因表現載體。該細胞基質可用體外(in vitro )、離體(ex vivo )或體內(in vivo )任一種方式表現該mir-302 miRNA/shRNA及其標的基因。實施例2及3描述用於mir-302 miRNA/shRNA建構及基因轉殖傳送的操作程序。In another preferred embodiment, the present invention provides a novel method for transforming mammalian somatic/cancer cells into ES-like pluripotent stem cells (Fig. 2A and Fig. 2B) using an isolated drug. Inducing a nucleic acid composition that can be transmitted, transcribed, and processed into mir-302 miRNA/shRNA molecules/homologs in mammalian cells; thus the development of mir-302 as indicated in such cells and Inducing a specific gene silencing effect in a differentiation-related gene, the method comprising the steps of: a) providing a cell matrix exhibiting a plurality of developmental and differentiation-related genes identified by the mir-302 family, and b) providing a recombinant nucleic acid composition A transcribed RNA encoding a plurality of non-coding mir-302 miRNAs/shRNAs or homologs thereof, which can be sequentially processed into mature mir-302 miRNAs/shRNAs or homologs thereof by intracellular mechanisms Thus, the function of the target gene in the cell matrix can be inhibited; c) the cell matrix is treated with the recombinant nucleic acid composition under conditions in which the function of the target gene in the cell matrix is inhibited. More preferably, the drug induces a recombinant nucleic acid composition- Tet-On vector comprising a recombinant mir-302 family gene group (mir-302s; SEQ.ID.NO.29-SEQ.ID.NO. a hybrid of 36) or an artificially-reset mir-302 shRNA homolog (ie, a hybrid of SEQ.ID.NO.27 and SEQ.ID.NO.28) inserted into SpRNAi-RGFP Colonization gene. In other words, the recombinant nucleic acid composition comprises a drug-evokable gene expression vector. Furthermore, the recombinant nucleic acid composition can 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 Tet-On or Tet-Off gene expression vector. The cell matrix can express the mir-302 miRNA/shRNA and its target gene in any manner in vitro , ex vivo or in vivo . Examples 2 and 3 describe procedures for mir-302 miRNA/shRNA construction and gene transfer delivery.

本發明利用細胞內剪接體、外體及NMD系統之優點來催化內含子mir-302 miRNA/shRNA因劑自該SpRNAi-RGFP 轉殖基因的釋放。在實施例1及圖2A與圖2B中分別描述,經由在該SpRNAi 之數個snRNP辨識位上之細胞內剪接體成分的一序列DNA重組[例如:snRNPs U1、U2及U4/U6.U5 tri-snRNP所需之結合基序,其包括:五端剪接位(SEQ.ID.NO.4)、分支點基序(BrP;SEQ.ID.NO.6)、多嘧啶段(PPT;SEQ.ID.NO.7或 SEQ.ID.NO.8)、及三端剪接位(SEQ.ID.NO.5)],用來將合成snRNP辨識元素組合到一SpRNAi 內含子以及將此一人工重組之SpRNAi 合併到一分離可誘發RGFP基因以形成該SpRNAi-RGFP 轉殖基因的方法。此外,該SpRNAi 進一步包含一內含子插入位,其位在用來選殖及表現一重組mir-302 miRNA/shRNA因劑的五端剪接位及BrP基序之間。實施例2及圖3B描述一重組mir-302家族基因群(mir-302s)或一人造重設mir-302 shRNA同源物之建構。實施例3及圖3C描述將該重組mir-302s miRNA/shRNA轉染成感興趣之細胞,以及成功基因轉殖細胞的篩選。換句話說,該多能性類幹細胞係選擇性地使用mir-302微型核醣核酸或Oct3/4作為標記來分離。實施例4-12描述用來評估將哺乳動物體細胞/癌細胞轉化為類ES多能性細胞的測驗。圖4至圖11顯示該等測驗之結果。The present invention utilizes the advantages of intracellular splices, exosomes, and NMD systems to catalyze the release of the intron mir-302 miRNA/shRNA agent from the SpRNAi-RGFP transgene. A sequence of DNA recombination via intracellular splice components on several snRNP recognition sites of the SpRNAi is described in Example 1 and Figures 2A and 2B, respectively [eg: snRNPs U1, U2 and U4/U6.U5 tri -snRNP required binding motif comprising: a five-terminal splice site (SEQ. ID. NO. 4), a branch point motif (BrP; SEQ. ID. NO. 6), a polypyrimidine segment (PPT; SEQ. ID.NO.7 or SEQ.ID.NO.8), and a three-terminal splice site (SEQ.ID.NO.5)] for combining synthetic snRNP recognition elements into an SpRNAi intron and this artificial The recombinant SpRNAi is incorporated into a method of isolating the RGFP gene to form the SpRNAi-RGFP transgene. In addition, the SpRNAi further comprises an intron insertion site between the five-terminal splice site and the BrP motif used to select and represent a recombinant mir-302 miRNA/shRNA agent. Example 2 and Figure 3B depict the construction of a recombinant mir-302 family of genes (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 transgenic 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 tests used to assess the conversion of mammalian somatic/cancer cells to ES-like pluripotent cells. Figures 4 through 11 show the results of these tests.

能表現類mir-302 miRNA或shRNA之一可誘發SpRNAi-RGFP 基因轉殖表現系統的設計及建構Design and construction of a transgenic expression system that can induce the expression of SpRNAi-RGFP gene by expressing one of the mir-302 miRNAs or shRNAs

本發明使用一可誘發Tet-On/Off 內含子miRNA/shRNA表現系統,即pTet-On-tTS-miR302s (圖3A),用以在去氧羥誘發之控制下並經由細胞內內含子miRNA生體合成之機制來觸發類mir-302基因靜默效應子的基因轉殖表現(圖1A)。實施例1及2描述pTet-On-tTS-miR302s 之建構。當pTet-On-tTS-miR302s 表現載體轉染成感興趣之其細胞後,其轉錄一TRE-Pol-II-驅動重組轉殖基因,即SpRNAi-RGFP ,其包含可生成內含子基因靜默效應子之一人工剪接勝任內含子(SpRNAi )(圖3A及圖3B),如類髮夾型miRNA及shRNA。實施例1中顯示藉由序列接合數個合成DNA序列的基因工程,將SpRNAi 合併到一紅色色偏螢光蛋白質基因(RGFP )。SpRNAi 包含一先驅miRNA或shRNA介子(插入位),其可由細胞內RNA剪接及處理機制釋出,如:剪接體、外顯子及NMD系統之成分,然後觸發一內含子RNA-介導基因靜默機制。其它可用來攜帶並生成SpRNAi 的RNA轉錄分子包括:hnRNA、mRNA、rRNA、tRNA、snoRNA、snRNA、smnRNA、病毒RNA、pre-microRNA及其先驅物與衍生物。The present invention uses a Tet-On/Off intron miRNA/shRNA expression system, pTet-On-tTS-miR302s (Fig. 3A), under the control of deoxyxylation and via intracellular introns. The mechanism of miRNA biosynthesis is to trigger the gene transfer performance of the mir-302 gene silencing effector (Fig. 1A). Examples 1 and 2 describe the construction of pTet-On-tTS-miR302s . When the pTet-On-tTS-miR302s expression vector is transfected into the cell of interest, it transcribes a TRE-Pol-II-driven recombinant transgene, the SpRNAi-RGFP , which contains a silencing effect that can generate an intron gene. One of the sub-splicing competent introns ( SspRNAi ) (Fig. 3A and Fig. 3B), such as hairpin-like miRNAs and shRNAs. In Example 1, it was shown that the SpRNAi was incorporated into a red-colored fluorescent protein gene ( RGFP ) by genetic engineering in which several synthetic DNA sequences were joined by a sequence. SpRNAi contains a precursor miRNA or shRNA meson (insertion site), which can be released by intracellular RNA splicing and processing mechanisms, such as: splices, exons, and components of the NMD system, and then triggers an intron RNA-mediated gene. Silence mechanism. Other RNA transcriptional molecules that can be used to carry and generate SpRNAi include: hnRNA, mRNA, rRNA, tRNA, snoRNA, snRNA, smnRNA, viral RNA, pre-microRNA, and precursors and derivatives thereof.

如實施例1所示,合成SpRNAi 並將其合併到一不含內含子之紅色色偏螢光蛋白質基因(RGFPrGFP )中,以形成一SpRNAi-RGFP 轉殖基因,其係自紫點海葵(Heteractis crispa )之HcRed1色素蛋白(chromoproteins)變異而成。由於所插入之SpRNAi 中斷了RGFP之功能性螢光蛋白質結構,故在一成功轉染之細胞或有機體中於570-nm波長下,吾人可經由該紅色螢光放射的再出現來判定內含子遭移除及RGFP -mRNA變異(圖1B)。此重組SpRNAi -RGFP轉殖基因之建構係基於一先驅訊息RNA(pre-mRNA)中之一剪接體內含子的天然結構。SpRNAi 之主要成分包括數個snRNP辨識位及鏈結子,如:在末端中用於精確酶切之五端及三端剪接位、用於剪接辨識之一分支點基序(BrP)、用於剪接體交互作用之一多嘧啶段(PPT)、用於連接每一該等 成分之鏈結子、以及用於所需之內含子插入的一些限制位。本發明之SpRNAi 包含,由五端往三端方向按結構依序列出:一五端剪接位、與類mir-302基因靜默效應子同源之一內含子介子、一分支點基序(BrP)、一多嘧啶段(PPT)、及一三端剪接位。此外,一些轉譯終止密碼子(T codon)可位在接近SpRNAi 之該三端剪接位的鏈接子序列中。As shown in Example 1, the SpRNAi was synthesized and incorporated into a red-free fluorescent protein gene ( RGFP or rGFP ) containing no intron to form a SpRNAi-RGFP transgene, which is derived from the purple spot. The HcRed1 chromoproteins of Heteractis crispa are mutated. Since the inserted SpRNAi interrupts the functional fluorescent protein structure of RGFP, in a successfully transfected cell or organism at 570-nm wavelength, we can determine the intron via the re-emergence of the red fluorescence emission. Removed and RGFP- mRNA mutations (Fig. 1B). The construction of this recombinant SpRNAi- RGFP transgene is based on the natural structure of one of the precursor RNAs (pre-mRNA) to splicing the intron. The main components of SpRNAi include several snRNP recognition sites and strands, such as five-terminal and three-terminal splice sites for precise digestion in the ends, one branch point motif (BrP) for splicing recognition, and splicing One of the body interactions is a polypyrimidine segment (PPT), a linker for attaching each of these components, and some restriction sites for the desired intron insertion. The SpRNAi of the present invention comprises: a five-terminal to three-terminal direction according to a structure: a five-terminal splice site, an intron of a mir-302-like gene silencing effector, and a branch point motif (BrP) ), a polypyrimidine moiety (PPT), and a three-terminal splice site. In addition, some translation stop codons (T codon) can be located in a linker subsequence that is close to the three-terminal splice site of SpRNAi .

一般而言,該五端剪接位係包含5’-GTAAGAGK-3’(SEQ.ID.NO.4)或GU(A/G)AGU基序任一者或與其同源的一核苷酸序列(如:5’-GTAAGAGGAT-3’(SEQ.ID.NO.37)、5’-GTAAGAGT-3’、5’-GTAGAGT-3’及5’-GTAAGT-3’),而該三端剪接位係包含GWKSCYRCAG(SEQ.ID.NO.5)或CT(A/G)A(C/T)NG基序任一者或與其同源的一核苷酸序列(如:5’-GATATCCTGC AG-3’(SEQ.ID.NO.42)、5’-GGCTGCAG-3’及5’-CCACAG-3’)。而且,一分支點基序係位在該五端剪接位及該三端剪接位之間,其包含與5’-TACTWAY-3’(SEQ.ID.NO.6)基序同源之同源物,如:5’-TACTAAC-3’及5’-TACTTAT-3’。該分支點序列之腺核苷「A」核苷酸在幾乎所有之剪接體內含子中以細胞(2’-5’)寡腺苷合成酶及剪接體形成一部分之(2’-5’)鏈結套馬索內含子RNA。此外,一多嘧啶段係位在接近該分支點及該三端剪接位之間,其包含與5’-(TY)m(C/-)(T)nS(C/-)-3’(SEQ.ID.NO.7)或5’-(TC)nNCTAG(G/-)-3’(SEQ.ID.NO.8)基序任一者同源之一高T或C含量的寡核苷酸序列。符號 「m」及「n」表示多個重覆,其1;更佳地,m的數量等於1~3且n的數量等於7~12。符號「-」係指在該序列中可被略過之一核苷酸。也有一些鏈接子核苷酸序列係用來連接所有該等內含子成分。根據37 CFR 1.822中關於用在核苷酸及/或氨基酸序列資料之符號及格式的準則,符號W係指一腺嘌呤(A)或胸腺嘧啶(T)/尿嘧啶(U),符號K係指一鳥嘌呤(G)或胸腺嘧啶(T)/尿嘧啶(U),符號S係指一胞嘧啶(C)或鳥嘌呤(G),符號Y係指一胞嘧啶(C)或胸腺嘧啶(T)/尿嘧啶(U),符號R係指一腺嘌呤(A)或鳥嘌呤(G),及符號N係指一腺嘌呤(A)、胞嘧啶(C)、鳥嘌呤(G)或胸腺嘧啶(T)/尿嘧啶(U)。關於上列所有的剪接體辨識成分,去氧胸苷(deoxythymidine,T)核苷酸可用尿核苷(uridine,U)代替。In general, the five-terminal splice site comprises a nucleotide sequence comprising or homologous to any of the 5'-GTAAGAGK-3' (SEQ. ID. NO. 4) or GU (A/G) AGU motifs. (eg 5'-GTAAGAGGAT-3' (SEQ.ID.NO.37), 5'-GTAAGAGT-3', 5'-GTAGAGT-3' and 5'-GTAAGT-3'), and the three-terminal splicing A nucleotide sequence comprising or homologous to any of the GWKSCYRCAG (SEQ.ID.NO.5) or CT(A/G)A(C/T)NG motifs (eg, 5'-GATATCCTGC AG -3' (SEQ. ID. NO. 42), 5'-GGCTGCAG-3' and 5'-CCACAG-3'). Moreover, a branch point motif is located between the five-terminal splice site and the three-terminal splice site, which comprises a homologous homologous to the 5'-TACTWAY-3' (SEQ.ID.NO.6) motif. Things 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'-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'( Oligonuclear high T or C content of one of the homologs of SEQ.ID.NO.7) or 5'-(TC)nNCTAG(G/-)-3' (SEQ.ID.NO.8) motif Glycosidic acid sequence. The symbols "m" and "n" indicate multiple repetitions, 1; More preferably, the number of m is equal to 1 to 3 and the number of n is equal to 7 to 12. The symbol "-" means that one nucleotide can be skipped in the sequence. There are also some linker nucleotide sequences that are used to link all of these intron components. According to the guidelines for the symbols and formats used in nucleotide and/or amino acid sequence data in 37 CFR 1.822, the symbol W refers to an adenine (A) or thymine (T) / uracil (U), symbol K Refers to a guanine (G) or thymine (T) / uracil (U), the symbol S refers to a cytosine (C) or guanine (G), and the symbol Y refers to a cytosine (C) or thymine (T) / uracil (U), symbol R refers to adenine (A) or guanine (G), and symbol N refers to adenine (A), cytosine (C), guanine (G) Or thymine (T) / uracil (U). With respect to all of the splicing recognition components listed above, deoxythymidine (T) nucleotides can be replaced with uridine (U).

為測試一經剪接SpRNAi 介子之功能,可將各種基因靜默效應子建構選殖至該重組SpRNAi-RGFP 轉殖基因之內含子插入位中。該內含子插入位包含多個限制及選殖位,其可藉由限制酶來辨識,該等限制酶係選自AatIIAccIAflII/IIIAgeIApaI/LIAseIAsp718IBamHIBbeIBclI/IIBglIIBsmIBsp120IBspHI/LU11I/120IBsrI/BI/GIBssHII/SIBstBI/U1/XIClaICsp6IDpnIDraI/IIEagIEcl136IIEcoRI/RII/47IIIEheIFspIHaeIIIHhaIHinPIHindIIIHinfIHpaI/IIKasIKpnIMaeII/IIIMfeIMluIMscIMseINaeINarINcoINdeINgoMINotINruINsiIPmlIPpu10IPstIPvuI/IIRsaISacI/IISalISau3AISmaISnaBISphISspIStuITaiITaqIXbaIXhoIXmaI 核酸內切酶及其組合組成之群。該等內含子介子係DNA模板(DNA template),其可轉錄成高度二級結構,該等結構係選自:套馬索型RNA(lariat-form RNA)、短臨時RNA(stRNA)、反義RNA、小干擾RNA(siRNA)、雙股RNA(dsRNA)、短髮夾型RNA(shRNA)、微型核醣核酸(miRNA)、Piwi交互作用RNA(piRNA)、核醣酵素、及同義或反義構造形態(conformation)任一種之其先驅物與衍生物、或以上兩者、及其組合組成之群。To test the function of a spliced SpRNAi meson, various gene silencing effectors can be constructed and inserted into the intron insertion site of the recombinant SpRNAi-RGFP transgene. The intron insertion site comprises a plurality of restriction and selection sites which can be identified by restriction enzymes selected from the group consisting of AatII , AccI , AflII/III , AgeI , ApaI/LI , AseI , Asp718I , BamHI. , BbeI, BclI / II, BglII , BsmI, Bsp120I, BspHI / LU11I / 120I, BsrI / BI / GI, BssHII / SI, BstBI / U1 / XI, ClaI, Csp6I, DpnI, DraI / II, EagI, Ecl136II, EcoRI / RII / 47III, EheI, FspI , HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI / II, KasI, KpnI, MaeII / III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI , NruI, NsiI, PmlI, Ppu10I , PstI, PvuI / II, RsaI, SacI / II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, and the endonuclease XmaI A group of combinations. 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- RNA, small interfering RNA (siRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), microRNA (miRNA), Piwi interaction RNA (piRNA), ribozyme, and synonymous or antisense constructs A group of any of its precursors and derivatives, or both, and combinations thereof.

為了方便基因轉殖傳送進感興趣之細胞或有機體,本發明之SpRNAi-RGFP 轉殖基因較佳地係合併在一表現勝任載體中,其係選自DNA轉殖基因、質體、反轉位子、轉位子、跳躍基因、病毒載體、及其組合組成之群。所得之此一基因轉殖表現載體較佳地係以一高效率基因傳送方法引入該細胞或有機體中,該基因傳送方法係選自化學/微脂體轉染法、電穿孔法、轉位子介導DNA重組、跳躍基因插入、病毒感染、顯微注射、基因槍穿透、及其組合組成之群。換句話說,該重組核酸組成物係以一基因傳送方法引進該哺乳動物細胞中,該基因傳送方法係選自微脂體轉染(liposomal transfection)、化學轉染(chemical transfection)、基因轉殖DNA重組(transgenic DNA recombination)、病毒感染(viral infection)、轉位子插入(transposon insertion)、跳躍基因插入(jumping gene insertion)、顯微注射(micro-injection)、電穿孔法 (electroporation)、基因槍穿透(gene-gun penetration)、及其組合組成之群。該載體可進一步包含至少一個病毒、Pol-II、或Pol-III啟動子、或其組合,用以表現該SpRNAi-RGFP 轉殖基因。更佳地係,以電穿孔法將該轉殖基因合併至一Tet-On/Off 載體中並基因轉殖地傳送入標的細胞內,如實施例3所示。此外,該載體可包含:用以增加真核細胞之轉譯效率之一Kozak一致性轉譯初始位(Kozak consensus translation initiation site)、該SpRNAi-RGFP 轉殖基因下游之多個SV40聚腺苷酸化訊息(SV40 polyadenylation signal)、用來在原核生物細胞內繁殖之一pUC複製來源(pUC origin of replication)、用來將該SpRNAi-RGFP 建構合併在該載體中之至少兩個限制位、用來在表現SV40 T抗原之哺乳動物細胞中複製的一任選SV40複製來源、以及用來在複製勝任原核生物細胞中表現一抗生素抗藥性基因的一任選SV40早熟啟動子(SV40 early promoter)。換句話說,該重組核酸組成物係選自一四環黴素反應元素、一病毒或第二型RNA聚合酶(Pol-II)啟動子、或以上兩者、一Kozak一致性轉譯起始位、聚腺苷酸化訊息、複數個限制/選殖位、及其組合組成之群。此外,該重組核酸組成物係選自一pUC複製來源、用來在複製勝任原核生物細胞中表現至少一個抗生素抗藥性基因的一SV40早熟啟動子、用來在哺乳動物細胞中複製的一任選SV40複製來源、及其組合組成之群。該抗生素抗藥性基因之表現可用來作為一選擇標記,其可用來分離具該基因轉 殖表現的成功選殖細胞。該等抗生素係選自盤尼西林G(penicillin G)、安比西林(ampicillin)、新黴素(neomycin)、巴龍黴素(paromycin)、康黴素(kanamycin)、鏈黴素(streptomycin)、紅黴素(erythromycin)、斯派克黴素(spectromycin)、霍火黴素(phophomycin)、四環黴素(tetracycline)、利福黴素(rifapicin)、兩性黴素B(amphotericin B)、健他黴素(gentamycin)、氯黴素(chloramphenicol)、頭孢黴素(cephalothin)、泰黴素(tylosin)、及其組合組成之群。In order to facilitate gene transfer into a cell or organism of interest, the SpRNAi-RGFP transgene of the present invention is preferably ligated into a competent vector selected from the group consisting of a DNA transgene, a plastid, and an inverted position. , a group of transposons, 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 selected from the group consisting of chemical/microlipid transfection, electroporation, transposition A group consisting of DNA recombination, skip gene insertion, viral infection, microinjection, gene gun penetration, and combinations thereof. In other words, the recombinant nucleic acid composition is introduced into the mammalian cell by a gene delivery method selected from the group consisting of liposomal transfection, chemical transfection, gene transfer. Transgenic DNA recombination, viral infection, transposon insertion, jumping gene insertion, micro-injection, electroporation, gene gun A group consisting of gene-gun penetration and its combination. The vector may further comprise at least one viral, Pol-II, or Pol-III promoter, or a combination thereof, to express the SpRNAi-RGFP transgene. More preferably, the transgenic gene is combined into a Tet-On/Off 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 eukaryotic cells, and a plurality of SV40 polyadenylation messages downstream of the SpRNAi-RGFP transgene ( SV40 polyadenylation signal), a pUC origin of replication for propagation in prokaryotic cells, at least two restriction positions for constructing the SpRNAi-RGFP construct in the vector, for expressing SV40 An optional SV40 replication source for replication in mammalian cells of the T antigen, 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 a tetracycline reaction element, a virus or a second type RNA polymerase (Pol-II) promoter, or both, and a Kozak consensus translation start position. , a polyadenylation message, a plurality of restriction/selection sites, and a combination thereof. Furthermore, the recombinant nucleic acid composition is selected from a source of pUC replication, an SV40 precocious promoter for expressing at least one antibiotic resistance gene in a competent prokaryotic cell, and an alternative for replication in mammalian cells. SV40 replication source, and a combination of its group. The expression of the antibiotic resistance gene can be used as a selection marker for isolating successful colonized cells with the gene's transcriptional performance. Such antibiotics are selected from the group consisting of penicillin G, ampicillin, neomycin, paromycin, kanamycin, streptomycin, and red mold. Erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamicin A group consisting of (gentamycin), chloramphenicol, cephalothin, tylosin, and combinations thereof.

已在一Tg(actin -GAL4:USA-gfp)品種斑馬魚中體內地測試使用該SpRNAi-RGFP 轉殖基因之內含子miRNA/shRNA的表現策略,以針對其綠色EGFP 基因表現來標的。如實施例6及圖1B中所示,表現一人工重組anti-EGFP pre-miRNA介子(第四列)之一SpRNAi-RGFP 質體的微脂體轉染,在EGFP 上顯示一極強之基因靜默效應(>80%基因減弱(gene knockdown)),然而在下列指出之自左列至右列的該等介子中並未觀察到其它靜默效應:(1)空白載體控制(Ctl);(2)標的HIV-p24 之pre-miRNA介子(mock);(3)不具髮夾型環狀結構之反義EGFP 介子(anti);及(5)反向之pre-miRNA序列,其與該anti-EGFP pre-miRNA完全互補(miR*)。並且,在非標的(off-target)基因上並未偵測到任何效應,如:標記RGFP及管家(house-keeping)肌動蛋白(β-actin)基因,其暗示此種內含子miRNA-介導基因靜默具有高度標的專一性(target-specific)。另外,藉由北方點墨分析法(圖1B,右方), 吾人觀察到小型內含子基因靜默效應子之生成僅源自該設計之SpRNAi-RGFP 基因轉錄分子(左列),而非源自不含內含子之RGFP 的一天然轉錄分子(中間列)或不含功能性五端剪接位之一缺陷性SpRNAi-RGFP 建構的一轉錄分子(右列),而經剪接之RGFP 外顯子係鏈接在一起以形成用來轉譯該標記紅色螢光蛋白質的成熟RNA。The expression strategy of the intron miRNA/shRNA using the SpRNAi-RGFP transgene has been tested in vivo in a Tg ( actin- GAL4: USA-gfp) variety zebrafish to target its green EGFP gene expression. As shown in Example 6 and Figure 1B, the liposome transfection of SpRNAi-RGFP plastid, which represents one of the artificially recombinant anti- EGFP pre-miRNA mesons (fourth column), showed a very strong gene on EGFP . Silence effect (>80% gene knockdown), however, no other silent effects were observed in the mesons from the left to the right listed below: (1) blank vector control (Ctl); Targeted HIV-p24 pre-miRNA meson (mock); (3) antisense EGFP meson (anti) without hairpin loop structure; and (5) inverted pre-miRNA sequence, and the anti- The EGFP pre-miRNA is fully complementary (miR*). Moreover, no effect was detected on the off-target gene, such as the labeling of RGFP and the house-keeping actin (β-actin) gene, suggesting that this intron miRNA- Mediating gene silencing has a high degree of target-specificity. In addition, by Northern blotting analysis (Fig. 1B, right), we observed that the generation of the small intron gene silencing effector was only derived from the designed SpRNAi-RGFP gene transcription molecule (left column), not the source. A natural transcriptional molecule (intermediate column) from intron-free RGFP or a transcriptional molecule constructed by defective SpRNAi-RGFP, which lacks one of the functional five-terminal splice sites (right column), and spliced RGFP The lines are linked together to form mature RNA for translating the labeled red fluorescent protein.

一重組mir-302家族基因群及一類mir-302 shRNA同源物的設計及建構Design and construction of a recombinant mir-302 family gene group and a class of mir-302 shRNA homologs

因為一些天然pre-miRNA的髮夾型環狀結構太大及/或太複雜,無法配合該SpRNAi-RGFP 轉殖基因,所以本發明者設計了一修飾後tRNAmet 環(即:5’-(A/U)UCCAAGGGGG-3’)(SEQ.ID.NO.43)來代替該等天然pre-miRNA環。該tRNAmet 環顯示能如同天然miRNAs般經由相同之Ran-GTP及Exportin-5傳送機制有效地促進將人造重設之miRNA自細胞核輸出至細胞質(Lin等人,2005)。有利地係,本發明目前使用一對人造改良之pre-mir-302環,包括:5’-GCTAAGCCAG GC-3’(SEQ.ID.NO.1)及5’-GCCTGGCTTA GC-3’(SEQ.ID.NO.2),其提供與天然pre-miRNAs相同的核輸出效率,且不會干擾tRNA輸出。並且,此改良加強了mir-302a-mir-302a*及mir-302c-mir-302c*雙鏈體之形成,其可穩定mir-302家族的整體功能。該等新穎pre-miRNA環之設計係以mir-302b/mir-302a之tRNAmet 環及短幹環的 結合來修飾,其高度地表現在胚胎幹細胞中而非在其它分化組織細胞中。因此,在mir-302家族中使用該等人造/人工pre-miRNA環將不會干擾我們身體中的天然miRNA路徑,因而細胞毒性較少且更加安全。Since some of the natural pre-miRNA hairpin-type loop structures are too large and/or too complex to fit the SpRNAi-RGFP transgene, the inventors designed a modified tRNA met loop (ie: 5'-( A/U) UCCAAGGGGG-3') (SEQ. ID. NO. 43) is substituted for these natural pre-miRNA loops. This tRNA met 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 pre-mir-302 rings, including: 5'-GCTAAGCCAG GC-3' (SEQ. ID. NO. 1) and 5'-GCCTGGCTTA GC-3' (SEQ. .ID.NO.2), which provides the same nuclear output 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* duplexes, 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 tRNA met loop and the short stem loop, which is highly expressed in embryonic stem cells but not in other differentiated tissue cells. Therefore, the use of such 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.

該等mir-302a、mir-302b、mir-302c及mir-302d之成熟序列分別為’-UAAGUGCUUC CAUGUUUUGG UGA-3’(SEQ.ID.NO.10)、5’-UAAGUGCUUC CAUGUUUUAG UAG-3’(SEQ.ID.NO.11)、5’-UAAGUGCUUC CAUGUUUCAG UGG-3’(SEQ.ID.NO.12)、及5’-UAAGUGCUUC CAUGUUUGAG UGU-3’(SEQ.ID.NO.13)。該等mir-302家族基因靜默效應子在其前十七個核苷酸中分享一高度一致性的五端區域(100%同源性),其與5’-UAAGUGCUUC CAUGUUU-3’(SEQ.ID.NO.3)完全相同。換句話說,該基因靜默效應子包含與SEQ.ID.NO.3同源或互補、或兩者皆是的一序列。在設計與該等mir-302序列同源的序列中,胸腺嘧啶(T)可用來代替尿嘧啶(U)。The mature sequences of these mir-302a, mir-302b, mir-302c and mir-302d are '-UAAGUGCUUC CAUGUUUUGG UGA-3' (SEQ.ID.NO.10), 5'-UAAGUGCUUC CAUGUUUUAG UAG-3', respectively. SEQ. ID. NO. 11), 5'-UAAGUGCUUC CAUGUUUCAG UGG-3' (SEQ. ID. NO. 12), and 5'-UAAGUGCUUC CAUGUUUGAG UGU-3' (SEQ. ID. NO. 13). These mir-302 family gene silencing effectors share a highly consistent five-terminal region (100% homology) in their first seventeen nucleotides, which is associated with 5'-UAAGUGCUUC CAUGUUU-3' (SEQ. ID.NO.3) is identical. In other words, the gene silencing effector comprises a sequence homologous or complementary to SEQ. ID. NO. 3, or both. In designing sequences homologous to these mir-302 sequences, thymine (T) can be used in place of uracil (U).

如實施例2中所述,家族mir-302 pre-miRNAs之基因群係以合成mir-302同源物之雜合及鏈結/接合來形成,其在一五端至三端方向包含四個部分:mir-302a、mir-302b、mir-302c及mir-302d pre-miRNA(圖3B)。所有該等人造重設之mir-302 miRNA/shRNA同源物在其前十七個核苷酸中具有一完全相同的五端[例如:5’-UAAGUGCUUC CAUGUUU-3’(SEQ.ID.NO.3)]。在其它實施例中,吾人可使用一人造重設之mir-302 shRNA來代替用於內含子插入的mir-302 pre-miRNA基因群。該重設之mir-302 shRNA係以合成同義mir-302家族(mir-302s-sense),5’-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA GCCAGGCACA CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3’(SEQ.ID.NO.27)及反義mir-302家族(mir-302s-antisense),5’-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3’(SEQ.ID.NO.28)之雜合來形成。此重設之mir-302 shRNA對所有天然之mir-302成員分享超過91%的同源性,並標的人類中相同的mir-302-標的基因。As described in Example 2, the gene group of the family mir-302 pre-miRNAs was formed by hybridization and linkage/joining of synthetic mir-302 homologues, which contained four in a five-terminal to three-terminal direction. Part: mir-302a, mir-302b, mir-302c and mir-302d pre-miRNA (Fig. 3B). All such artificially-reset mir-302 miRNA/shRNA homologs have an identical five-terminal in their first seventeen nucleotides [eg: 5'-UAAGUGCUUC CAUGUUU-3' (SEQ. ID. NO. 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 system synthesizes the synonymous mir-302 family (mir-302s-sense), 5'-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA GCCAGGCACA CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3' (SEQ.ID.NO.27) and The mir-302 family (mir-302s-antisense), 5'-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3' (SEQ. ID. NO. 28) was formed by hybridization. This reset mir-302 shRNA shares more than 91% homology to all native mir-302 members and targets the same mir-302-target gene in humans.

將可誘發mir-302-表現SpRNAi-RGFP 轉殖基因傳送進人類正常頭髮毛囊(hHFC)及癌症黑色素癌Colo細胞之原生培養中Transfer of mir-302-expressing SpRNAi-RGFP transgenic genes into native cultures of human normal hair follicles (hHFC) and cancer melanoma Colo cells

由於該重組SpRNAi-RGFP 轉殖基因之內含子插入位在其五端及三端分別以一PvuI 及一MluI 限制/選殖位為側翼,故該原生介子可簡單地以各種pre-miRNA/shRNA介子(例如:mir-302 pre-miRNA/shRNA)加以移除及取代,其具有配合該等PvuI 及一MluI 限制位的相配粘著端。藉由針對各種基因轉錄分子來改變該等內含子的介子,該內含子miRNA/shRNA表現系統可用作體外及體內地誘發標的基 因靜默的一有力工具。在實驗中,首先將該mir-302 pre-miRNA/shRNA插入該SpRNAi-RGFP 轉殖基因中,然後將該轉殖基因合併到該pTet-On-tTS 載體之該選殖位(即:一XhoI-ClaI 位)內,因而形成一pTet-On-tTS-miR302s 轉殖基因表現載體(圖3A)。其後,將該pTet-On-tTS-mir302 載體(10-30μg)與該等宿主細胞(200-2000)在一低滲性PH緩衝液(400μl;Eppendorf)中混合,且在400-450伏特下實行電穿孔法100微秒(μsec)以將該轉殖基因傳送進該等宿主細胞基因體內。在72小時後分離並收集基因轉殖成功之細胞,使用FACS流式細胞計數儀經anti-RGFP及anti-Oct3/4單株抗體來篩選(圖3C)。此新穎mir-302s基因轉殖方法的成功率經測量超過91%。因為該SpRNAi-RGFP 轉殖基因係以一同源區域為側翼而重組插入至不包含基因的一特定基因體位(圖4A),故該SpRNAi-RGFP 轉殖基因所編碼之mir-302 miRNA/shRNA效應子之表現全依照該pTet-On-tTS 載體之TRE-CMV 啟動子的Dox誘發活化而定。該pTet-On-tTS 載體包含一CMV驅動之tTS 抑制劑基因,以去活化該轉殖基因之TRE-CMV 啟動子。當去氧羥(Dox)存在時,tTS之功能被Dox抑制住,因此表現出該SpRNAi-RGFP 轉殖基因及其編碼mir-302家族(圖4B)。Since the intron insertion site of the recombinant SpRNAi-RGFP transgene is flanked by a PvuI and a MluI restriction/selection at its five and three ends, the native meson can simply be a variety of pre-miRNA/ The shRNA meson (eg, mir-302 pre-miRNA/shRNA) is removed and replaced with a matching adhesion end that matches the PvuI and a MluI restriction sites. By changing the mesons of these introns for various gene transcriptional molecules, the intron miRNA/shRNA expression system can be used as a powerful tool for inducing target gene silencing in vitro and in vivo. In the experiment, the mir-302 pre-miRNA/shRNA was first inserted into the SpRNAi-RGFP transgene, and then the transgene was incorporated into the selection site of the pTet-On-tTS vector (ie: an XhoI). Within the -ClaI position, a pTet-On-tTS-miR302s transgenic gene expression vector was thus formed (Fig. 3A). Thereafter, the pTet-On-tTS-mir302 vector (10-30 μg) was mixed with the host cells (200-2000) in a hypotonic pH buffer (400 μl; Eppendorf) at 400-450 volts. An electroporation method was performed for 100 microseconds (μsec) to deliver the transgene into the host cell gene. After 72 hours, the cells successfully transfected were isolated and collected, and screened by anti-RGFP and anti-Oct3/4 monoclonal antibodies using a FACS flow cytometer (Fig. 3C). The success rate of this novel mir-302s gene transfer method was measured over 91%. Since the SpRNAi-RGFP transgene is flanked by a homologous region and inserted into a specific gene locus containing no gene (Fig. 4A), the mir-302 miRNA/shRNA encoded by the SpRNAi-RGFP transgene is included. The expression of the effector is based on the Dox-induced activation of the TRE-CMV promoter of the pTet-On-tTS vector. The pTet-On-tTS vector contains a CMV-driven tTS inhibitor gene to deactivate the TRE-CMV promoter of the transgene. When deoxyl (Dox) is present, the function of tTS is inhibited by Dox, thus exhibiting the SpRNAi-RGFP transgene and its coding mir-302 family (Fig. 4B).

在一不含哺乳細胞培養條件下將人類正常及癌症體細胞轉化成一類ES狀態Converting normal human and cancer somatic cells into a class of ES states in a culture containing no mammalian cells

如實施例1-2及圖3A-3B所述,吾人已設計並建構一可誘發SpRNAi-RGFP 轉殖基因,其編碼一人造鏈結之mir-302a-mir-302b-mir-302c-mir-302d(mir-302s)pre-miRNA或一重設之類mir-302 shRNA[例如:一類髮夾型序列,包含5’-UAAGUGCUUC CAUGUUUUAG UGU-3’(SEQ.ID.NO.9)],然後測試該轉殖基因以用來靜默各種體細胞及癌細胞(如人類正常頭髮毛囊細胞(hHFC)及癌症黑色素癌Colo細胞)中之發育及分化相關標的基因。As described in Examples 1-2 and Figures 3A-3B, we have designed and constructed a miRNA-302a-mir-302b-mir-302c-mir- which can induce a SpRNAi-RGFP transgene encoding an artificial link. 302d (mir-302s) pre-miRNA or a mir-302 shRNA such as a reset [eg, a type of hairpin sequence, including 5'-UAAGUGCUUC CAUGUUUUAG UGU-3' (SEQ.ID.NO.9)], and then tested The transgene is used to silence genes involved in development and differentiation in various somatic cells and cancer cells such as human normal hair follicle cells (hHFC) and cancer melanoma Colo cells.

依循圖2A及圖2B中所示之程序,本發明者分別將該重組mir-302s pre-miRNA基因轉殖地傳送入hHFC細胞內,以及將該重設之mir-302 shRNA同源物(SEQ.ID.NO.9)基因轉殖地傳送入Colo細胞內。換句話說,該基因靜默效應子係一重組類髮夾型RNA,其包括與SEQ.ID.NO.9同源的一序列。在以Dox誘發異位mir-302表現後,所有該等mir-302轉導/誘發多能性幹(mirPS)細胞株的形態從梭形(spindle)轉形為圓形(round)(下方圖),這表示其不只可能會喪失遷移之能力,亦可能具有一類似ES細胞生長之極慢的細胞再生率(圖5A)。比較DNA含量與一細胞週期階段之流式細胞儀分析(上方圖;實施例7)進一步顯示:在mirPS細胞之有絲分裂細胞群體中有超過67%的減少,這暗示該等細胞增殖率遠較其身體/癌症來源之增殖率為 低。平均來說,該等細胞群體在一不含哺乳細胞之培養條件下每20-24小時分裂一次,其包含在37℃及5% CO2 下之DMEM/F12或RPMI 1640/B27培養液,其補充成分為10%炭吸附(charcoal-stripped)胎牛血清(FBS)、4mM L-麩醯胺(glutamine)、1mM丙酮酸鈉(sodium pyruvate)、5ng/ml活化素(activin)、5ng/ml Noggin蛋白、3ng/ml bFGF、以及0.5μM Y-27632與0.5μM GSK-3抑制劑XV之一等量混合物(即:mirPS細胞培養基)。換句話說,可在DMEM/F12或RPMI 1640/B27培養液中不含哺乳細胞地培養該等多能性類幹細胞,該培養液的補充成分為10%炭吸附FBS、4mM L-麩醯胺、1mM丙酮酸鈉、5ng/ml活化素、3ng/ml bFGF、以及0.5μM Y-27632與0.5μM GSK-3抑制劑XV之一等量混合物。流式細胞計數儀圖表之第一(左)及第二(右)波峰表示在該整個測試受之細胞群體中休眠之G0/G1及有絲分裂M期之細胞群體的含量。在該mir-302s轉染後,該有絲分裂細胞群體(M期)在hHFC中自41%減少到11%且在Colo細胞中自36%減少到11%,然而在以一空SpRNAi-RGFP 載體及Dox(+Dox)或不含Dox之一mir-302表現SpRNAi-RGFP 載體(+mir-302s-Dox)轉染後,其細胞形態或細胞增殖率皆不會有明顯的改變。根據該等發現,吾人展示異位mir-302s表現能將正常及癌症人類體細胞轉化為一類ES細胞的形態及細胞分裂率。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.NO.9) The gene is transfected into Colo cells. In other words, the gene silencing effector is a recombinant hairpin RNA comprising a sequence homologous to SEQ. ID. NO. After the expression of ectopic mir-302 induced by Dox, the morphology of all these mir-302 transduced/induced pluripotent stem (mirPS) cell lines was transformed from a spindle to a round (bottom) ), which means that it is not only likely to lose the ability to migrate, but may also have a very slow rate of cell regeneration similar to ES cell growth (Fig. 5A). Comparison of DNA content and flow cytometry analysis at one cell cycle stage (top panel; Example 7) further showed that there was a 67% reduction in the mitotic cell population of mirPS cells, suggesting that these cell proliferation rates are much higher than their The proliferation rate of body/cancer sources is low. On average, the cell populations are split every 20-24 hours under culture conditions without mammalian cells, including DMEM/F12 or RPMI 1640/B27 medium at 37 ° C and 5% CO 2 . Supplementary ingredients are 10% charcoal-stripped fetal bovine serum (FBS), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/ml activin, 5 ng/ml Noggin protein, 3 ng/ml bFGF, and an equal mixture of 0.5 μM Y-27632 and 0.5 μM GSK-3 inhibitor XV (ie: mirPS cell culture medium). In other words, the pluripotent stem cells can be cultured in the DMEM/F12 or RPMI 1640/B27 culture medium without the mammalian cells, and the supplement of the culture solution is 10% carbon adsorption FBS, 4 mM L-glutamate , 1 mM sodium pyruvate, 5 ng/ml activin, 3 ng/ml bFGF, and an equal mixture of 0.5 μM Y-27632 and 0.5 μM GSK-3 inhibitor XV. The first (left) and second (right) peaks of the flow cytometer chart represent the content of the G0/G1 and mitotic M phase cell populations that are dormant in the entire population of cells subjected to the test. After transfection of this mir-302s, the mitotic cell population (M phase) decreased from 41% to 11% in hHFC and from 36% to 11% in Colo cells, whereas in an empty SpRNAi-RGFP vector and Dox (+Dox) or one of Dox-free mir-302 showed no significant change in cell morphology or cell proliferation rate after transfection with SpRNAi-RGFP vector (+mir-302s-Dox). 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.

源自各種mirPS細胞之類胚胎體的形成From the formation of embryonic bodies such as various mirPS cells

所有該等mirPS細胞皆能形成源自人類胚胎幹(ES)細胞之類胚胎體(EB)的緊密細胞叢(圖5B)。換句話說,這證明本發明所轉化之該等多能性類幹細胞可形成類胚胎體。當以胰蛋白酶EDTA(trypsin-EDTA)及膠原蛋白酶IV(collagenase IV)之一混合物分離該等類EB細胞,然後在僅具有10%FBS補充成分之RPMI 1640培養液中培養該等類EB細胞後,該等類EB細胞會分化成神經元源祖細胞(progenitor cell),其中有許多該等神經元源祖細胞表現神經元標記Tuj1及/或ABCA2。在限數稀釋法及進一步在該不含哺乳細胞之DMEM/F12培養液(補充成分為10%炭吸附FBS、4mM L-麩醯胺、1mM丙酮酸鈉、5ng/ml活化素、3ng/ml bFGF、以及0.5μM Y-27632與0.5μM GSK-3抑制劑XV之一等量混合物)中培養後,每一mirPS細胞可繼續形成用來繼代培養(sub-culturing)及/或移植/植入測驗的一純類胚胎體(圖5C)。鑑於該等類ES幹細胞特性,吾人接著在該等mirPS細胞中檢驗mir-302s及ES細胞標記的表現,並與人類ES WA01-H1及WA09-H9細胞作比較。All of these mirPS cells were able to form 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 Tuj1 and/or ABCA2. In the limiting dilution method and further in the DMEM/F12 culture medium containing no lactating cells (the supplement is 10% carbon adsorption FBS, 4 mM L-glutamate, 1 mM sodium pyruvate, 5 ng/ml activin, 3 ng/ml After culturing bFGF, and 0.5 μM Y-27632 in an equal mixture of 0.5 μM GSK-3 inhibitor XV, each mirPS cell can continue to be formed for sub-culturing and/or transplantation/planting. A pure embryonic body of the test (Fig. 5C). In view of these ES stem cell characteristics, we then examined 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.

在mirPS細胞中之mir-302s表現的微型核醣核酸(miRNA)微陣列分析MicroRNA (miRNA) microarray analysis of mir-302s in mirPS cells

為確認該等mirPS細胞中的基因轉殖mir-302表現,吾人實行實施例9中所述之微型核醣核酸(miRNA)微陣列分析。如圖6A所示,miRNA微陣列分析顯示,與原始體 細胞(控制組)相較,在Dox處理(100μM)後所有mir-302成員之表現率(最右下方,白色方框中之圓圈)在該等mirPS-hHFC細胞中明顯地增加。mir-302s在mirPS細胞中的表現程度與Dox誘發之濃度成比例對應(圖4B),如北方點墨法所判定般。在mirPS-Colo細胞中也觀察到相同的結果(Lin等人,2008b)。在早期EB階段,使用一mir VanaTM miRNA分離工具(Ambion Inc.,Austin,TX)自每一細胞株分離出多個小型RNA。該等分離之小型RNA的純度及品質係使用3.5%甲醛-洋菜膠電泳及光譜儀測量(Bio-Rad,Hercules,CA)來評估,然後立刻送交至LC Sciences(San Diego,CA)進行微陣列分析。在Cy3及Cy5強度影像(藍背景)中,當訊號強度自第1級增加至第65,535級時,對應之色彩由藍色轉變成綠色、黃色,再到紅色。在Cy5/Cy3比之影像(黑背景)中,當Cy3級高於Cy5時,色彩為綠色;當Cy3級等於Cy5級時,色彩為黃色;及當Cy5級高於Cy3級時,色彩為紅色。由於在天然mir-302家族成員及本發明之人造重設mir-302 pre-miRNA/shRNA因劑之間的該等成熟RNA序列分享極高的同源性(>91%),此結果表示該等重設mir-302因劑可取代天然mir-302s之功能。To confirm the gene transfer mir-302 expression in these mirPS cells, we performed the microRNA (miRNA) microarray analysis described in Example 9. As shown in Figure 6A, miRNA microarray analysis showed the rate of performance of all mir-302 members after Dox treatment (100 μM) compared to the original somatic cells (control group) (bottom right, circle in white box) Significantly increased in these mirPS-hHFC cells. The degree of expression of mir-302s in mirPS cells was proportional to the concentration induced by Dox (Fig. 4B), as determined by the Northern blotting method. The same results were also observed in mirPS-Colo cells (Lin et al., 2008b). EB at an early stage, the use of a separating tool mir Vana TM miRNA (Ambion Inc., Austin, TX) a plurality of small RNA was isolated from each cell line. The purity and quality of these isolated miniRNAs were assessed using 3.5% formaldehyde-cabbage electrophoresis and spectrometer measurements (Bio-Rad, Hercules, CA) and immediately sent to LC Sciences (San Diego, CA) for microscopy. Array 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 sequence of the natural mir-302 family member and the artificially-reset mir-302 pre-miRNA/shRNA agent of the present invention, this result indicates that The reset mir-302 agent can replace the function of natural mir-302s.

根據此結果,吾人也發現mir-302表現之提高可進一步地增加一些其它miRNA的表現,如:mir-92、mir-93、mir-200c、mir-367、mir-371、mir-372、mir-373、mir-374、及整個mir-520家族成員。根據對該等miRNAs之預測標的基因的分析,使用連結至Sanger miRBase::Sequences網 站(http://microrna.sanger.ac.uk/)之「TARGETSCAN」(http://www.targetscan.org/vert_42/)及「PICTAR-VERT」(http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi?)程式展示mir-302與該等miRNA分享超過400株標的基因,這表示該等miRNA在維持幹細胞多能性與再生方面亦可能扮演了重要的角色。該等一致性標的基因包括(但不限於):RAB/RAS-相關致癌基因之成員、ECT相關致癌基因、型態多樣化腺瘤基因(pleiomorphic adenoma genes)、E2F轉錄因子、細胞週期素cyclin D結合類Myb轉錄因子、HMG-box轉錄因子、Sp3轉錄因子、轉錄因子類CP2蛋白質、NFkB活化蛋白基因、細胞週期素相關激酶(CDK)、MAPK相關激酶、SNF相關激酶、肌凝蛋白輕鏈激酶(myosin light chain kinases)、TNF-α-誘發蛋白基因、DAZ相關蛋白基因、LIM相關同位序列基因、DEAD/H box蛋白基因、叉頭盒(forkhead box)蛋白基因、BMP調節子、Rho/Rac鳥嘌呤核苷酸交換因子、IGF受體、內皮素(endothelin)受體、左右決定因子、細胞週期素、p53可誘發核蛋白基因、類RB 1、RB結合蛋白基因、最大結合蛋白基因(Max-binding protein genes)、免疫識別c-MIR細胞調節子(c-MIR cellular modulator of immune recognition)、類Bcl2凋亡促進子(apoptosis facilitator)、連接蛋白(protocadherins)、整合蛋白(integrin)ß4/ß8、抑制素(inhibin)、錒克力(ankyrins)、SENP1、NUFIP2、FGF9/19、SMAD2、CXCR4、EIF2C、PCAF、MECP2、組織蛋白乙 醯轉化酶(histone acetyltransferase)MYST3、細胞核核醣核蛋白H3(nuclear RNP H3)、以及許多細胞核受體與因子。大多數之該等基因與腫瘤/癌症之胚胎發育及/或腫瘤發生高度地相關。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-371, mir-372, mir -373, mir-374, and members of the entire mir-520 family. Based on the analysis of predicted genes for these miRNAs, use the link to Sanger miRBase::Sequences Station (http://microrna.sanger.ac.uk/) "TARGETSCAN" (http://www.targetscan.org/vert_42/) and "PICTAR-VERT" (http://pictar.bio.nyu. The edu/cgi-bin/PicTar_vertebrate.cgi?) program shows that mir-302 shares more than 400 target genes with these miRNAs, suggesting 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 RAB/RAS-related oncogenes, ECT-related oncogenes, pleiomorphic adenoma genes, E2F transcription factors, and cyclin D Binding of Myb transcription factor, HMG-box transcription factor, Sp3 transcription factor, transcription factor CP2 protein, NFkB activation protein gene, cyclin-related kinase (CDK), MAPK-related kinase, SNF-related kinase, myosin light chain kinase (myosin light chain 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 binding protein gene (Max -binding protein genes), c-MIR cellular modulator of immune recognition, Bcl2 apoptosis promoter, connective egg (Protocadherins), integrins (integrin) ß4 / ß8, inhibin (inhibin), actinium grams (ankyrins), SENP1, NUFIP2, FGF9 / 19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, histone B Histone acetyltransferase MYST3, nuclear RNP H3, and many nuclear receptors and factors. Most of these genes are highly associated with embryonic development and/or tumorigenesis of tumors/cancers.

標準人類ES標記表現之識別,即:Oct3/4、SSEA-3、SSEA-4、Sox2及NanogIdentification of standard human ES marker expressions: Oct3/4, SSEA-3, SSEA-4, Sox2, and Nanog

如圖6B及9B所示,該等mirPS細胞強烈地表現許多標準人類ES細胞標記,如:Oct3/4、SSEA-3、SSEA-4、Sox2及Nanog,然而在原始體細胞(hHFC控制組)中未偵測到任何該等標記,且該等體細胞係以一空SpRNAi-RGFP 載體及去氧羥(hHFC+Dox)或是不含去氧羥之一mir-302s載體(mirPS-Dox)來轉染。如北方及西方點墨分析法所判定之mRNA及蛋白質含量,該等ES標記之表現型式極類似於人類ES WA01-H1及WA09-H9細胞之表現型式。此等結果表示mir-302s之異位表現能將成人體細胞/癌細胞轉化為類ES多能性幹細胞,其呈現許多標準人類ES標記。在mirPS-Colo細胞中也觀察到相同的結果(圖8C)。As shown in Figures 6B and 9B, these mirPS cells strongly express many standard human ES cell markers, such as: Oct3/4, SSEA-3, SSEA-4, Sox2, and Nanog, whereas in primitive somatic cells (hHFC control group). No such markers were detected, and the somatic cell lines were either an empty SpRNAi-RGFP vector and deoxyhydroxyl (hHFC+Dox) or a mir-302s vector (mirPS-Dox) without deoxyhydroxyl Transfection. The mRNA and protein content determined by the Northern and Western blotting assays are very similar to the expression patterns of human ES WA01-H1 and WA09-H9 cells. These results 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(也稱為Oct-3或Oct-4)係該等POU轉錄因子其一,其主要並高度地表現在全能之胚胎幹及生殖細胞中(Scholer等人,(1989)EMBO J. 8 :2543-2550;Rosner等人,(1990)Nature 345 :686-692)。吾人需要一臨界含量之Oct3/4表現來維持幹細胞自我再生及多能性。Oct3/4之抑低調節(down-regulation)導致胚胎幹細胞分化進入分歧發育程 序。SSEA蛋白、SSEA-1、SSEA-3及SSEA-4原先係以單株抗體來識別,該等單株抗體辦識在著床前期階段之鼠科動物胚胎及畸形癌(teratocarcinoma)幹細胞之表面上的lacto-及globo-醣脂類,但不辨識在其分化衍生物上的lacto-及globo-醣脂類(Solter等人,(1978)Proc.Natl.Acad.Sci.USA 75 :5565-5569)。未分化之靈長類胚胎幹(ES)細胞、人類胚胎癌(embryonic cancer,EC)及ES細胞全都表現SSEA-3及SSEA-4,但未表現SSEA-1(Thomson等人,(1998)Science 282 :1145-1147)。SSEA-3及SSEA-4係於卵子生成期間合成,並主要呈現在卵母細胞、接合子及早期酶切階段胚胎之該等膜中(Shevinsky等人,(1982)Cell 30 :697-705)。在維持多能性方面,Sox2具有如一核轉錄因子之功能,但此功能並非專對胚胎幹細胞(Boyer等人,(2005)Cell 122 :947-956)。因此,根據此種認知,該等mirPS細胞很可能呈現該等人類ES標記之所有特性。Oct3/4 (also known as Oct-3 or Oct-4) is one of these POU transcription factors, which are predominantly and highly expressed in pluripotent embryonic stem and germ cells (Scholer et al., (1989) EMBO J. 8 : 2543-2550; Rosner et al. (1990) Nature 345 : 686-692). We need a critical amount of Oct3/4 performance to maintain stem cell self-regeneration and pluripotency. The down-regulation of Oct3/4 causes embryonic stem cells to differentiate into divergent developmental programs. SSEA protein, SSEA-1, SSEA-3, and SSEA-4 were originally identified by monoclonal antibodies, which were found on the surface of murine embryos and teratocarcinoma stem cells in the pre-implantation stage. Lacto- and globo-glycolipids, but not recto- and globo-glycolipids on their differentiated derivatives (Solter et al., (1978) Proc. Natl. Acad. Sci. USA 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) Science 282 : 1145-1147). SSEA-3 and SSEA-4 are synthesized during oogenesis and are predominantly present in oocytes, zygotes, and early embryos of the enzyme stage (Shevinsky et al. (1982) Cell 30 : 697-705). . In terms of maintaining pluripotency, Sox2 functions as a nuclear transcription factor, but this function is not specific to embryonic stem cells (Boyer et al. (2005) Cell 122 : 947-956). Therefore, based on this recognition, these mirPS cells are likely to exhibit all of the characteristics of these human ES markers.

基因體DNA去甲基化(轉化)之評估Evaluation of demethylation (transformation) of genetic DNA

後天修飾之改變特別強調ES細胞之另一獨特的功能:基因體去甲基化(Hochedlinger等人,(2006)Nature 441 :1061-1067)。為了將一細胞轉化成其ES狀態,許多胚胎基因需要藉由DNA去甲基化來再次活化,如:Oct3/4 。為了評估在該等mirPS細胞中之此種後天效應,吾人首先以Hpall 進行整個基因體解消,Hpall 係一對CpG甲基化敏感的限制酶,並僅切開一未甲基化之CCGG位而非一甲 基化之CCGG位。圖7A顯示來自體細胞控制組之解消的DNA片段,其比來自該等mirPS細胞之解消DNA片段超過兩倍大,這表示整個mirPS細胞基因體係高度地去甲基化。使用二亞硫酸鹽-基因體PCR及核酸定序來實行Oct3/4 基因啟動子區域中的進一步評估(Takahashi及Yamanaka,2006)。二亞硫酸鹽可將所有未甲基化之胞嘧啶轉換為尿嘧啶。因為未甲基化之ACGT位會被改變成AUGT位,ACGT-切割限制酶之解消無法切開在該等mirPS細胞基因體中的分離區域(圖7B)。由該二亞硫酸鹽DNA定序來顯示之詳細去甲基化映像圖進一步展示該Oct3/4 基因啟動子區域在該等mirPS細胞中喪失超過90%之甲基化位,其類似在該等人類ES WA09-H9細胞中所發生的狀況(圖7C),這表示的確發生一泛基因體轉化事件以再活化該Oct3/4 基因表現。實施例8顯示上述之CpG去甲基化實驗。The alteration of acquired modifications places particular emphasis on another unique function of ES cells: genomic demethylation (Hochedlinger et al. (2006) Nature 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: Oct3/4 . To assess this effect in those acquired in the mirPS cells, firstly we Hpall for Canceled entire genome, a system Hpall CpG methylation-sensitive restriction enzymes, and only a cut unmethylated CCGG site instead of One methylated CCGG position. Figure 7A shows the depleted DNA fragment from the somatic cell control group, which is more than twice as large as the depleted DNA fragment from the mirPS cells, indicating that the entire mirPS cell gene system is highly demethylated. Further evaluation in the promoter region of the Oct3/4 gene was carried out using disulfite-genosome PCR and nucleic acid sequencing (Takahashi and Yamanaka, 2006). Disulfite converts all unmethylated cytosines to uracil. Since the unmethylated ACGT position was changed to the AUGT position, the release of the ACGT-cleavage restriction enzyme could not cut the isolated region in the miRNA of the mirPS cells (Fig. 7B). The detailed demethylation map shown by the disulfite DNA sequencing further demonstrates that the Oct3/4 gene promoter region loses more than 90% of the methylation sites in the mirPS cells, which is similar in these The condition that occurs in human ES WA09-H9 cells (Fig. 7C), which indicates that a pan-genome transformation event does occur to reactivate the Oct3/4 gene expression. Example 8 shows the above CpG demethylation experiment.

在判定Oct3/4 基因啟動子中之該等去甲基化位的實驗中,吾人首先以二亞硫酸鹽(CpGenome DNA修飾工具,Chemicon,CA)處理該等分離之基因體DNA,其將所有未甲基化之胞嘧啶轉換成尿嘧啶,然後使用聚合酶鏈反應來分離Oct3/4 5’ -上游啟動子區域(長模板PCR延伸工具,Roche,IN)。其後,以多個ACGT切割限制酶之一相等混合物(每一限制酶5U)來收集並解消PCR產物,該等ACGT切割限制酶包含:AclI (AACGTT)、BmgBI (CACGTC)、PmlI (CACGTG)、SnaBI (TACGTA)及HpyCH4IV (ACGT)。因為在此區域中之該等未甲基化之ACGT位係以二亞硫酸鹽改 變成AUGT位,其無法用該等ACGT切割限制酶來切開,故圖7B之結果指出在該控制組hHFC、PC3及Colo細胞中超過四個甲基化ACGT位被改變成在對應之mirPS細胞中的去甲基位。因此,該Oct3/4 基因啟動子之此mir-302-介導去甲基化可促成在該等mirPS細胞中之Oct3/4 基因表現的再活化。In experiments to determine such demethylation sites in the promoter of Oct3/4 gene, we first treated the isolated genomic DNA with disulfite (CpGenome DNA Modification Tool, Chemicon, CA), which will Unmethylated cytosine was converted to uracil and then polymerase chain reaction was used to isolate the Oct3/4 5' -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) comprising: AclI (AACGTT), BmgBI (CACGTC), PmlI (CACGTG) , SnaBI (TACGTA) and HpyCH4IV (ACGT). Since the unmethylated ACGT positions in this region are changed to the AUGT position with disulfite, which cannot be cleaved with the ACGT cleavage restriction enzymes, the results of Figure 7B indicate that hHFC in the control group, More than four methylated ACGT positions in PC3 and Colo cells were altered to the demethylation position in the corresponding mirPS cells. Thus, this mir-302-mediated demethylation of the Oct3/4 gene promoter contributes to reactivation of the Oct3/4 gene expression in these mirPS cells.

源自轉移性癌症之該等mirPS細胞中遷移能力之喪失Loss of migration capacity in these mirPS cells derived from metastatic cancer

人類ES細胞不會遷移。源自快速轉移性癌症細胞株之該等mirPS細胞(如:mirPS-PC3細胞)中常可觀察到細胞失去遷移能力。由於ES細胞傾向在一處休眠並在原位(in situ )形成類胚胎體,此可解釋為何轉移性人類前列腺癌PC3細胞在異位mir-302轉染後會喪失其遷移能力。在其它實施例中,mir-302可將某些細胞遷移相關基因靜默,以避免正常細胞遷移及癌細胞侵入,該等細胞遷移相關基因如:細微管相關蛋白質基因1B(MAP1B )、類肌動蛋白蛋白質(ACTL6A )、錒克力2(ANK2 )、澱粉樣蛋白ß先驅物A4(APP )、肌凝蛋白輕多肽激酶(myosin light polypeptide kinase,MYLK )。如圖7D及實施例12中所示,轉移性PC3細胞很快地隨時間而遷移,然而mirPS-PC3細胞保持靜止不動。在所有其它控制組中沒有觀察到任何形態上的改變。因此,本發明之基因轉殖mir-302s表現足以將人類癌症細胞轉形成一更類似ES之細胞形態及細胞分裂率,這暗示一種在癌症治療上極有益的利用。此結果暗示用來將該等mir-302傳送 進癌症/腫瘤細胞中的一潛在治療應用,其不僅可將該等惡性之癌症/腫瘤細胞轉化為有用之類ES幹細胞,也可減少癌症轉移的機會。更有利的係,由於該等mirPS細胞係由病人自身之細胞生成,且因此其和病人具有免疫相容性,故可利用該等mirPS細胞來發展一種新穎移植治療,以修補該等癌症/腫瘤受損組織而不會有免疫排斥之風險。Human ES cells do not migrate. The ability of cells to lose migration is often observed in such mirPS cells (eg, mirPS-PC3 cells) derived from rapidly metastatic cancer cell lines. Since ES cells tend to be dormant and form embryoid bodies in situ , this may explain why metastatic human prostate cancer PC3 cells lose their ability to migrate after ectopic mir-302 transfection. In other embodiments, mir-302 may silence certain cell migration-associated genes to prevent normal cell migration and cancer cell invasion, such as microtubule-associated protein gene 1B ( MAP1B ), and muscle-like movement. Protein protein ( ACTL6A ), Acrylic 2 ( ANK2 ), amyloid ß precursor A4 ( APP ), myosin light polypeptide kinase ( MYLK ). 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 all other control groups. Thus, the gene transgenic mir-302s of the present invention behaves sufficiently to transform human cancer cells into a more ES-like cell morphology and cell division rate, suggesting a very beneficial utilization in cancer therapy. This result suggests a potential therapeutic application for the delivery of these mir-302s into cancer/tumor cells, which not only converts these malignant cancer/tumor cells into useful ES stem cells, but also reduces cancer metastasis. opportunity. More advantageously, since the mirPS cell lines are produced by the patient's own cells, and thus are immunologically compatible with the patient, the mirPS cells can be utilized to develop a novel transplantation treatment to repair the cancer/tumor Damaged tissue without the risk of immune rejection.

使用基因微陣列分析識別整體ES標記表現Identification of overall ES marker performance using gene microarray analysis

泛基因體基因圖譜可提供對與該mir-302-介導轉化事件有關之基因變化的理解。在確認標準ES細胞標記及基因轉殖mir-302s的共同表現後,吾人進一步在該異位mir-302表現之前及之後,在該等細胞中以及在該等mirPS及其它人類ES細胞(如:WA01-H1及WA09-H9)間進行人類基因體微陣列分析來篩選泛基因體基因表現型式的變化。實施例10中顯示詳細之操作程序。使用Affymetrix基因微陣列(GeneChip U133A&B及U133 plus 2.0陣列)來評估超過47,000種人類基因表現型式之改變。吾人首先使用相同之mirPS樣本來複製該等微陣列測試,並從該等測試中之一選擇兩百株最可變之基因(白點)以供進一步之比較。如圖8A(mirPS-Colo)及圖9A(mirPS-hHFC)所示,複製測試之整體變化少於一倍(最左側),這表示背景變化係極有限的。根據所有微陣列識別之基因的分散型式,吾人接著計算兩組比較轉錄體資料庫之結果間的相關係數(correlation coefficiency,CC)。再提供一CC率以顯示在該 泛基因體基因表現型式中之相似度百分比,該等泛基因體基因表現型式具有的臨限值僅有1倍的表現量差異(one-fold change)。在此種嚴謹之CC率定義下,吾人發現mirPS細胞之該等基因表現型式極類似於人類ES WA01-H1(>89%)及WA09-H9(>86%)細胞之表現型式,然而在該等mirPS細胞及其體細胞/癌細胞來源之間僅顯示一低的47%-53% CC率。在人類ES及mirPS細胞間之此種強大基因相關性暗示mir-302s可能需要改變數千種細胞基因表現,其包括將一體細胞/癌細胞轉化為一類ES mirPS細胞的過程。例如,如圖8B所示,在該等mirPS及人類ES細胞結果中持續並同時觀察到,多個ES基因表現提高以及許多個致癌、發育與mir-302標的細胞週期相關的基因停擺。參考圖9B,針對該等mirPS細胞之基因表現型式而論,也可注意到SSEA-1適度地表現在該等mirPS細胞中,而Klf4則無。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 performed before and after the ectopic mir-302 expression in the cells and in the 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 over 47,000 human gene expression patterns. 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), indicating 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 a CC rate to display in the The percent similarity in the ubiquitin gene phenotype, which has a one-fold change in the threshold of the ubiquitin gene phenotype. Under the definition of such a strict 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 mirPS cells and their somatic/cancer cell sources. This strong genetic correlation between human ES and mirPS cells suggests that mir-302s may need to alter thousands of cellular gene expression, including the process of converting a single cell/cancer cell 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 oncogenic, developmental gene arrests associated with the cell cycle of mir-302. 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.

圖8B顯示在Colo及mirPS-Colo細胞之間之一些主要分化表現基因的清單。在圖8B中,吾人注意到細胞週期檢查點基因(即:CDK2、細胞週期素cyclin D1及D2)以及DNA甲基促進子(即:MECP2及MECP1-p66)全經確認為mir-302s之強力標的。換句話說,該等多能性類幹細胞表現豐富之mir-302微型核醣核酸及Oct4,但僅表現有限之CDK2、cyclin D1、MECP1-p66及MECP2。在圖8C及圖9B中也觀察到相同的結果。已知cyclin E相關CDK2係進入S期細胞週期所必需的,且抑制CDK2會造成G1期檢 查點停滯,然而,cyclin D1在回應DNA損害時可無視於G1期的停滯。根據此原則,在mirPS細胞中CDK2及cyclin D1兩者之抑制揭露一項事實:mir-302-轉染癌症細胞之細胞週期可達到一極慢的細胞分裂率,如圖5A所示。因此,此種癌細胞-幹細胞週期轉換的結果對癌症治療可有極大的好處。此外,抑制MECP2及MECP1-p66活動的結果與圖7A-C的該等結果一致,這表示惡性癌症細胞後天轉化為良性mirPS細胞。可以想見從病人處所得之該等mirPS細胞可進一步地幫助修補腫瘤/癌症之組織損害。總結來說,所有該等發現顯示本發明之mir-302基因轉殖方法可用來將人類體細胞/癌細胞之基因圖譜轉化成一高度類ES表現型式,其類似人類ES細胞之表現型式。Figure 8B shows a list of some of the major differentiation-expressing genes between Colo and mirPS-Colo cells. In Figure 8B, we noticed that the cell cycle checkpoint genes (ie, CDK2, cyclin D1 and D2) and DNA methyl promoters (ie, MECP2 and MECP1-p66) were all confirmed to be the strength of mir-302s. Subject. In other words, these pluripotent stem cells are abundant in mir-302 microRNA and Oct4, but only have limited CDK2, cyclin D1, MECP1-p66 and MECP2. The same results were also observed in Figures 8C and 9B. It is known that the cyclin E-related CDK2 line is required for the S phase of the cell cycle, and inhibition of CDK2 will cause G1 phase examination. Checkpoint stagnation, however, cyclin D1 can ignore the G1 stagnation in response to DNA damage. According to this principle, inhibition of both CDK2 and cyclin D1 in mirPS cells reveals the fact that the cell cycle of mir-302-transfected cancer cells can reach a very slow rate of cell division, as shown in Figure 5A. Therefore, the results of such cancer cell-stem cell cycle switching can be of great benefit to cancer treatment. Furthermore, the results of inhibition of MECP2 and MECP1-p66 activity are consistent with those of Figures 7A-C, which indicates that malignant cancer cells are acquired into benign mirPS cells. It is conceivable that the mirPS cells obtained from the patient can further help repair tissue damage of the tumor/cancer. In summary, all of these findings show that the mir-302 gene transfer method of the present invention can be used to transform the genetic map of human somatic/cancer cells into a highly ES-like phenotype that resembles the phenotype of human ES cells.

MirPS細胞之多能性Pluripotency of MirPS cells

多能性定義一ES細胞最重要之特性。經由以不同因子及/或賀爾蒙之體外操縱,人類ES細胞可分化成三層胚胎生殖層(外胚層、中胚層及決定性之內胚層),其係所有成人組織之基礎。在不進行任何處理下,將該等mirPS衍生類胚胎體異種移植至雌性假性懷孕免疫功能不足之SCID-beige小鼠的子宮或腹腔中可形成類畸胎瘤之組織囊腫(圖10)。在其它組織位置中未觀察到該等囊腫。然而,不同於畸胎瘤,該等組織囊腫會對其周圍組織形成一極佳且清楚之邊界。並且,在小鼠中之該等囊腫結構的成長在移植後約2.5週時減緩。此似乎有一自我調節之機制可體 內地限制該等mirPS細胞的隨機生長。此種自我調節機制也可避免從該等mirPS細胞形成腫瘤,這提供了在臨床試驗及治療上用來設計及發育不含腫瘤之多能性幹細胞的一種手段。Pluripotency defines the most important characteristics of an ES cell. Human ES cells can differentiate into three layers of embryonic germ layer (ectoderm, mesoderm, and definitive endoderm), which are the basis of all adult tissues, via manipulation with different factors and/or hormones. Xenograft-like tissue cysts were formed in the uterus or peritoneal cavity of SCID-beige mice with insufficient immune function in female pseudopregnancy without any treatment (Fig. 10). These cysts were not observed in other tissue locations. However, unlike teratomas, these tissue cysts form an excellent and clear boundary to their surrounding tissue. Moreover, the growth of these cyst structures in mice slowed down about 2.5 weeks after transplantation. This seems to have a self-regulating mechanism The Mainland limits the random growth of these mirPS cells. This self-regulating mechanism also avoids the formation of tumors from such mirPS cells, which provides a means of designing and developing tumor-free pluripotent stem cells in clinical trials and treatments.

MirPS細胞分化之體外分子導引In vitro molecular guidance of MirPS cell differentiation

在定義上,一多能性幹細胞可分化為類似源自胚胎外胚層、中胚層及/或內胚層之該等組織細胞的各種細胞類型。例如,使用各種生長因子及/或賀爾蒙之體外處理,吾人可導引類ES mirPS細胞分化為數種身體及/或生殖細胞系組織細胞類型,包括:神經元源祖(圖5B)、類精原細胞(圖11A-E)、纖維母細胞(圖11F-J)、及軟骨細胞(圖11K-O)。已利用免疫組織化學(immunohistochemical,IHC)偵測來識別該等特別組織譜系之標記,其分別顯示神經元特異性之Tuj1與ABCA2、生殖細胞系特異性之Dazla與EE2、纖維組織母細胞特異性atlastin1與第一類原膠原蛋白(COL1A1)、及軟骨細胞特異性原彈性蛋白與第二類原膠原蛋白(COL2A1)。換句話說,本發明所生成之多能性類幹細胞可分化為一類生殖細胞系細胞、類精原細胞、正常體細胞、纖維組織母細胞、軟骨細胞、及其一組合。在該等分化之mirPS細胞中沒有觀察到腫瘤形成的跡象。事實上,根據Sanger網站之「TARGETSCAN」及「PICTAR-VERT」程式的預測,已知許多致癌基因係mir-302s之標的。此外,mir-302s可壓抑細胞週期素相關 激酶2(CDK2)、細胞週期素D1與D2以避免腫瘤細胞之快速生長(Lin等人,2008b)。該等發現表示該等mirPS細胞之不含腫瘤的多能性。可以想見,可使用各種分子處理從該等mirPS細胞中誘導出更多組織細胞類型。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 body and/or germ cell lineage tissue types, including: neuronal ancestors (Fig. 5B), classes Spermatogonia cells (Fig. 11A-E), fibroblasts (Fig. 11F-J), and chondrocytes (Fig. 11K-O). Immunohistochemical (IHC) detection has been used to identify markers of these particular tissue lineages, which show neuron-specific Tuj1 and ABCA2, germ cell line-specific Dazla and EE2, and fibrous tissue blast cell specificity. Atlastin1 and the first type of procollagen (COL1A1), and chondrocyte-specific tropoelastin and the second type of procollagen (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, many of the oncogenes mir-302s are known to be based on the predictions of the "TARGETSCAN" and "PICTAR-VERT" programs on the Sanger website. In addition, mir-302s can suppress cyclin-related Kinase 2 (CDK2), cyclins D1 and D2 avoid rapid growth of tumor cells (Lin et al., 2008b). These findings represent tumor-free pluripotency of these mirPS cells. It is envisioned that various tissue cell types can be induced from such mirPS cells using various molecular treatments.

在一不含哺乳細胞培養條件下體外地進行實驗,顯示吾人已成功地導引mirPS細胞分化成三種相對同質性之體細胞類型(圖11A-O及實施例11)。首先,藉由以一天然雄激素-二氫睪固酮(dihydrotestosterone,DHT 50ng/ml)在一不含哺乳細胞培養皿中處理該等mirPS細胞達六小時,然後將該等已處理之細胞(105 )體內移植到一六週大雌性免疫不全SCID-beige小鼠的子宮內,於一週後在該移植位上生成類精原細胞之一囊腫(圖11A-E)。其次,藉由以轉形生長因子β1(TGF-ß1 100ng/ml)來處理該等mirPS細胞達12小時,然後再依循相同之移植程序,該等mirPS細胞分化為纖維母細胞並在僅一週內開始分泌膠原蛋白(圖11F-J)。最後,藉由以骨成形蛋白4(bone morphogenetic protein 4,BMP4 100ng/ml)處理該等mirPS細胞達12小時,然後將該等細胞異體移植到一六週大之免疫功能不足SCID-beige小鼠的肝臟內,該等mirPS細胞分化為以鈣化沈澱物圍繞的軟骨細胞(圖11K-O)。該免疫功能不足之裸鼠係用來提供模擬移植治療之一體內環境。該等發現提供強而有力的證據:吾人已成功地利用本發明之mir-302基因轉殖方法來生成新的類ES多能性幹細胞株,其可在一不含哺乳細胞的條件下體外及體內地導引成多種組織細胞 類型。因此,本發明不僅能將分化後之體細胞/癌細胞轉化成一類ES狀態,同時也能在一不含哺乳細胞培養條件下維持類ES之再生與多能性。Experiments in vitro in a mammalian-free culture condition showed that we have successfully directed mirPS cells to differentiate into three relatively homogeneous somatic cell types (Figures 11A-O and Example 11). First, the mirPS cells were treated with a natural androgen-dihydrotestosterone (DHT 50 ng/ml) in a non-dairy cell culture dish for six hours, and then the treated cells were treated (10 5 The body was transplanted 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). Secondly, the mirPS cells were treated with the transforming growth factor β1 (TGF-ß1 100 ng/ml) for 12 hours, and then followed by the same transplantation procedure, the mirPS cells were differentiated into fibroblasts and only within one week. Collagen secretion began (Fig. 11F-J). Finally, the mirPS cells were treated with bone morphogenetic protein 4 (BMP4 100 ng/ml) for 12 hours, and then the cells were allografted to a six-week-old immunodeficiency SCID-beige mouse. Within the liver, the mirPS cells differentiated into chondrocytes surrounded by calcified precipitates (Fig. 11K-O). This immunocompromised nude mouse is used to provide an in vivo environment for mimicking transplant therapy. These findings provide strong evidence that we have successfully used the mir-302 gene transfer method of the present invention to generate a novel ES-like pluripotent stem cell line that can be cultured in vitro without a mammalian cell. Guided into a variety of tissue cell types in vivo. 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.

因此,利用一可誘發mir-302表現轉殖基因,本發明提供一有力的新工具及策略,其可用來生成類ES mir-302-誘發多能性幹細胞(mirPS),特別是源自人類體細胞及癌細胞之原生培養的mirPS細胞。更佳地,該等mirPS細胞係得自人類正常頭髮毛囊,因為其較容易取得。由於該內含子miRNA生體合成路徑係以多個細胞內監測系統良好地調節,包括:mRNA轉錄之成分、RNA剪接、外體處理及NMD機制,因此本發明之內含子mir-302表現可視為較原先之Pol-III(U6/H1)驅動的siRNA/shRNA表現系統更加安全。事實上,本發明者已觀察到該Pol-III驅動之表現系統傾向於過度表現mir-302,並引起細胞週期在G1期停滯及死亡。本發明在結合一藥物可誘發(Tet-On/Off )載體下可進一步提供一種可控制手段,其不僅可將哺乳動物體細胞/癌細胞轉化為類ES mirPS細胞,且可導引其形成有用的正常組織細胞。因為已發現mir-302係一強力之腫瘤抑制基因,故本發明所生成之該等mirPS細胞沒有腫瘤形成的風險。Thus, the present invention provides a powerful new tool and strategy for inducing mir-302-expressing transgenic genes, which can be used to generate ES-like mir-302-induced pluripotent stem cells (mirPS), particularly from humans. Native cultured mirPS cells of 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 miRNA biosynthesis pathway is well regulated by multiple intracellular monitoring systems, including: mRNA transcription components, RNA splicing, exosome processing, and NMD mechanisms, the intron mir-302 of the present invention behaves It can be considered safer than the original Pol-III (U6/H1) driven siRNA/shRNA expression system. In fact, the inventors have observed that the Pol-III driven expression system tends to overexpress mir-302 and cause cell cycle arrest and death in the G1 phase. The present invention further provides a controllable means for converting mammalian somatic cells/cancer cells into ES-like mirPS cells in combination with a drug-inducible ( Tet-On/Off ) vector, and can be useful for guiding them to form useful Normal tissue cells. Since mir-302 has been found to be a potent tumor suppressor gene, the mirPS cells produced by the present invention are not at risk of tumor formation.

本發明具有至少五個有益之突破。第一,一株mir-302表現轉殖基因可代替在該等先前之iPS方法中所用的所有四株大型轉錄因子基因,用以生成更具同源性的類ES多 能性幹細胞(僅源自病人之少數體細胞),此可改善病人之免疫系統的幹細胞純度及相容性。第二,因為該mir-302表現轉殖基因之總長度相對地較小(約一千個鹼基),與該等先前iPS方法中之基因轉殖傳送最大值為2%相較,本方法之基因轉殖傳送係極高的(成功率超過91%)。第三,mirPS細胞之生成及培養完全係在一不含哺乳細胞的條件下進行,此避免了哺乳細胞抗原污染的風險。第四,未使用致癌基因,此避免了細胞變異及腫瘤形成的風險。最後,本發明使用電穿孔法來代替反轉錄病毒感染以傳送該單一mir-302表現轉殖基因,此避免了隨機反轉錄病毒插入宿主細胞基因體的風險,其通常會引起插入突變(insertional mutagenesis)。總結來說,該等優點解決了該等先前iPS方法之三個主要問題,其避免了反轉錄病毒感染、致癌基因突變及不確定之腫瘤發生的風險。The invention has at least five beneficial breakthroughs. First, a mir-302-expressing gene can replace all four large transcription factor genes used in these previous iPS methods to generate more homologous ES-like genes. The ability of stem cells (only from a few somatic cells of the patient) can improve the stem cell purity and compatibility of the patient's immune system. Second, because the mir-302 exhibits a relatively small total length of the transgenic gene (about one thousand bases), compared to the maximum gene transfer delivery rate of 2% in the previous iPS methods, the method 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 mutagenesis (insert mutagenesis) ). In summary, these advantages address the three main problems of these prior iPS methods, which avoid the risks of retroviral infection, oncogene mutations, and uncertain tumorigenesis.

A.定義A. Definition

為方便理解本發明,以下定義一些術語:核苷酸(Nucleotide) :一單分子之去氧核醣核酸(DNA)或核醣核酸(RNA),其包含:一醣部份體(戊醣pentose)、一磷酸根(phosphate)及一含氮雜環鹼基(nitrogenous heterocyclic base)。該鹼基係經由醣苷碳(glycosidic carbon,該戊醣之1端碳)與該醣部份體鏈結,且該鹼基及醣的組合係一核苷(nucleoside)。包含與該戊醣之三端與五 端位置鍵結之至少一個磷酸基的一核苷係一核苷酸(nucleotide)。For ease of 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 pentose), Phosphate and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via glycosidic carbon (the terminal carbon of the pentose), and the combination of the base and the sugar is a nucleoside. A nucleoside nucleotide comprising at least one phosphate group bonded to the three-terminal and five-terminal positions of the pentose sugar.

寡核苷酸(Oligonucleotide): 包含兩個以上之去氧核醣核酸或核醣核酸的一分子,其較佳地係超過三個,而通常超過十個。其確切長度取決於許多因素,其依次係依照該寡核苷酸之最佳功能或用途而定。該寡核苷酸可用任何方式生成,包括:化學合成、DNA複製、反轉錄、或其一組合。 Oligonucleotide: A molecule comprising more than two deoxyribonucleic acids or ribonucleic acids, 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 oligonucleotide can be produced in any manner, including: chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

核酸(Nucleic Acid): 核苷酸(nucleotide)之一聚合物,其可為單股或雙股。 Nucleic Acid: A polymer of nucleotides, which may be single or double stranded.

核苷酸相似物(Nucleotide Analog): 一嘌呤(purine)或嘧啶(pyrimidine)核苷酸,在結構上A、T、G、C或U不同但足夠相似,因此可在一核酸分子中取代正常核苷酸。 Nucleotide Analog: A purine or pyrimidine nucleotide that differs in structure A, T, G, C, or U but is sufficiently similar to replace normal in a nucleic acid molecule. Nucleotide.

核酸組成物(Nucleic Acid Composition): 一核酸組成物係指多核苷酸(polynucleotide),如:去氧核醣核酸(DNA)或核醣核酸(RNA),其可以單股或雙股分子結構的形式存在。 Nucleic Acid Composition: A nucleic acid composition refers to a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may exist in the form of a single or double stranded molecular structure. .

基因(Gene): 一核酸,其核苷酸序列係針對一RNA及/或一多肽類(蛋白質)編碼。一基因可以是RNA或DNA。 Gene: A nucleic acid whose nucleotide sequence is encoded for an RNA and/or a polypeptide (protein). A gene can be RNA or DNA.

鹼基對(Base Pair,bp): 於一雙股DNA分子中之胸腺嘧啶(thymine,T)與腺嘌呤(adenine,A),或鳥嘌呤(guanine,G)與胞嘧啶(cytosine,C)之聯合(partnership)。在 RNA中,尿嘧啶(uracil,U)取代胸腺嘧啶(thymine,T)。一般來說,該聯合係透過氫鍵(hydrogen bonding)來連接。 Base Pair (bp): thymine (T) and adenine (A), or guanine (G) and cytosine (C) in a double-stranded DNA molecule. The partnership. In RNA, uracil (U) replaces thymine (T). Generally, the joint is connected by hydrogen bonding.

先驅訊息核醣核酸(Precursor messenger RNA,pre-mRNA): 一基因之前驅核醣核酸轉錄分子(primary ribonucleotide transcript),其經由一細胞內機制在真核細胞中以第二型RNA聚合酶(Pol-II)結構產生,該機制稱為轉錄(transcription)。一pre-mRNA序列包含:五端非轉譯區(5’-end untranslated region)、三端非轉譯區(3’-end untranslated region)、外顯子(exon)、及內含子(intron)。 Precursor messenger RNA (pre-mRNA): a gene primo ribonucleotide transcript that uses a second type of RNA polymerase (Pol-II) in eukaryotic cells via an intracellular mechanism. Structure generation, 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.

內含子(Intron) :一基因轉錄分子序列之一部分或多個部分,其編碼非蛋白質讀框,如:同一讀框內含子(in-frame intron)、五端非轉譯區(5’-UTR)及三端非轉譯區(3’-UTR)。 Intron : A part or multiple 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).

外顯子(Exon) :一基因轉錄分子序列之一部分或許多部分,其編碼蛋白質讀框。 Exon : A portion or portion of a gene transcriptional molecule sequence that encodes a protein reading frame.

訊息核醣核酸(Messenger RNA,mRNA) :pre-mRNA外顯子之組合,其在以核內剪接結構除去內含子之後形成,並作為用於蛋白質合成的一蛋白質編碼RNA。 Message RNA (mRNA) : A combination of pre-mRNA exons that is formed after removal of introns by nuclear splicing constructs and as a protein-encoding RNA for protein synthesis.

互補去氧核醣核酸(cDNA) :與一mRNA序列互補之單股DNA,且其不含任何內含子序列。 Complementary Deoxyribonucleic Acid (cDNA) : A single strand of DNA complementary to an mRNA sequence and which does not contain any intron sequences.

同義核酸(Sense) :一核酸分子,其序列順序及組成與同源之mRNA相同。以「+」、「s」或「sense」符號來表示此同義核酸構造形態。 Synonymous nucleic acid (Sense) : A nucleic acid molecule whose sequence and composition are identical to homologous mRNA. The synonymous nucleic acid structure is represented by the "+", "s" or "sense" symbols.

反義核酸(Antisense) :與個別mRNA分子互補之一核酸分子。以「-」符號來表示此反義核酸構造形態,或是在DNA或RNA之前加上「a」或「antisense」,例如:「aDNA」或「aRNA」。 Antisense : A nucleic acid molecule that is complementary to an individual mRNA molecule. Use the "-" symbol to indicate the structure of the antisense nucleic acid, or add "a" or "antisense" before the DNA or RNA, for example: "aDNA" or "aRNA".

五端(5’-end) :在連續核苷酸之5端位置處缺少一核苷酸的一端,其中,一個核苷酸之五端氫氧基係以一磷酸二酯鏈接連接至下一個核苷酸之三端氫氧基。在該端可以有其它基,如一或多個磷酸根。 5'-end : one end lacking a nucleotide at the 5-terminal position of a contiguous nucleotide, wherein the five-terminal hydroxyl group of one nucleotide is linked to the next by a phosphodiester link. The three-terminal hydroxyl group of the nucleotide. There may be other groups at this end, such as one or more phosphates.

三端(3’-end) :連續核苷酸之3端位置處缺少一核苷酸的一端,其中,一個核苷酸之五端氫氧基係以一磷酸二酯鏈接連接至下一個核苷酸之三端氫氧基。在該端可以有其它基,通常係一氫氧根。 3'-end : A terminal lacking a nucleotide at the 3-terminal position of a contiguous nucleotide, wherein the five-terminal hydroxyl group of one nucleotide is linked to the next nucleus by a phosphodiester link. The three terminal hydroxyl group of the glucosinolate. There may be other groups at this end, usually a hydroxide.

模板(Template) :能以一核酸聚合酶拷貝之一核酸分子。根據不同的聚合酶,一模板可為單股、雙股或部分雙股。合成後之拷貝係與該模板、或一雙股或部分雙股模板中之至少一股互補。RNA與DNA皆在五端到三端的方向合成。一核酸雙鏈體之兩股總是排列在一起,使得該等兩股之五端係在該雙鏈體之相對端上(必要的話,該等兩股之三端亦然)。 Template : A nucleic acid molecule can be copied by a nucleic acid polymerase. Depending on the polymerase, a template can be single-stranded, double-stranded or partially double-stranded. The synthesized copy is complementary to the template, or at least one of a double-stranded or partially double-stranded template. Both RNA and DNA are synthesized in a five-terminal to three-terminal direction. 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 as well).

核酸模板(Nucleic Acid Template) :一雙股DNA分子、雙股RNA分子、雜合分子(如:DNA-RNA或RNA-DNA雜合物)、或單股DNA或RNA分子。 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-stranded DNA or RNA molecule.

一致性(Conserved) :若一核苷酸序列係與一預選(參考)序列之確切互補物非隨機地雜合,則兩者的序列為一致性。 Conserved : If a nucleotide sequence is non-randomly heterozygous with the exact complement of a preselected (reference) sequence, the sequences of the two are consistent.

互補(Complemetary或complementarity或complementation): 用來參照依該等鹼基對原則(base-pairing rule)關聯之多核苷酸(即:一序列之核苷酸)。例如:序列「A-G-T」與序列「T-C-A」及「T-C-U」互補。互補可以在兩股DNA之間、一股DNA與一股RNA之間、或是在兩股RNA之間。互補可以是「部分」或「完全」或是「整體」的。僅當一些核酸鹼基係根據該等鹼基對原則相配對時才會發生部分互補(partial complementarity或complementation)。完全或整體互補則是當該等鹼基在該等核酸股之間完全相配時才會發生。在核酸股之間的互補程度對於核酸股之間的雜合效率及強度有重大之影響。此對於擴增(amplification)反應特別重要,且對於根據核酸間之鍵合(binding)的偵測方法也很重要。互補率(Percent complementarity或complementation)係指在該核酸之一股中失配鹼基數與全部鹼基數的比。因此,50%的互補率意指一半的鹼基失配,而另一半的鹼基相配對。即使核酸之兩股與鹼基數不同,核酸之兩股也能互補。在此情況下,互補發生於部分之較長股間,該較長股之鹼基與較短股之鹼基成對。 Complement (Complemetary or complementarity or complementation): used to refer to a polynucleotide (ie, a sequence of nucleotides) associated with the base-pairing rule. For example, the sequence "AGT" is complementary to the sequences "TCA" and "TCU". Complementation can be between two strands of DNA, between one strand of DNA and one strand of RNA, or between two strands of RNA. Complementarity can be "partial" or "complete" or "whole". Partial complementarity or complementation occurs only when some of the nucleic acid bases are paired according to the principles of the base pairing. Complete or complete complementation occurs when the bases are perfectly matched between the nucleic acid strands. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important for amplification reactions and is also important for detection methods based on binding between nucleic acids. Percent 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. Thus, a 50% complementarity means half of the base mismatch and the other half of the bases are paired. 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, complementation occurs between a portion of the longer strands, the bases of the longer strands being paired with the bases of the shorter strands.

同源(homologous或homology) :意指一多核苷酸序列,其與一基因或mRNA序列相似。例如:一核酸序列可 能部分或完全與一特定基因或mRNA序列同源。同源也可用全部核苷酸數與相似核苷酸數的比率所定的一百分比表示。 Homologous or homology : means a polynucleotide sequence that is similar to a gene or mRNA sequence. For example, a nucleic acid sequence may be partially or completely homologous to a particular gene or mRNA sequence. Homology can also be expressed as a percentage of the ratio of all nucleotide numbers to similar nucleotide numbers.

互補鹼基(complemetary base) :當DNA或RNA形成一雙股配置時正常配對之核苷酸。 Complementary base : A nucleotide that normally pairs when DNA or RNA forms a double stranded configuration.

互補核苷酸序列(Complemetary Nucleotide Sequence) :在一單股分子之DNA或RNA中的一核苷酸序列,其足以與在另一單股上的核苷酸序列互補,以在該等兩股之間用所需之氫鍵專對地雜合。 Complemetary Nucleotide Sequence : A nucleotide sequence in a DNA or RNA of a single strand that is sufficient to complement a nucleotide sequence on another single strand to serve in the two strands Interspersed with the desired hydrogen bond.

雜合(Hybirdize及Hybridization) :在核苷酸序列之間雙鏈體的形成,其充分地互補以藉由鹼基配對形成複合體。其中一引子(或剪接模板)與標的(模板)「雜合」,因此複合體(或雜合物)係充分地穩定以提供一DNA聚合酶引發DNA合成所需的引子功能。在兩條互補多核苷酸之間有一可競爭性抑制(competitively inhibited)的特殊交互作用(即:非隨機)。 Hybirdize and Hybridization : The formation of duplexes between nucleotide sequences that are sufficiently complementary to form a complex by base pairing. One of the primers (or splicing templates) is "hybrid" with the target (template), so the complex (or hybrid) is sufficiently stable to provide the primer function required for DNA polymerase to initiate DNA synthesis. There is a special interaction (ie, non-random) between the two complementary polynucleotides that is competitively inhibited.

後轉錄基因靜默(Posttranscriptional Gene Silencing) :在mRNA降解或轉譯抑制階段下之一標的基因剔除(knockout)或減弱(knockdown)效應,其通常以外來/病毒DNA轉殖基因或小型抑制性RNA任一者來觸發。 Posttranscriptional Gene Silencing : A knockout or knockdown effect at the mRNA degradation or translational inhibition stage, usually any of the foreign/viral DNA transgenes or small inhibitory RNAs. To trigger.

核醣核酸干擾(RNA interference,RNAi) :一種在真核細胞中之後轉錄基因靜默機制,其可用小RNA分子觸發, 如:微型核醣核酸(miRNA)、小髮夾型RNA(shRNA)及小干擾核醣核酸(siRNA)。該等小RNA分子通常可作為基因靜默子,其干擾細胞內基因的表現,包含對該等小RNA的完全或部分互補。 RNA interference (RNAi) : A mechanism of transcription of a transcriptional gene in eukaryotic cells that can be triggered by small RNA molecules such as microRNAs (miRNAs), small hairpin RNAs (shRNAs), and small interfering ribose Nucleic acid (siRNA). Such small RNA molecules are typically used as gene silencers that interfere with the expression of genes within the cell, including complete or partial complementation of the small RNAs.

非編碼RNA(Non-coding RNA) :無法用來經由細胞內轉譯機制合成肽類或蛋白質的一RNA轉錄分子。 Non-coding RNA : An RNA transcription molecule that cannot be used to synthesize peptides or proteins via an intracellular translation mechanism.

微型核醣核酸(MicroRNA,miRNA) :能夠與對該miRNA部分互補之標的基因轉錄分子鍵合的一單股RNA。MiRNA通常係長約17到27個寡核苷酸,並能依照在該miRNA與其標的mRNA之間的互補程度來直接降解其細胞內之mRNA標的,或抑制其標的mRNA之蛋白質轉譯。幾乎能在所有真核細胞中發現天然的miRNAs,其如對抗病毒感染之一防衛物般作用,並允許在動植物發育期間調節基因表現。 MicroRNA (miRNA) : A single-stranded RNA that is capable of binding to a target gene transcription molecule that is partially complementary to the miRNA. MiRNAs are typically about 17 to 27 oligonucleotides in length and are capable of directly degrading the mRNA target in their cells or inhibiting the protein translation of their target mRNA, depending on the degree of complementarity between the miRNA and its target mRNA. Natural miRNAs can be found in almost all eukaryotic cells, acting as a defense against one of the viral infections, and allowing regulation of gene expression during plant and animal development.

先驅微核醣核酸(Pre-miRNA) :類髮夾型單股RNAs,其包含用來與細胞內RNaseIII內切酶交互作用的幹臂(stem-arm)及幹環(stem-loop)區域,以產生一或多個微型核醣核酸(miRNAs),其可靜默與該等微型核醣核酸序列互補之標的基因。一pre-miRNA的幹臂結構可形成一完美(100%)或部分(失配)之雜合雙鏈體,而其幹環結構係連接至該幹臂雙鏈體之一端以形成一圓形或一髮夾型環狀構造形態。Precursor microRNA (Pre-miRNA) : hairpin-type single-stranded RNAs containing stem-arm and stem-loop regions for interaction with intracellular RNaseIII endonucleases One or more microRNAs (miRNAs) are produced which silence the target gene complementary to the miniribonucleic acid sequences. A dry-arm structure of a pre-miRNA can form a perfect (100%) or partial (mismatched) hybrid duplex, and its dry loop structure is attached to one end of the dry arm duplex to form a circular shape Or a hairpin type annular structure.

小干擾核醣核酸(small interfering RNA,siRNA) :短雙股核醣核酸,其係約18到25個完美鹼基對之核醣核苷酸雙鏈體,並可降解具幾乎完美互補的標的基因轉錄分子。 Small interfering RNA (siRNA) : short double-stranded ribonucleic acid, which is a ribonucleotide duplex of about 18 to 25 perfect base pairs, and degrades the target gene transcription molecule with almost perfect complementarity. .

小髮夾型或短髮夾型RNA(small hairpin或short hairpin RNA,shRNA) :單股核醣核酸,其包含一對部分或完全相配之幹臂核苷酸序列,該序列係以一失配環狀寡核苷酸分割而形成一類髮夾型結構。許多天然miRNAs係源自類髮夾型RNA先驅物,即:先驅微核型醣核酸(pre-miRNA)。 Small hairpin or short hairpin RNA (shRNA) : a single-stranded ribonucleic acid comprising a pair of partially or fully matched stem-arm nucleotide sequences in a mismatched loop The oligonucleotides are divided to form a hairpin type structure. Many natural miRNAs are derived from hairpin-like RNA precursors, namely the precursor micronucleic acid (pre-miRNA).

載體(Vector): 能於不同基因環境中移動或滯留之一重組核酸組成物,如:重組DNA(rDNA)。一般來說,另一核酸分子係在此操作性地鏈結。該載體能於一細胞中自動複製,其中該載體及所貼附之片段也會複製。一類型之較佳載體係一游離基因組(episome),即:可染色體外複製的一核酸分子。較佳的載體係可自動複製及表現核酸的該等載體。能夠引導為一或多個多肽及/或非編碼RNA編碼之表現基因的載體在此稱為「表現載體(expression vector)」。特別重要之載體允許由使用一反轉錄酶所製造之mRNAs來選殖cDNA。 Vector: A recombinant nucleic acid composition, such as recombinant DNA (rDNA), that can move or retain in a different genetic environment. Generally, another nucleic acid molecule is operatively linked here. The vector is capable of being automatically replicated in a cell, wherein the vector and the attached fragment 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. A vector capable of directing a gene encoding one or more polypeptides and/or non-coding RNAs is referred to herein as an "expression vector." A particularly important vector allows for the selection of cDNA by mRNAs produced using a reverse transcriptase.

作用子(Cistron) :在一DNA分子中之一核苷酸序列,其編碼一胺基酸殘基序列並包括上游及下游DNA表現控制元素。 Cistron : A nucleotide sequence in a DNA molecule that encodes an amino acid residue sequence and includes upstream and downstream DNA expression control elements.

啟動子(Promoter) :一核酸,其由一聚合酶分子所辨識 (或與其鍵合)並引發合成。針對本發明之目的,一啟動子可以係一已知之聚合酶鍵合位、一增強子及類似者,以及任何序列可用一所需聚合酶引發合成。 Promoter : A nucleic acid that is recognized (or bonded) by a polymerase molecule and initiates synthesis. For the purposes of the present invention, a promoter can be a known polymerase binding site, an enhancer and the like, and any sequence can be initiated by a desired polymerase.

抗體(Antibody) :一肽類或蛋白質分子,其具有一預選之一致範圍結構,該結構編碼一可鍵合一預選配位子(ligand)的受體。 Antibody : A peptide or protein molecule having a preselected uniform range structure that encodes a receptor that binds to a preselected ligand.

B.組成物B. Composition

一重組核酸組成物,其用來表現一分離之mir-302因劑,然後在哺乳動物細胞中誘發mir-302介導基因靜默,該重組核酸組成物包含:a)一重組轉殖基因,其中該轉殖基因編碼與mir-302家族之成員同源的一重組非編碼RNA;及b)一表現勝任載體,其中該載體可用來傳送並表現哺乳動物細胞中之重組轉殖基因。A recombinant nucleic acid composition for expressing an isolated mir-302 agent and then inducing mir-302-mediated gene silencing in a mammalian cell, the recombinant nucleic acid composition comprising: a) a recombinant transgene, wherein The transgene encodes a recombinant non-coding RNA homologous to a member of the mir-302 family; and b) a competent competent vector, wherein the vector can be used to transmit and express recombinant transgenic genes in mammalian cells.

上述之重組轉殖基因進一步包含:a)複數個外顯子,其中該等外顯子可鏈結以形成具有一所需功能之一基因轉錄分子;及b)至少一個內含子,其中該內含子包含一重組mir-302同源物,並可經由細胞內RNA剪接及處理機制來切除該等外顯子。The recombinant transgenic gene described above 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 intron, wherein The intron contains a recombinant mir-302 homolog and can be excised via intracellular RNA splicing and processing mechanisms.

上述之重組轉殖基因的內含子進一步包含:a)用於剪接體鍵合之一五端受體剪接位;b)與該mir-302家族之成員同源的一基因靜默效應子介子;c)用於剪接體辨識之一分支點基序;d)用於剪接體交互作用之一多嘧啶段;e)用於剪接體鍵合之一三端受體剪接位;及f)複數個鏈接子,其用來在一五端至三端方向連接每一該等上述成分。The intron of the above recombinant gene further comprises: a) one of the five-terminal receptor splice sites for splice-binding; b) a gene silencing effector meson homologous to a member of the mir-302 family; c) one of the branch point motifs for splice identification; d) one of the polypyrimidine segments for splice ligand interaction; e) one for the splice junction, and one for the three-terminal acceptor splice sites; A linker for connecting each of the above components in a five-terminal to three-terminal direction.

較佳地,該基因靜默效應子編碼與5’-UAAGUGCUUCCAUGUUU-3’(SEQ.ID.NO.3)同源之一核酸組成物。該五端受體剪接位係包含5’-GTAAGAGK-3’(SEQ.ID.NO.4)或GU(A/G)AGU基序任一者或與其同源的一核苷酸序列(如:5’-GTAAGAGGAT-3’(SEQ.ID.NO.37)、5’-GTAAGAGT-3’、5’-GTAGAGT-3’及5’-GTAAGT-3’),而該三端受體剪接位係包含GWKSCYRCAG(SEQ.ID.NO.5)或CT(A/G)A(C/T)NG基序任一者或與其同源的一核苷酸序列(如:5’-GATATCCTGC AG-3’(SEQ.ID.NO.42)、5’-GGCTGCAG-3’及5’-CCACAG-3’)。此外,一分支點序列係位在該五端剪接位及該三端剪接位之間,其包含與5’-TACTWAY-3’(SEQ.ID.NO.6)同源之一基序,如:5’-TACTAAC-3’及5’-TACTTAT-3’。另外,一多嘧啶段係 位在接近該分支點及三端剪接位之間,其包含與5’-(TY)m(C/-)(T)nS(C/-)-3’(SEQ.ID.NO.7)或5’-(TC)nNCTAG(G/-)-3’(SEQ.ID.NO.8)任一者同源的一高T或C含量序列。符號「m」及「n」表示多個重覆,其1;更佳地,m的數量等於1~3且n的數量等於7~12。符號「-」係指在該序列中可被略過之一核苷酸。根據37 CFR 1.822中關於用於核苷酸及/或氨基酸序列資料之符號及格式的準則,符號W係指一腺嘌呤(adenine,A)或胸腺嘧啶(thymine,T)/尿嘧啶(uracil,U),符號K係指一鳥嘌呤(guanine,G)或胸腺嘧啶(T)/尿嘧啶(U),符號S係指一胞嘧啶(cytosine,C)或鳥嘌呤(G),符號Y係指一胞嘧啶(C)或胸腺嘧啶(T)/尿嘧啶(U),符號R係指一腺嘌呤(A)或鳥嘌呤(G),及符號N係指一腺嘌呤(A)、胞嘧啶(C)、鳥嘌呤(G)或胸腺嘧啶(T)/尿嘧啶(U)。關於上列所有的剪接體辨識成分,去氧胸苷(thymine/deoxythymidine,T)核苷酸可用尿核苷(uridine,U)代替。Preferably, the gene silencing effector encodes a nucleic acid composition homologous to 5'-UAAGUGCUUCCAUGUUU-3' (SEQ. ID. NO. 3). The five-terminal receptor splice site comprises a nucleotide sequence of any of 5'-GTAAGAGK-3' (SEQ.ID.NO.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 A nucleotide sequence comprising or homologous to any of the GWKSCYRCAG (SEQ.ID.NO.5) or CT(A/G)A(C/T)NG motifs (eg, 5'-GATATCCTGC AG -3' (SEQ. ID. NO. 42), 5'-GGCTGCAG-3' and 5'-CCACAG-3'). Furthermore, a branch point sequence is ligated between the five-terminal splice site and the three-terminal splice site, which comprises a motif homologous to 5'-TACTWAY-3' (SEQ. ID. NO. 6), such as : 5'-TACTAAC-3' and 5'-TACTTAT-3'. 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.NO.7) or a high T or C content sequence homologous to any of 5'-(TC)nNCTAG(G/-)-3' (SEQ.ID.NO.8). The symbols "m" and "n" indicate multiple repetitions, 1; More preferably, the number of m is equal to 1 to 3 and the number of n is equal to 7 to 12. The symbol "-" means that one nucleotide 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 adenine (A) or thymine (T) / uracil (uracil, U), the symbol K refers to a guanine (G) or thymine (T) / uracil (U), the symbol S refers to a cytosine (C) or guanine (G), symbol Y Refers to cytosine (C) or thymine (T) / uracil (U), symbol R refers to an adenine (A) or guanine (G), and symbol N refers to an adenine (A), cell Pyrimidine (C), guanine (G) or thymine (T) / uracil (U). For all of the splicing recognition components listed above, thymine/deoxythymidine (T) nucleotides can be replaced with uridine (U).

C.方法C. Method

一種用來將哺乳動物細胞轉化為多能性幹細胞之方法,其使用能夠在由mir-302所標的之基因上誘發特定基因靜默效應的一重組核酸組成物,該方法包含以下步驟:a)提供:i)一細胞基質,其表現複數個由mir-302所標的之發育及細胞分化相關基因,及ii)一重組核酸組成 物,其可被傳送、轉錄並處理成在該細胞基質中與mir-302同源的非編碼RNA;b)在該細胞基質中之mir-302-標的基因功能受抑制的條件下,以該重組核酸組成物處理該細胞基質。A method for transforming mammalian cells into pluripotent stem cells using a recombinant nucleic acid composition capable of inducing a specific gene silencing effect on a gene designated by mir-302, the method comprising the steps of: a) providing :i) a cellular matrix that exhibits a plurality of genes involved in developmental and cell differentiation as defined by mir-302, and ii) a recombinant nucleic acid a substance which can be transmitted, transcribed and processed into a non-coding RNA homologous to mir-302 in the cell matrix; b) under conditions in which the function of the mir-302-target gene in the cell matrix is inhibited The recombinant nucleic acid composition processes the cell matrix.

更佳地係,該重組核酸組成物係可表現一重組轉殖基因之一藥物可誘發Tet-OnTet-Off 載體,其編碼一重組mir-302家族基因群(pre-mir-302s;SEQ.ID.NO.29-SEQ.ID.NO.36之鏈結雜合物)或一人造重設之mir-302 shRNA同源物(SEQ.ID.NO.27及SEQ.ID.NO.28之雜合物)。該細胞基質可用體外(in vitro )、離體(ex vivo )或體內(in vivo )任一種方式來表現pre-mir-302s/mir-302 shRNA及其標的基因。More preferably, the recombinant nucleic acid composition can express a Tet-On or Tet-Off vector, which encodes a recombinant mir-302 family gene (pre-mir-302s; SEQ) .ID.NO.29-linked hybrid of SEQ.ID.NO.36) or an artificially-reset mir-302 shRNA homolog (SEQ.ID.NO.27 and SEQ.ID.NO.28) Hybrid). The cell matrix can be used to express pre-mir-302s/mir-302 shRNA and its underlying genes in either in vitro , ex vivo or in vivo .

實施例Example

以下實施例係作為舉例說明本發明之某些較佳具體實施例及態樣,其不應視為限制本發明之範疇。The following examples are intended to illustrate certain preferred embodiments and aspects of the invention and are not to be construed as limiting the scope of the invention.

在以下揭示之實驗文件中,所用的簡稱如下:M(莫耳,molar);mM(毫莫耳,millimolar);μm(微莫耳,micromolar);mol(摩耳,moles);pmol(微微莫耳,picomolar);gm(公克,grams);mg(毫克,milligrams);μg(微克,micrograms);ng(奈克,nanograms);L(公升,liters);ml(毫升,milliliters);μl(微升,microliters);℃(攝氏度, degrees Centigrade);cDNA(DNA之拷貝或互補;copy or complementary DNA);DNA(去氧核醣核酸,deoxyribonucleic acid);ssDNA(單股DNA,single stranded DNA);dsDNA(雙股DNA,double-stranded DNA);dNTP(去氧核苷酸三磷酸,deoxyribonucleotide triphosphate);RNA(核醣核酸,ribonucleic acid);PBS(磷酸鹽緩衝液,phosphate buffered saline);NaCl(氯化鈉,sodium chloride);HEPES(N-2-羥乙基哌嗪-N-2-乙烷磺酸,N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid);HBS(HEPES緩衝液,HEPES buffered saline);SDS(十二烷基硫酸鈉,sodium dodecylsulfate);Tris-HCl(三羥甲基胺基甲烷-氫氯化物,tris-hydroxymethylaminomethane-hydrochloride);及ATCC(美國菌種培養中心,American Type Culture Collection,Rockville,MD)。In the experimental documents disclosed below, the abbreviations used are as follows: M (mole, molar); mM (millollar); μm (micromolar); mol (moles, moles); pmol (pico Momo, picomolar); gm (grams, grams); mg (milligrams); μg (micrograms); ng (nograms); L (liters, liters); ml (ml, milliliters); μl (microliters, microliters); °C (degrees Celsius, Degrees Centigrade); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA, double-stranded DNA) dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); PBS (phosphate buffered saline); NaCl (sodium chloride); HEPES (N 2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; HBS (HEPES buffered saline); SDS (dodecyl sulfate) Sodium, sodium dodecylsulfate); Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride); and ATCC (American Type Culture Collection, Rockville, MD).

實施例1Example 1 包含SpRNAi 重組RGFP基因(SpRNAi-RGFP )之建構Construction of a recombinant RGFP gene ( SspRNAi-RGFP ) containing SpRNAi

以下列出用來生成包含同義、反義或髮夾型EGFP 介子之SpRNAi 內含子的合成寡核苷酸:同義N1(N1-sense),5’-GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3’(SEQ.ID.NO.14);反義N1(N1-antisense),5’-CGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGGATCC TCTTAC-3’ (SEQ.ID.NO.15);同義N2,5’-GTAAGAGGAT CCGATCGCTT GAAGAAGATG GTGCGCTCCT GA-3’(SEQ.ID.NO.16);反義N2,5’-CGCGTCAGGA GCGCACCATC TTCTTCAAGC GATCGGATCC TCTTAC-3’(SEQ.ID.NO.17);同義N3,5’-GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GTTAACTTGA AGAAGATGGT GCGCTCCTGA-3’(SEQ.ID.NO.18);反義N3,5’-CGCGTCAGGA GCGCACCATC TTCTTCAAGT TAACTTGAAG AAGATGGTGC GCTCCTGCGA TCGGATCCTC TTAC-3’(SEQ.ID.NO.19);同義N4,5’-CGCGTTACTA ACTGGTACCT CTTCTTTTTT TTTTTGATAT CCTGCAG-3’(SEQ.ID.NO.20);反義N4,5’-GTCCTGCAGG ATATCAAAAA AAAAAGAAGA GGTACCAGTT AGTAA-3’(SEQ.ID.NO.21)。從SEQ.ID.NO.14-SEQIDNO.21所列出之所有序列在其五端皆經磷酸根化。The synthetic oligonucleotides used to generate the SpRNAi introns containing the synonymous, antisense or hairpin EGFP mesons are listed below: Synonymous N1 (N1-sense), 5'-GTAAGAGGAT CCGATCGCAG GAGCGCACCA TCTTCTTCAA GA-3' (SEQ .ID.NO.14); antisense N1 (N1-antisense), 5'-CGCGTCTTGA AGAAGATGGT GCGCTCCTGC GATCGGATCC TCTTAC-3'(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 CTTCTTTTTT 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. 21 were phosphated at their five ends.

此外,兩個RGFP外顯子係藉由在一紅色螢光RGFP 基因(SEQ.ID.NO.22)之第208個核苷酸(nt)位酶切Drall 限制酶來生成。該五端RGFP 外顯子進一步地以T4 DNA聚合酶來使端點鈍化(blunt-ended)。關聯一新穎紅色色偏螢光色素蛋白質基因之RGFP 係藉由自紫點海葵(Heteractis crispa ,BD Biosciences,CA)分離之HcRed1色素蛋白質的第69個胺基酸(amino acid,a.a.)位插入一額外之天門冬胺 酸(aspartate)來生成,其造成較少之聚集以及幾乎兩倍強度之波長570-nm的紅外線螢光放射。Furthermore, two RGFP exons were generated by digesting the Drall restriction enzyme at the 208th nucleotide (nt) position of the red fluorescent RGFP gene (SEQ. ID. NO. 22). The 5-terminal RGFP exons further to T 4 DNA polymerase to make the end passivated (blunt-ended). The RGFP line associated with a novel red-chromophoric pigment protein gene was inserted into the 69th amino acid (aa) position of the HcRed1 pigment protein isolated from Heteractis crispa (BD Biosciences, CA). An additional aspartate is formed which results in less aggregation and infrared fluorescein emission at a wavelength of 570-nm of almost twice the intensity.

同義N1-sense至反義N1-antisense、同義N2-sense至反義N2-antisense、同義N3-sense至反義N3-antisense、以及同義N4-sense至反義N4-antisense之雜合分別以下列步驟實行:將每一同義及反義(1:1)序列之互補混合物加熱至94℃兩分鐘,然後置於70℃的1倍PCR緩衝液中10分鐘(例如:50mM Tris-HCl,pH 9.2,25℃;16mM(NH4 )2 SO4 ;1.75mM MgCl2 )。之後立即在1小時期間內,藉由把N1+N4、N2+N4或N3+N4(1:1)雜合之混合物從50℃分別逐漸地冷卻至10℃,以執行N1、N2或N3雜合物與N4雜合物的序列接合,然後將T4 DNA接合酶及緩衝液(Roche,IN)加進該混合物中並在12℃下一起培育12小時。此可形成該等SpRNAi 內含子。其後,將該等兩個RGFP 外顯子加進該反應中(1:1:1),並相應地調整T4 DNA接合酶及緩衝液以在12℃下再次進行該接合反應12小時。Synonymous from N1-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 Steps: Heat each complementary mixture of synonymous and antisense (1:1) sequences to 94 °C for two minutes, then place in 1x PCR buffer at 70 °C for 10 minutes (eg 50 mM Tris-HCl, pH 9.2) 25 ° C; 16 mM (NH 4 ) 2 SO 4 ; 1.75 mM MgCl 2 ). Immediately thereafter, the mixture of N1+N4, N2+N4 or N3+N4 (1:1) hybrids was gradually cooled from 50 °C to 10 °C over a period of 1 hour to perform N1, N2 or N3 N4 engagement thereof with the sequence of the hybrid, then the T 4 DNA enzymes engagement buffer (Roche, iN) added to the mixture and incubated at 12 ℃ 12 hours. This can form these SpRNAi introns. Thereafter, the outer two RGFP exons, etc. The reaction is added (1: 1: 1), and adjust the T 4 DNA enzymes engagement buffer for the re-engaged at 12 ℃ 12 hours.

關於選殖正確重組之SpRNAi 插入RGFP (SpRNAi-RGFP )基因方面,以一對RGFP 特異性引子5’-CTCGAGCATG GTGAGCGGCC TGCTGAA-3’(SEQ.ID.NO.23)及5’-TCTAGAAGTT GGCCTTCTCG GGCAGGT-3’(SEQ.ID.NO.24)藉由PCR來擴增10ng之接合序列,其係在94℃進行一分鐘,在52-57℃進行一分鐘,然後在68℃進行兩分鐘,共進行25至30個循環。將所得 之PCR產物在一2%洋菜膠上層析,然後使用一膠體萃取工具(Qiagen,CA)來萃取及純化一900到1100鹼基對(bp)之核苷酸序列。藉由定序進一步確認此~1-kb的SpRNAi-RGFP 基因之組成物。較佳地,在沒有內含子插入下,該SpRNAi 內含子序列之同義股係5’-GTAAGTGGTC CGATCGTCGC GACGCGTCAT TACTAACTAT CAATATCTTA ATCCTGTCCC TTTTTTTTCC ACAGTAGGAC CTTCGTGCA-3’(SEQ.ID.NO.25),而該SpRNAi 內含子序列之反義股係5’-TGCACGAAGG TCCTACTGTG GAAAAAAAAG GGACAGGATT AAGATATTGA TAGTTAGTAA TGACGCGTCG CGACGATCGG ACCACTTAC-3’(SEQ.ID.NO.26)。For the insertion of the correctly recombined SpRNAi into the RGFP ( SpRNAi-RGFP ) gene, a pair of RGFP- specific primers 5'-CTCGAGCATG GTGAGCGGCC TGCTGAA-3' (SEQ.ID.NO.23) and 5'-TCTAGAAGTT GGCCTTCTCG GGCAGGT- 3' (SEQ. ID. NO. 24) 10 ng of the junction sequence was amplified by PCR, which was carried out at 94 ° C for one minute, at 52-57 ° C for one minute, and then at 68 ° C for two minutes for a total of 25 to 30 cycles. The resulting PCR product was chromatographed on a 2% agar extract, and then a nucleotide sequence of 900 to 1100 base pairs (bp) was extracted and purified using a colloidal extraction tool (Qiagen, CA). The composition of the ~1-kb SpRNAi-RGFP gene was further confirmed by sequencing. Preferably, in the absence of intron insertion, the SpRNAi intron sequence has the synonym strand 5'-GTAAGTGGTC CGATCGTCGC GACGCGTCAT TACTAACTAT CAATATCTTA ATCCTGTCCC TTTTTTTTCC ACAGTAGGAC CTTCGTGCA-3' (SEQ.ID.NO.25), and the SpRNAi The antisense strand of the intron sequence 5'-TGCACGAAGG TCCTACTGTG GAAAAAAAAG GGACAGGATT AAGATATTGA TAGTTAGTAA TGACGCGTCG CGACGATCGG ACCACTTAC-3' (SEQ. ID. NO. 26).

在其它實施例中,該重組SpRNAi-RGFP 轉殖基因可藉由接合具有DraII 切開RGFP 外顯子之限制片段之SEQ.ID.NO.25與SEQ.ID.NO.26的雜合物(SpRNAi )來直接製成,並依以上所示之相同操作程序處理。依此方式形成用以測試該人造重設之mir-302 shRNA介子(編碼SEQ.ID.NO.9)的SpRNAi-RGFP 轉殖基因。In other embodiments, the recombinant SpRNAi-RGFP transgene can be hybridized with SEQ. ID. NO. 25 and SEQ. ID. NO. 26 by ligating a restriction fragment having a DraII cleavage RGFP exon ( SpRNAi ) is made directly and processed according to the same operating procedure as shown above. In this manner, a SpRNAi-RGFP transgene was generated to test the artificially-reset mir-302 shRNA meson (coding SEQ. ID. NO. 9).

由於該重組SpRNAi-RGFP 轉殖基因在其五端及三端分別具有一XhoI 及一XbaI 限制位,其可容易地被選殖至具有黏著至該等XhoIXbaI 選殖位之黏著端的一載體中。該載體必須是一表現勝任有機體或子有機體,其係選自DNA轉殖基因、質體、跳躍基因、轉位子及病毒載體 組成之群。此外,由於在該內含子內之插入位係分別在其五端及三端以一PvuI 及一MluI 限制位為側翼,吾人可移除及取代具有另一不同之插入序列的內含子介子,該不同之插入序列具有黏著至該等PvuIMlul 選殖位之黏著端。該插入序列較佳地係類髮夾型基因靜默效應子,其對一標的基因具有高互補度,該標的基因係選自螢光蛋白質(GFP )基因、螢光酵素基因、lac-Z乳糖表現基因、病毒基因、細菌基因、植物基因、動物基因及人類基因組成之群。 在該基因靜默效應子及其標的基因間的互補及/或同源比率係在約30%-100%的範圍內,更佳地係用於一髮夾型shRNA介子之範圍35%-49%,及用於同義RNA及反義RNA介子兩者之範圍90%-100%。Because the recombinant SpRNAi-RGFP transfected colonization genes each having a XhoI and an XbaI restriction site at its 5-terminal and three-terminal, which can easily be cloned into having a carrier adhered to the adhesive side of the cloning site of such XhoI and XbaI in. 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. In addition, since the insertional positions in the intron are flanked by a PvuI and a MluI restriction at their five and three ends, respectively, we can remove and replace introns with another different insertion sequence. The different insertion sequence has an adhesion end that adheres to the PvuI and Mlul 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%-49% of a hairpin type shRNA meson. And for both synonymous RNA and antisense RNA mesons ranging from 90% to 100%.

實施例2Example 2 將該等SpRNAi-RGFP 基因選殖至一表現勝任載體中以及將重組mir-302同源物插入該SpRNAi-RGFP 基因中The SpRNAi-RGFP gene was cloned into a competent vector and the recombinant mir-302 homolog was inserted into the SpRNAi-RGFP gene.

由於該重組SpRNAi-RGFP 轉殖基因在其五端及三端分別具有一XhoI 及一XbaI 限制位,其可輕易地被選殖進一載體中,該載體具備黏著至該等XhoIXbaI 限制位的相對黏著端。如圖3A所示,吾人將該SpRNAi-RGFP 轉殖基因以1:1重量比(w/w)合併至一XhoI/XbaI 線性化(linearized)~6,900-bppTet-On-tTS 的質體中,將該混合物在50分鐘之一期間內自65℃冷卻至15℃,然後相應地將T4 接合酶及緩衝液加進該混合物中以在12℃下接合12小 時。此形成了一可誘發SpRNAi-RGFP 表現載體。以RGFP 特異性引子SEQ.ID.NO.23及SEQ.ID.NO.24藉由PCR來確認該載體之組成物,其在94℃下進行一分鐘,然後在68℃下進行兩分鐘,共進行30個循環,再進一步地定序。在選殖至一病毒載體中方面,除了使用一XhoI/XbaI -線性化pLNCX2 反轉錄病毒載體(BD Clontech)來代替之外,其餘進行相同之限制及接合程序。由於該SpRNAi 內含子之插入位在其五端及三端分別以一PvuI 及一MluI 限制位為側翼,吾人可移除及取代具有一人造重設mir-302 shRNA介子之anti-EGFP shRNA介子,該人造重設mir-302 shRNA介子具有黏著至該等PvuIMlul 選殖位的黏著端。該重設mir-302 shRNA介子包含與5’-UAAGUGCUUC CAUGUUU-3’(SEQ.ID.NO.3)同源的一序列,其包括:5’-UAAGUGCUUC CAUGUUUUAG UGU-3’(SEQ.ID.NO.9)、5’-UAAGUGCUUC CAUGUUUUGG UGA-3’(SEQ.ID.NO.10)、5’-UAAGUGCUUC CAUGUUUUAG UAG-3’(SEQ.ID.NO.11)、5’-UAAGUGCUUC CAUGUUUCAG UGG-3’(SEQ.ID.NO.12)、或5’-UAAGUGCUUC CAUGUUUGAG UGU-3’(SEQ.ID.NO.13)。更佳地係,該重設mir-302 shRNA介子包含5’-UAAGUGCUUC CAUGUUUUAG UGU-3’(SEQ.ID.NO.9)。換句話說,該基因靜默效應子包括至少一個重組RNA序列,其與SEQ.ID.NO.9、SEQ.ID.NO.10、SEQ.ID.NO.11、SEQ.ID.NO.12、SEQ.ID.NO.13、及其一組 合組成之群同源。Since the recombinant SpRNAi-RGFP transgene has a XhoI and an XbaI restriction at its five- and three-terminal ends, respectively, it can be easily cloned into a vector having adhesion to the XhoI and XbaI restriction sites. Relatively sticky end. As shown in Figure 3A, we cloned the SpRNAi-RGFP transgenic gene in a 1:1 weight ratio (w/w) to a XhoI/XbaI linearized ~6,900-bp pTet-On-tTS plastid. The mixture was cooled from 65 ° C to 15 ° C in one of 50 minutes, and then T 4 ligase and buffer were added to the mixture correspondingly to be joined at 12 ° C for 12 hours. This induces formation of an SpRNAi-RGFP expression vector. The composition of the vector was confirmed by PCR using RGFP- specific primers SEQ.ID.NO.23 and SEQ.ID.NO.24, which were carried out at 94 ° C for one minute and then at 68 ° C for two minutes. Perform 30 cycles and further sequence. In terms of selection into a viral vector, the same restriction and ligation procedures were performed except that a XhoI/XbaI -linearized pLNCX2 retroviral vector (BD Clontech) was used instead. Since the insertion site of the SpRNAi intron is flanked by a PvuI and a MluI restriction at its five and three ends, we can remove and replace the anti- EGFP shRNA meson with an artificially reset mir-302 shRNA meson. the artificial reset mir-302 shRNA mesons having adhered to the adhesive side PvuI and Mlul cloning these bits. The reset mir-302 shRNA meson comprises a sequence homologous to 5'-UAAGUGCUUC CAUGUUU-3' (SEQ. ID. NO. 3), which comprises: 5'-UAAGUGCUUC CAUGUUUUAG UGU-3' (SEQ.ID. NO.9), 5'-UAAGUGCUUC CAUGUUUUGG UGA-3' (SEQ.ID.NO.10), 5'-UAAGUGCUUC CAUGUUUUAG UAG-3' (SEQ.ID.NO.11), 5'-UAAGUGCUUC CAUGUUUCAG UGG- 3' (SEQ. ID. NO. 12), or 5'-UAAGUGCUUC CAUGUUUGAG UGU-3' (SEQ. ID. NO. 13). More preferably, the reset mir-302 shRNA meson comprises 5'-UAAGUGCUUC CAUGUUUUAG UGU-3' (SEQ. ID. NO. 9). In other words, the gene silencing effector comprises at least one recombinant RNA sequence which is SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ. ID. NO. 13, and a group of its constituents are homologous.

以下列出用於該重組mir-302家族pre-miRNA或該人造重設mir-302 shRNA介子之DNA重組的合成寡核苷酸:同義mir-302a,5’-GTCCGATCGT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATCTCGAGCT C-3’(SEQ.ID.NO.29);反義mir-302a,5’-GAGCTCGAGA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GACGATCGGA C-3’(SEQ.ID.NO.30);同義mir-302b,5’-ATCTCGAGCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTAG CGCTA-3’(SEQ.ID.NO.31);反義mir-302b,5’-TAGCGCTAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGCTC GAGAT-3’(SEQ.ID.NO.32);同義mir-302c,5’-CGCTAGCGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3’(SEQ.ID.NO.33);反義mir-302c,5’-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCGCTAGCG-3’(SEQ.ID.NO.34);同義mir-302d,5’-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGACGCGTC AT-3’(SEQ.ID.NO.35);反義mir-302d,5’-ATGACGCGTC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3’(SEQ.ID.NO.36);及同義miR-302s,5’-GTCCGATCGT CATAAGTGCT TCCATGTTTT AGTGTGCTAA GCCAGGCACA CTAAAACATG GAAGCACTTA TCGACGCGTC AT-3’(SEQ.ID.NO.27);反義mir-302s,5’-ATGACGCGTC GATAAGTGCT TCCATGTTTT AGTGTGCCTG GCTTAGCACA CTAAAACATG GAAGCACTTA TGACGATCGG AC-3’(SEQ.ID.NO.28)(Sigma-Genosys,MO)。所有該等合成序列係在接合之前以PAGE膠體萃取來純化。換句話說,該基因靜默效應子係以SEQ.ID.NO.29、SEQ.ID.NO.30、SEQ.ID.NO.31、SEQ.ID.NO.32、SEQ.ID.NO.33、SEQ.ID.NO.34、SEQ.ID.NO.35、SEQ.ID.NO.36、及其一組合的雜合物之接合鏈結來形成。The synthetic oligonucleotides 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-302a, 5'-GAGCTCGAGA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GACGATCGGA C-3' (SEQ.ID.NO.30); synonymous mir- 302b,5'-ATCTCGAGCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTAG CGCTA-3' (SEQ.ID.NO.31); antisense mir-302b, 5'-TAGCGCTAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGCTC GAGAT-3' (SEQ.ID.NO.32); synonymous mir-302c, 5'-CGCTAGCGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3' (SEQ.ID.NO.33); antisense mir-302c, 5'-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCGCTAGCG-3' (SEQ.ID.NO.34); synonymous mir-302d, 5'-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGACGCGTC AT-3' (SEQ.ID.NO.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.

該重組mir-302家族pre-miRNA基因群係以四組mir-302a-d雜合物之鏈結來形成,包括:同義mir-302a與反義mir-302a、同義mir-302b與反義mir-302b、同義 mir-302c與反義mir-302c、以及同義mir-302d與反義mir-302d。該等mir-302a、mir-302b、mir-302c、及mir-302d之雜合物分別以PvuI /XhoI、XhoI /NheI、NheI /XbaI 、及XbaI /MluI 限制酶來解消,並在35μl高壓蒸氣滅菌二次水(autoclaved ddH2 O)中藉由一膠體萃取過濾柱(gel extraction filter column)(Qiagen,CA)收集在一起。之後,立即將該等混合之雜合物接合以形成具T4 DNA接合酶之mir-302家族pre-miRNA介子的一基因群(Roche,20U),並進一步地插入該等PvuI/MluI 線性化SpRNAi-RGFP 表現載體中。在其它實施例中,該人造重設mir-302 shRNA係藉由雜合SEQ.ID.NO.27及SEQ.ID.NO.28的兩個合成序列來製成,然後以PvuI/MluI 限制酶切開以插入該等PvuI/MluI 線性化SpRNAi-RGFP 表現載體中。依循圖2A及圖2B所列之程序,包含該重組mir-302家族pre-miRNA(即:pTet-On-tTS-mir302s )之可誘發SpRNAi-RGFP 表現載體係基因轉殖地被傳送進hHFC細胞內,然而包含該重設mir-302 shRNA之載體則係被引進Colo 829細胞內。The recombinant mir-302 family pre-miRNA gene group is formed by four groups of mir-302a-d hybrids, including: synonymous mir-302a and antisense mir-302a, synonymous mir-302b and antisense mir -302b, synonymous mir-302c and antisense mir-302c, and synonymous mir-302d and antisense mir-302d. The hybrids of mir-302a, mir-302b, mir-302c, and mir-302d were decomposed with PvuI / XhoI, XhoI / NheI, NheI / XbaI , and XbaI / MluI restriction enzymes, respectively, and 35 μl of high pressure vapor. The sterilized secondary water (autoclaved ddH 2 O) was collected by a gel extraction filter column (Qiagen, CA). Thereafter, the hybrids of the Mixed immediately joined to form a group of genes with T 4 DNA ligase of the mir-302 familial pre-miRNA meson (Roche, 20U), and is further inserted such PvuI / MluI linear SpRNAi-RGFP expression vector. 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, followed by PvuI/MluI restriction enzyme The plasmid was inserted into the PvuI/MluI linearized SpRNAi-RGFP expression vector. Following the procedures set forth in Figures 2A and 2B, the inducible SpRNAi-RGFP expression vector gene containing the recombinant mir-302 family pre-miRNA (i.e., pTet-On-tTS-mir302s ) was transfected into hHFC cells. However, the vector containing the mir-302 shRNA was introduced into Colo 829 cells.

該等SpRNAi-RGFP 表現載體可繁殖進包含100μg/ml之安比西林的E.coli DH5α LB培養液中(Sigma Chemical,St.Louis,MO)。該等繁殖之SpRNAi-RGFP 表現載體係使用一小量製備(mini-prep)或大量製備(maxi-prep)之質體萃取工具(Qiagen,CA)來分離及純化。在pLNCX2 反轉錄病毒載體方面,吾人也可使用一包裝細胞株GP2-293(Clontech,CA)以製造有感染力但無法自我複製 (replication-incompetent)的病毒。該等轉染之GP2-293細胞係在37℃及5% CO2 下於1倍之DMEM培養液中生長,該培養液的補充成分為10%炭吸附胎牛血清(FBS),其含有4mM L-麩醯胺、1mM丙酮酸鈉、100μg/ml硫酸鏈黴素(streptomycin sulfate)及50μg/ml新黴素(Sigma Chemical,MO)。依照一retro-X qRT-PCR滴定工具(Clontech,CA)的操作程序,病毒滴定量在轉染前經測量係超過感染複數(multiplicity of infection,MOI)30。The SpRNAi-RGFP expression vectors were propagated into E. coli DH5α LB medium (Sigma Chemical, St. Louis, MO) containing 100 μg/ml of ampicillin. The propagated SpRNAi-RGFP expression vector was isolated and purified using a mini-prep or maxi-prep plastid extraction tool (Qiagen, CA). In the case of the pLNCX2 retroviral vector, we can also use a packaging cell line GP2-293 (Clontech, CA) to create an infectious but incapable of replication-incompetent virus. The transfected GP2-293 cell line was grown in 1x DMEM medium at 37 ° C and 5% CO 2 . The supplement of the culture solution was 10% carbon adsorbed fetal bovine serum (FBS) containing 4 mM. L-bromoamide, 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate, and 50 μg/ml neomycin (Sigma Chemical, MO). According to the operating procedure of a retro-X qRT-PCR titration tool (Clontech, CA), the virus titer was measured by the multiplicity of infection (MOI) 30 before transfection.

實施例3Example 3 mir-302s之細胞培養及基因轉殖傳送Cell culture and gene transfer of mir-302s

人類癌症PC3及Colo 829細胞株係取自美國菌種培養中心(ATCC,Rockville,MD),而hHFC及hpESC細胞係分別以膠原蛋白酶/胰蛋白酶(4:1)分解2至10個頭髮毛囊根或外殖自本發明者之頭髮或手臂的2mm3 皮膚來製備。在37℃及5% CO2 下之RPMI 1640培養液中培養該等細胞,該培養液的補充成分為10%胎牛血清(FBS)、4mM L-麩醯胺、1mM丙酮酸鈉及100μg/ml健他黴素(Sigma Chemical,MO)。藉由將細胞暴露在trypsin-EDTA溶液1分鐘並以RPMI淋洗一次,以及將該等分離之細胞在1:10稀釋度下於新鮮的培養基(growth medium)中重新覆蓋(replate),來製成70%~80%滿盤的培養物。關於以電穿孔法基因轉殖mir-302s傳送方面,該pTet-On-tTS-mir302s 載體(10-30μg)與該等宿主細胞(200-2000)在一低滲性PH緩 衝液(400μl;Eppendorf)中混合,且電穿孔法係在400-450伏特下實行100微秒以將該載體傳送進該等宿主細胞基因體內。在72小時後分離並收集正向基因轉殖的mirPS細胞,使用FACS流式細胞計數儀經anti-RGFP單株抗體來篩選細胞(圖3C)。此新穎mir-302s基因轉殖方法的成功率經測量超過91%。Human cancer PC3 and Colo 829 cell lines were obtained from the American Culture Center (ATCC, Rockville, MD), while hHFC and hpESC cell lines decomposed 2 to 10 hair follicle roots with collagenase/trypsin (4:1), respectively. Or prepared from 2 mm 3 of skin of the inventor's hair or arm. The cells were cultured in RPMI 1640 medium at 37 ° C and 5% CO 2 supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamate, 1 mM sodium pyruvate and 100 μg/ M. Gentamicin (Sigma Chemical, MO). The cells were prepared by exposing the cells to trypsin-EDTA solution for 1 minute and rinsing once with RPMI, and re-reserving the isolated cells in a fresh medium (growth medium) at a 1:10 dilution. A 70% to 80% plate full of culture. Regarding the electroporation gene transfer mir-302s delivery, the p Tet-On-tTS-mir302s vector (10-30 μg) and the host cells (200-2000) in a hypotonic pH buffer (400 μl; Eppendorf) was mixed and the electroporation method was carried out at 400-450 volts for 100 microseconds to deliver the vector into the host cell genes. After 72 hours, positive gene-transferred mirPS cells were isolated and collected, and 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 over 91%.

在其它實施例中,在反轉錄病毒載體傳送方面,吾人首先培養pVSV-G 共轉染GP2-293細胞(Clontech,CA),其具有包含一重組mir-302家族pre-miRNA介子之SpRNAi-RGFP 插入pLNCX2 反轉錄病毒載體。在37℃及5% CO2 下培育36小時後,過濾該等GP2-293細胞之培養基(每一培養基10ml)(0.25μm)並於37℃及5% CO2 下將其直接轉移到該等受測試之細胞培養物內持續12小時。其後,加入新鮮之mirPS細胞培養基來代替該病毒培養基,每三天置換一次。由於該等培養基包含極高之設計反轉錄病毒載體的滴定量,幾乎所有的受測細胞(99.4%-99.8%)係由該等載體基因轉殖地感染,並在24小時內開始表現該等內含子介子及RGFP。在感染後24小時分離並收集正向基因轉殖的mirPS細胞,使用FACS流式細胞計數儀經anti-RGFP單株抗體(Clontech,CA)來分類細胞。In other embodiments, in terms of retroviral vector delivery, we first cultured pVSV-G co-transfected GP2-293 cells (Clontech, CA) with SpRNAi-RGFP comprising a recombinant mir-302 family pre-miRNA meson The pLNCX2 retroviral vector was inserted. After incubation for 36 hours at 37 ° C and 5% CO 2 , the medium of the GP2-293 cells (10 ml per medium) (0.25 μm) was filtered and transferred directly to these at 37 ° C and 5% CO 2 . The cell cultures tested were continued for 12 hours. Thereafter, fresh mirPS cell culture medium was added instead of the virus medium, and every three days. Since these media contain very high titers of the designed retroviral vector, almost all of the cells tested (99.4% - 99.8%) are transfected with these vector genes and begin to exhibit within 24 hours. Intron meson and RGFP. Positive gene-transferred mirPS cells were isolated and collected 24 hours after infection, and cells were sorted by anti-RGFP monoclonal antibody (Clontech, CA) using a FACS flow cytometer.

實施例4Example 4 北方點墨分析法Northern dot blot analysis

在1%甲醛-洋菜膠上分餾所有RNAs(20μg)並轉移至 尼龍膜(nylon membrane,Schleicher & Schuell,Keene,NH)上。合成LNA-DNA探針(Sigma-Genosys,MO)可補足位於在該等RGFP 五端外顯子之間或在所設計之pre-miRNA/shRNA介子處側面的75-bp接面序列,或補足一標的基因轉錄分子,該等合成之LNA-DNA探針係用Prime-It II工具(Stratagene,La Jolla,CA)在[32 P]-dATP出現下以隨機引子延伸來標示([32 P]-dATP>3000Ci/mM,Amersham International,Arlington Heights,IL),並以10bp-cutoff Micro Bio-Spin層析管柱(Bio-Rad,Hercules,CA)來純化。雜合係在50%的新鮮去離子甲醯胺(freshly deionized formamide,pH 7.0)、5倍Denhardt溶液、0.5% SDS、4倍SSPE及250mg/mL的變性鮭魚精子DNA片段(18小時,42℃)的混合物中進行。在自動放射照相術(autoradiography)之前將膜在2倍SSC、0.1%的SDS(15分鐘,25℃)中連續清洗兩次,並在0.2倍SSC、0.1%的SDS(45分鐘,37℃)中清洗一次。圖4B及6B中顯示該等結果。All RNAs (20 μg) were fractionated on 1% formaldehyde-cabbage and transferred to a nylon membrane (nylon membrane, Schleicher & Schuell, Keene, NH). A synthetic LNA-DNA probe (Sigma-Genosys, MO) complements the 75-bp junction sequence located between the five-terminal exons of the RGFP or at the side of the designed pre-miRNA/shRNA meson, or complements A target gene transcriptional molecule, these synthetic LNA-DNA probes were identified by random primer extension in the presence of [ 32 P]-dATP using the Prime-It II tool (Stratagene, La Jolla, CA) ([ 32 P] -dATP > 3000 Ci/mM, Amersham International, Arlington Heights, IL) and purified on a 10 bp-cutoff Micro Bio-Spin chromatography column (Bio-Rad, Hercules, CA). Hybrids were in 50% freshly deionized formamide (pH 7.0), 5 times Denhardt's solution, 0.5% SDS, 4 times SSPE and 250 mg/mL denatured salmon sperm DNA fragments (18 hours, 42 ° C In the mixture. The membrane was washed twice in 2 times SSC, 0.1% SDS (15 min, 25 °C) before autoradiography and at 0.2 times SSC, 0.1% SDS (45 min, 37 ° C) Clean once. These results are shown in Figures 4B and 6B.

實施例5Example 5 SDS-PAGE及西方點墨分析法SDS-PAGE and Western dot blot analysis

對於標的蛋白質之免疫點墨法(immunoblotting)(圖8C及9B),在移除培養基之後以冰冷PBS淋洗約~70%滿盤之分離細胞,然後溶解一CelLytic-M溶解/萃取試劑(lysis/extraction reagent,Sigma-Aldrich,MO),其補充成分為蛋白酶抑制劑(protease inhibitor)、Leupeptin、TLCK、 TAME及PMSF。該等細胞係在室溫下於一震動器(shaker)上培育15分鐘,之後將其刮到微量離心管(microtube)內,然後在12,000 xg下離心5分鐘以將該細胞碎片製成粒狀。收集包含蛋白質之細胞溶解產物並在-70℃下儲存備用。以SOFTmax套裝軟體在一E-max微量盤測讀儀(microplate reader,Molecular Devices,Sunnyvale,CA)上測量蛋白質定量。在還原(reducing,+50mM DTT)及非還原(non-reducing,無DTT)的條件下,將每30μg之細胞溶解產物加入SDS-PAGE樣本緩衝液中,並在裝載到6%-8%之聚丙烯醯胺凝膠上之前沸騰3分鐘;分子量係藉由與標準蛋白質(Bio-Rad,Hercules,CA)的比較來測定。根據該等標準操作程序來實行SDS-聚丙烯醯胺凝膠電泳法。以PAGE分解之蛋白質在一硝化纖維素膜上電漬,並在室溫下於Odyssey阻斷試劑(Odyssey blocking reagent)(Li-Cor Biosciences,Lincoln,NB)中培育2小時。然後,吾人在該試劑中施加一原生抗體並在4℃下培育該混合物。原生抗體使用包括: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)、及RGFP(1:1000,Clontech)。經過一夜後,以TBS-T淋洗該膜三次,然後在室溫下暴露在羊抗小鼠IgG(goat anti-mouse IgG)中達1小時,該羊抗小鼠IgG和Alexa Fluor 680反應性染料(1:2,000;Invitrogen-Molecular Probes)結合形成二 級抗體。在三次額外的TBS-T淋洗後,使用Li-Cor Odyssey紅外線成像器(Infrared Imager)及Odyssey軟體第10版來管理該免疫點墨法之螢光掃描及影像分析。For the immunoblotting of the target protein (Fig. 8C and 9B), after removing the medium, the cells were washed with ice-cold PBS for about ~70% full plate, and then a CelLytic-M dissolution/extraction reagent was dissolved. /extraction reagent, Sigma-Aldrich, MO), supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF. The cell lines were incubated on a shaker for 15 minutes at room temperature, then scraped into a microtube and centrifuged at 12,000 xg for 5 minutes to pellet the cell debris. . Cell lysates containing proteins were collected and stored at -70 °C until use. Protein quantification was measured on a E-max microplate reader (Molecular Devices, Sunnyvale, CA) using SOFTmax kit software. Under the conditions of reduction (+50 mM DTT) and non-reducing (no DTT), 30 μg of cell lysate was added to SDS-PAGE sample buffer and loaded at 6%-8%. The polyacrylamide 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 the 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 Dye (1:2,000; Invitrogen-Molecular Probes) combines to form two Grade antibody. After three additional TBS-T rinses, the Li-Cor Odyssey Infrared Imager (Infrared Imager) and Odyssey Software version 10 were used to manage the fluorescent scanning and image analysis of the immunospot ink method.

實施例6Example 6 斑馬魚中之內含子RNA介導基因靜默Intron RNA-mediated gene silencing in zebrafish

在轉染期間,Tg(actin -GAL4:UAS-gfp)品種之斑馬魚幼蟲係在含10ml之0.2倍不含血清RPMI 1640培養液的一魚缸中養育。藉由將60μl之FuGene微脂體轉染試劑(Roche Biochemicals,Indianapolis,IN)小心地溶解在1ml之1倍不含血清的RPMI 1640培養液中製備一轉染預混物。接著,如實施例1-2所示,將具一anti-EGFP pre-miRNA介子之該等SpRNAi-RGFP 載體(20μg)與該預混溶液混合,然後置於冰上30分鐘再直接施加在該魚缸內的該Tg(actin -GAL4:UAS-gfp)魚幼蟲上。在12小時之間隔中給予全部三種劑量(共60μg)。在首次轉染60小時後收集樣本。圖1B顯示該結果。During transfection, the zebrafish larvae of the Tg ( actin- GAL4: UAS-gfp) variety were reared in an aquarium containing 10 ml of 0.2-fold serum-free RPMI 1640 medium. A transfection premix was prepared by carefully dissolving 60 [mu]l of FuGene liposome transfection reagent (Roche Biochemicals, Indianapolis, IN) in 1 ml of 1 serum-free RPMI 1640 medium. Next, as shown in Examples 1-2, the SpRNAi-RGFP vectors (20 μg) with an anti- EGFP pre-miRNA meson were mixed with the premixed solution, then placed on ice for 30 minutes and then directly applied thereto. The Tg ( actin- GAL4: UAS-gfp) fish larvae in the aquarium. All three doses (60 μg total) were administered at 12 hour intervals. Samples were collected 60 hours after the first transfection. Figure 1B shows the result.

實施例7Example 7 流式細胞計數儀測驗Flow cytometry test

在所需之實驗後,將細胞胰蛋白酶水解、形成粒狀,並於-20℃下將其再懸浮於1ml之PBS中(含預冷的70%甲醇)中,固定該等細胞1小時。將該等細胞形成粒狀並以1ml之PBS清洗一次。將該等細胞再次形成粒狀並於37℃ 下再懸浮於1ml之PBS溶液(含1mg/ml碘化丙啶(propidium iodide)、0.5mg/ml核醣核酸苷(RNase))中達30分鐘。然後,在BD FACSCalibur流式細胞計數儀(San Jose,CA)上分析約15,000個細胞。藉由繪製脈衝寬度對脈衝面積的圖並圈選該等單一細胞來排除細胞雙元體。使用套裝軟體Flowjo以「Watson Pragmatic」演算法來分析所收集之資料。如圖5A所示,該流式細胞計數儀圖表之第一(左)及第二(右)波峰表示在整個受測試之細胞群體中休眠G0/G1及有絲分裂M期之細胞群體的含量。After the desired experiment, the cells were trypsin hydrolyzed, pelletized, and resuspended in 1 ml of PBS (containing pre-cooled 70% methanol) at -20 ° C, and the cells were fixed for 1 hour. The cells were pelleted and washed once with 1 ml of PBS. The cells were again pelleted and at 37 ° C It was resuspended in 1 ml of PBS solution (containing 1 mg/ml propidium iodide, 0.5 mg/ml RNase) for 30 minutes. Then, about 15,000 cells were analyzed on a BD FACSCalibur flow cytometer (San Jose, CA). Cell dimers were excluded by plotting pulse width versus pulse area and circled the single cells. Use the package software Flowjo to analyze the collected data using the "Watson Pragmatic" algorithm. As shown in Figure 5A, the first (left) and second (right) peaks of the flow cytometer chart represent the amount of cell populations that dormant G0/G1 and mitotic M phases throughout the population of cells tested.

實施例8Example 8 DNA去甲基化測驗DNA demethylation test

來自約兩百萬株細胞之基因體DNA係以一DNA分離工具(DNA isolation kit,Roche)來分離,並分成兩個小劑量。該DNA小劑量之一(2μg)以一CCGG切割限制酶HpaII 來解消,然後以1%之洋菜膠電泳法來評估,以判定泛基因體的去甲基化(圖7A)。在二亞硫酸鹽修飾之前及之後,另一小劑量(2μg)係用於PCR選殖該Oct3/4 啟動子之整個9,400鹼基對(bp)五端調節區域(NT_007592核苷酸21992184-22001688)。二亞硫酸鹽修飾係以一CpGenome DNA修飾工具(Chemicon,CA)來實行。對DNA作二亞硫酸鹽處理以將所有未甲基化之胞嘧啶轉換成尿嘧啶,而已甲基化之胞嘧啶則仍維持為胞嘧啶。例如:將未甲基化之ACGT位(而非已甲基化之ACGT)改變成AUGT位。在二 亞硫酸鹽修飾之前及之後已設計並測試了專對標的Oct3/4 五端啟動子區域的PCR引子,包括:兩前向引子5’-GAGGAGTTGA GGGTACTGTG-3’(SEQ.ID.NO.44)(經二亞硫酸鹽修飾後之DNA)與5’-GAGGAGCTGA GGGCACTGTG-3’(SEQ.ID.NO.45)(未修飾之DNA)以及一反向引子5’-GTAGAAGTGC CTCTGCCTTC C-3’(SEQ.ID.NO.46)。在PCR選殖方面,不論是已作二亞硫酸鹽處理或未作處理之該等基因體DNA(50ng),首先在1倍PCR緩衝液中與該等引子(共150pmole)混合,再加熱至94℃持續4分鐘,然後立即於冰上冷卻。其後,進行25個如下之PCR循環:在92℃下進行1分鐘,在55℃下進行1分鐘,然後在70℃下進行5分鐘,其係使用一長模板PCR延伸工具(Roche)。以一PCR純化工具(PCR purification kit,Qiagen)收集所得之產物,且以多種ACGT切割限制酶的均量混合物(每一種5U)來解消2μg之DNA,該等限制酶包含:AclI (AACGTT)、BmgBI (CACGTC)、PmlI (CACGTG)、SnaBI (TACGTA)及HpyCH4IV (ACGT)。然後,使用3%洋菜膠電泳法來評估該等已解消之片段(圖7B)。The genomic DNA from about two million cells was isolated using a DNA isolation kit (Roche) and divided into two small doses. One of the small doses of DNA (2 μg) was abolished with a CCGG cleavage restriction enzyme HpaII and then evaluated by 1% agaric gel electrophoresis to determine the demethylation of the pan-genosomes (Fig. 7A). Before and after the disulfite modification, another small dose (2 μg) was used to PCR the entire 9,400 base pair (bp) five-terminal regulatory region of the Oct3/4 promoter (NT_007592 nucleotide 21992184-22001688) ). The disulfite modification was carried out using a CpGenome DNA modification tool (Chemicon, CA). The DNA is disulfite treated to convert all unmethylated cytosines to uracil, while the methylated cytosine remains as cytosine. For example: changing the unmethylated ACGT position (rather than the methylated ACGT) to the AUGT position. The PCR primers for the target Oct3/4 five-terminal promoter region were designed and tested before and after the disulfite modification, including: two forward primers 5'-GAGGAGTTGA GGGTACTGTG-3' (SEQ.ID.NO. 44) (DNA modified with dithionite) and 5'-GAGGAGCTGA GGGCACTGTG-3' (SEQ.ID.NO.45) (unmodified DNA) and a reverse primer 5'-GTAGAAGTGC CTCTGCCTTC C-3 '(SEQ.ID.NO.46). In the case of PCR colonization, whether or not these genomic DNAs (50 ng) have been treated with or without disulfite, first mixed with the primers (150 pmole) in 1× PCR buffer, and then heated to 94 ° C for 4 minutes, then immediately cooled on ice. Thereafter, 25 PCR cycles were carried out: 1 minute at 92 ° C, 1 minute at 55 ° C, and 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 2 μg of DNA was depleted with a mixture of various ACGT cleavage restriction enzymes (5 U each) containing: AclI (AACGTT), BmgBI (CACGTC), PmlI (CACGTG), SnaBI (TACGTA) and HpyCH4IV (ACGT). These eclipsed fragments were then evaluated using 3% acacia gel electrophoresis (Fig. 7B).

在二亞硫酸鹽DNA定序分析(圖7C)方面,吾人進一步使用定量PCR(qPCR)擴增一467-bp標的區域,其係在Oct3/4 轉錄起始位(NT_007592核苷酸21996577-21997043)之側翼。所用之引子係一前向引子5’-GAGGCTGGAG TAGAAGGATT GCTTTGG-3’(SEQ.ID.NO.47)及一反向引 子5’-CCCTCCTGAC CCATCACCTC CACCACC-3’(SEQ.ID.NO.48)。將以上選殖之9,400-bpOct3/4 五端啟動子區域(50ng)與該等qPCR引子(總共100pmole)混合在1倍PCR緩衝液中,加熱至94℃持續2分鐘,然後立即於冰上冷卻。其後,進行20個如下之PCR循環:在94℃下進行30秒,在68℃下進行1分鐘,其係使用一高準確度PCR延伸工具(high-fidelity PCR extension kit,Roche)。具一正確467-bp長度之擴增後DNA產物進一步地以3%洋菜膠電泳法來分餾,再以一膠體萃取工具(Qiagen)純化,然後用於DNA定序。該等DNA甲基化位之一詳細圖譜係藉由比較在該已作二亞硫酸鹽修飾之DNA序列中之未改變的胞嘧啶、與在該未作二亞硫酸鹽修飾之DNA序列中之未改變的胞嘧啶來產生。In the disulfite DNA sequencing analysis (Fig. 7C), we further amplified a 467-bp target region by quantitative PCR (qPCR), which is in the Oct3/4 transcription start site (NT_007592 nucleotide 21996577-21997043). The flank. The primers used were a forward primer 5'-GAGGCTGGAG TAGAAGGATT GCTTTGG-3' (SEQ. ID. NO. 47) and a reverse primer 5'-CCCTCCTGAC CCATCACCTC CACCACC-3' (SEQ. ID. NO. 48). The above-selected 9,400-bp Oct3/4 five-terminal promoter region (50 ng) was mixed with the qPCR primers (100 pmole total) in 1× PCR buffer, heated to 94 ° C for 2 minutes, and immediately on ice. cool down. Thereafter, 20 PCR cycles were performed: 30 seconds at 94 ° C and 1 minute at 68 ° C 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 the DNA methylation positions is obtained by comparing the unmodified cytosine in the disulfite-modified DNA sequence with the DNA sequence modified without the disulfite. Unmodified cytosine is produced.

實施例9Example 9 微型核醣核酸(miRNA)微陣列分析MicroRNA (miRNA) microarray analysis

在70%滿盤下,使用mir VanaTM miRNA分離工具(miRNA isolation kit,Ambion)自每一細胞培養物中分離小RNAs。使用1%甲醛-洋菜膠電泳及光譜儀測量(Bio-Rad)來評估該等分離之小RNAs的純度及數量,然後立刻以乾冰冷涷並送往LC Sciences(San Diego,CA)進行miRNA微陣列分析。每一微陣列晶片分別與標示為Cy3或Cy5之一單一樣本雜合,或是與標示為Cy3與Cy5之一對樣本雜合。進行背景相減及正規化(normalization)。在一雙重樣本 測驗方面,執行一p 值計算並產生超過3倍之分化表現轉錄分子的一列表。在圖6A之Cy3及Cy5強度影像(藍背景)中,當訊號強度自第1級增加至第65,535級時,對應之色彩由藍色轉變成綠色、黃色,再到紅色。訊息強度在23,000級之上被視為基因表現中的正向呼叫。在Cy5/Cy3比之影像(黑背景)中,當Cy3高於Cy5級時,色彩為綠色;當Cy3級等於Cy5級時,色彩為黃色;及當Cy5級高於Cy3級時,色彩為紅色。At 70% of full disk, using separating tool mir Vana TM miRNA (miRNA isolation kit, Ambion) isolated small RNAs from each cell culture. The purity and quantity of these isolated small RNAs were assessed using 1% formaldehyde-cabbage electrophoresis and spectrometer measurements (Bio-Rad), then immediately chilled on dry ice and sent to LC Sciences (San Diego, CA) for miRNA microscopy. Array analysis. Each microarray wafer is hybridized with a single sample labeled Cy3 or Cy5, or with one of the samples labeled Cy3 and Cy5. Perform background subtraction and normalization. In a double sample test, a p- value calculation was performed and a list of more than three times the differentially expressed transcriptional molecules 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 red. Message strength above 23,000 is considered a forward call in gene performance. In Cy5/Cy3 than in the image (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 .

實施例10Example 10 整體細胞基因表現型式之泛基因體微陣列分析Pan-genome microarray analysis of whole cell gene expression patterns

包含超過54,000個寡核苷酸探針之人類基因體GeneChip U133A&B與plus 2.0陣列(Affymetrix,Santa Clara,CA)係用來偵測在mirPS細胞中之泛基因體47,000個人類基因轉錄體的表現型式,如圖8A及9A所示。每一樣本係在第三份複本中測試,並重覆相同實驗四次。來自每一受測樣本之全部RNA係使用RNeasy離心管柱(spin columns,Qiagen)來分離。為製備用於微陣列雜合之標定的探針,使用Superscript選擇系統(Invitrogen)將該等萃取之全部RNA(2μg)轉換成雙股cDNA,其具有一合成之oligo(dT)24 -T7啟動引子5'-GGCCAGTGAA TTGTAATACG ACTCACTATA GGGAGGCGG-(dT)24 -3'(SEQ.ID.NO.49)。將所得之cDNAs以苯酚/氯仿萃取來純化,以乙醇沈澱析出,並在0.5μg/μl之濃度下再懸浮於以焦碳酸二乙酯 (diethyl pyrocarbonate,DEPC)處理之二次水(ddH2 O)中。然後,進行體外轉錄,其包含:1μg之dsDNAs、7.5mM未標定ATP與GTP、5mM未標定UTP與CTP、及2mM生物素標定CTP與UTP(biotin-11-CTP、biotin-16-UTP,Enzo Diagnostics),以及20U之T7 RNA聚合酶。反應在37℃下進行4小時,然後將所得之cRNAs以RNeasy離心管柱(Qiagen)純化。在一1%之洋菜膠上分離一部分之cRNA樣本以檢查長度範圍,然後藉由在94℃下於pH 8.0的40mM三醋酸鹽(Tris-acetate)、100mM KOAc/30mM MgOAc中加熱35分鐘,將10μg之cRNAs隨機分餾成50個鹼基的一平均長度。在200μl之AFFY緩衝液(Affymetrix)中於40℃下持續地混合16小時以完成雜合。在雜合之後,以200μl之6倍SSPE-T緩衝液(1倍之0.25M氯化鈉/15mM磷酸鈉,pH 7.6/1mM EDTA/0.005%氚核)淋洗陣列三次,然後以200μl之6倍SSPE-T在50℃下清洗1小時。該等陣列另外再以0.5倍之SSPE-T淋洗兩次,並以0.5倍之SSPE-T在50℃下清洗15分鐘。然後,以2μg/ml之卵白素-藻紅蛋白(streptavidin-phycoerythrin,Invitrogen-Molecular Probes)及1mg/ml之醯化BSA(Sigma)在6倍之SSPE-T(pH 7.6)中完成染色測驗。該等陣列係以一共焦掃描器(confocal scanner,Molecular Dynamics)在7.5μm下讀取。The human genome GeneChip U133A&B and the plus 2.0 array (Affymetrix, Santa Clara, CA) containing more than 54,000 oligonucleotide probes were 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 μg) was converted to a double-stranded cDNA using a Superscript selection system (Invitrogen) with a synthetic oligo(dT) 24 -T7 start-up 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 μg/μl in secondary water (ddH 2 O treated with diethyl pyrocarbonate (DEPC). )in. Then, in vitro transcription was performed, which included: 1 μg of dsDNAs, 7.5 mM uncalibrated ATP and GTP, 5 mM uncalibrated UTP and CTP, and 2 mM biotin-labeled CTP and UTP (biotin-11-CTP, biotin-16-UTP, Enzo Diagnostics), and 20 U of T 7 RNA polymerase. The reaction was carried out at 37 ° C for 4 hours, and the resulting cRNAs were then purified on an RNeasy centrifugal column (Qiagen). A portion of the cRNA sample was separated on a 1% agar extract to examine the length range, and then heated by trifluoroacetic acid (Tris-acetate), 100 mM KOAc/30 mM MgOAc at pH 8.0 at 94 ° C for 35 minutes. 10 μg of cRNAs were randomly fractionated into an average length of 50 bases. Hybridization was continued in 200 μl of AFFY buffer (Affymetrix) at 40 ° C for 16 hours to complete the hybridization. After hybridization, the array was rinsed three times with 200 μl of 6-fold SSPE-T buffer (1×0.25 M sodium chloride/15 mM sodium phosphate, pH 7.6/1 mM EDTA/0.005% nucleus), and then 200 μl. The SSPE-T was washed at 50 ° C for 1 hour. The arrays were additionally rinsed twice with 0.5 times SSPE-T and washed with 0.5 times SSPE-T at 50 °C for 15 minutes. Then, the staining test was completed in 6-fold SSPE-T (pH 7.6) with 2 μg/ml of streptavidin-phycoerythrin (Invitrogen-Molecular Probes) and 1 mg/ml of deuterated BSA (Sigma). The arrays were read at 7.5 μm using a confocal scanner (Molecular Dynamics).

為識別該背景之變化,吾人使用相同之樣本來複製該等微陣列測試,並選擇兩百株基因(圖8A之白點)以作進一 步之比較,其略為在該等測試之一側呈現。使用完美相配探針及失配探針之間的總平均差將該等樣本訊號正規化。之後,使用Affymetrix Microarray Suite 5.0版、Expression ConsoleTM 1.1.1版(Affymetrix)及Genesprings(Silicon Genetics)軟體來分析整個泛基因體基因表現型式之變化(圖8A之綠點)。超過一倍(one-fold)之基因表現率的變化被視為陽性分化基因(positive differential genes)。在基因叢集測驗中,使用一外掛程式Genetrix(Epicenter Software)與Affymetrix軟體配合。以在每一微陣列中之內部管家基因控制平均正規化該樣本之訊號。在正規化之後,當訊號強度由第1級增加至第65,535級時,該對應之色彩由綠色轉變成黑色,再到紅色。在第23,000級(紅色)之上的訊息強度視為一正向呼叫,其中一北方墨點分析可偵測為陽性。To identify this change in background, we used the same sample to replicate the microarray tests and selected two hundred genes (white dots in Figure 8A) for further comparison, which was slightly presented on one side of the tests. These sample signals are normalized using the total average difference between the perfect matching probe and the mismatched probe. Thereafter, using Affymetrix Microarray Suite 5.0 Edition, Expression Console TM 1.1.1 Edition (the Affymetrix) and Genesprings (Silicon Genetics) software to analyze changes in gene expression patterns of the entire genome pan (FIG. 8A of the green dot). Changes in the one-fold gene expression rate are considered to be positive differential genes. In the gene cluster test, a plug-in Genetrix (Epicenter Software) was used with the Affymetrix software. The signal of the sample is averaged normalized by the internal housekeeping gene in each microarray. After normalization, when the signal strength is increased from level 1 to level 65, 535, the corresponding color changes from green to black to red. The intensity of the message above level 23,000 (red) is considered a forward call, and a northern ink dot analysis can be detected as positive.

實施例11Example 11 細胞分化及免疫偵測測驗Cell differentiation and immune detection test

在不進行任何處理下,除了不含哺乳細胞之mirPS培養基的處理以外,將該等mirPS衍生類胚胎體異種移植至一六週大雌性假性懷孕免疫功能不足之SCID-beige小鼠的子宮或腹腔中可形成類畸胎瘤之囊腫(圖10)。該免疫功能不足之裸鼠係用來提供模擬移植治療的一體內環境。製造該假性懷孕小鼠的方法為:在腹膜內注射1IU之人類停經後促性腺激素(human menopausal gonadotrophin,HMG)兩天,然後再注射人類絨毛膜性腺激素(human chorionic gonadotrophin,hCG)一天。在體外分子引導幹細胞分化成生殖細胞系譜系方面,mirPS細胞在37℃及5% CO2 下保持在DMEM/F12(1:1;高葡萄糖)培養液中之聚鳥胺酸/基膜素覆蓋皿(polyornithine/laminin-coated)上12小時,該培養液的補充成分為10%炭吸附FBS、4mM L-麩醯胺、1mM丙酮酸鈉、5ng/ml活化素及50ng/ml之二氫睪固酮(dihydrotestosterone,DHT)。之後,胰蛋白酶水解該等細胞,以1倍PBS清洗,並收集在四個小劑量之冷凍Matrige(每一個劑量100μl)及一個小劑量之100μl的1倍PBS中。之後,吾人隨即將該等細胞轉殖到六週大之免疫功能不足SCID-beige裸鼠的後肢肌肉、腹膜、子宮、皮下頸部皮膚(含Matrigel)及尾靜脈(含PBS)中。在實驗處理期間以二乙醚(diethyl ether)麻醉該小鼠。一週後,僅在子宮區域發現類精原細胞。在纖維母細胞分化方面,吾人在異體移植之前按照以上所示之相同程序,但使用標準不含酚紅之DMEM培養液進行6小時,該培養液的補充成分為10% FBS、4mM L-麩醯胺、1mM丙酮酸鈉、5ng/ml Noggin蛋白及100ng/ml轉形生長因子-β1(TGF-β1)。一週後在子宮中發現纖維母細胞。在軟骨細胞分化方面,吾人如先前般實行相同程序,但使用標準RPMI 1640培養液進行6小時,該培養液的補充成分為10% FBS、4mM L-麩醯胺、1mM丙酮酸鈉及100ng/ml骨成形蛋白4(bone morphogenetic protein 4,BMP4)。僅在肝臟區域發現軟骨細胞。The mirPS-derived embryoid bodies were xenografted to the uterus of a SCID-beige mouse with insufficient immune function of one to six weeks of large female pseudopregnancy without any treatment except for the treatment of mirPS medium containing no mammalian cells. A teratoma-like cyst can be formed in the abdominal cavity (Fig. 10). This immunocompromised nude mouse is used to provide an integrated internal environment for mimicking transplant therapy. The pseudopregnant mouse was prepared by intraperitoneal injection of 1 IU of human menopausal gonadotrophin (HMG) for two days, followed by injection of human chorionic gonadotrophin (hCG) for one day. In vitro molecular-guided stem cell differentiation into germline lineage, mirPS cells maintained poly-ornithine/basementin coverage in DMEM/F12 (1:1; high glucose) medium at 37 ° C and 5% CO 2 On a dish (polyornithine/laminin-coated) for 12 hours, the supplement of the culture solution was 10% carbon adsorption FBS, 4 mM L-glutamate, 1 mM sodium pyruvate, 5 ng/ml activin and 50 ng/ml dihydrosterolone. (dihydrotestosterone, DHT). Thereafter, the cells were hydrolyzed by trypsin, washed with 1× PBS, and collected in four small doses of frozen Matrige (100 μl per dose) and a small dose of 100 μl of 1×PBS. Afterwards, we immediately transferred the cells to the hind limb muscle, peritoneum, uterus, subcutaneous neck skin (including Matrigel) and tail vein (PBS) of the six-week-old SCID-beige nude mice. 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 standard DMEM medium without phenol red for 6 hours. The supplement of the culture solution was 10% FBS, 4 mM L-breast. Indoleamine, 1 mM sodium pyruvate, 5 ng/ml Noggin protein and 100 ng/ml transforming growth factor-β1 (TGF-β1). Fibroblasts were found in the uterus one week later. In terms of chondrocyte differentiation, we performed the same procedure as before, but used standard RPMI 1640 medium for 6 hours. The supplement of the medium was 10% FBS, 4 mM L-glutamate, 1 mM sodium pyruvate and 100 ng/ Mp bone morphogenetic protein 4 (BMP4). Chondrocytes are found only in the liver area.

在特定組織標記之免疫偵測方面,該等組織樣本係在4℃下於4%三聚甲醛(paraformaldehyde)中固定一整夜。在將該等樣品嵌入石蠟中之前,先將該等樣品相繼地以1倍PBS、甲醇、異丙醇及四氫萘(tetrahydronaphthalene)清洗。然後在一微切片機(microtome)上以7-10μm之厚度切割該等嵌入之樣本並固定在乾淨的TESPA塗布玻片上。然後,以二甲苯(xylene)去除該等玻片之蠟並使用封片膠(mounting media;Richard Allan Scientific,Kalamazoo,MI)固定在蓋玻片下,再以蘇木精(hematoxylin)及曙紅(eosin)(H&E,Sigma)染色以供形態觀察之用。免疫組織化學(IHC)染色工具係購自Imgenex(San Diego,CA)。根據製造商的建議實行抗體稀釋及免疫染色法之程序。所用之原生抗體包括:Tuj1(1:500,Abcam Inc.,Cambridge,MA)、ABCA2(1:100,Santa Cruz Biotechnology,Santa Cruz,CA)、Dazla(1:100,Abcam)、EE2(1:100,Santa Cruz)、atlastin1(1:200,Santa Cruz)、COL1A1(1:500,Santa Cruz)、COL2A1(1:500,Santa Cruz)、tropoelastin(1:200,Abcam),及RGFP(1:500,Clontech)。以螢光染色標定之羊抗兔(goat anti-rabbit)或馬抗小鼠(horse anti-mouse)的抗體係用來作為二級抗體(1:2,000,Invitrogen-Molecular Probes)。在具全場掃描之一100倍顯微鏡下觀察到陽性結果,並以一Metamorph影像處理程式(Nikon 80i及TE2000顯微鏡定量分析系統)在200倍或400倍之放大倍率下測量以作定量分析。For tissue detection of specific tissue markers, the 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 paraffin. The embedded samples were then cut at a thickness of 7-10 μm on a microtome and mounted on a clean TESPA coated slide. Then, the wax of the slides was removed with xylene and fixed under a cover glass using a mounting medium (Richard Allan Scientific, Kalamazoo, MI), followed by hematoxylin and blush (eosin) (H&E, Sigma) staining 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: Tuj1 (1:500, Abcam Inc., Cambridge, MA), ABCA2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), Dazla (1:100, Abcam), EE2 (1: 100, Santa Cruz), atlastin1 (1:200, Santa Cruz), COL1A1 (1:500, Santa Cruz), COL2A1 (1:500, Santa Cruz), tropoelastin (1:200, Abcam), and RGFP (1: 500, Clontech). A goat anti-rabbit or horse anti-mouse anti-system 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.

實施例12Example 12 細胞遷移測驗Cell migration test

在一96孔培養盤中,吾人在每一孔中將一PC3及一mirPS-PC3細胞放置在一起,然後記錄其移動及交互作用。該等細胞皆在37℃及5% CO2 下於RPMI 1640培養液中生長,該培養液的補充成分為10%炭吸附FBS、4mM L-麩醯胺、1mM丙酮酸鈉、5ng/ml活化素、5ng/ml Noggin蛋白、3ng/ml bFGF及0.5μM GSK-3抑制劑XV。在一TE2000倒立式顯微鏡系統(invert microscopic system,Nikon)下使用一成對之MO-188NE 3D液壓式精密微操縱器(hydraulic fine micromanipulators)來個別地分離並收集該等細胞,該微操縱器具有一細胞固持器(cell holder)。整個微操縱器及顯微鏡系統皆放置在一防震桌上。每15秒記錄該等相片(放大倍率400x及600x),持續6小時。藉由追蹤在該等相片中之細胞移動及該細胞之形態判定該細胞遷移。如圖7D所示,轉移性癌症PC3細胞呈現如ATCC所述之一快速梭形移動,其中該mirPS-PC3細胞保持在該放置位置中,其顯現一圓形休眠之表現型。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-glutamate, 1 mM sodium pyruvate, 5 ng/ml activation. , 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. The photos were recorded every 15 seconds (magnification 400x and 600x) for 6 hours. The cell migration is determined by tracking cell movement 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.

實施例13Example 13 統計分析Statistical Analysis

該等結果以平均值±標準差(mean±SE)表示。資料之統計分析係以單因子變異數分析(one-way ANOVA)來計算。 當主要效應明顯時,使用Dunnett事後測試法(Dunnett's post-hoc test)來識別與控制組有明顯差異之群。在兩組處理群之間進行比對時,使用雙尾studentt 測試(two-tailed studentt test)。對於包含超過兩組處理群之實驗,則依照一事後多範圍測試(post-hoc multiple range test)實行變異數分析(ANOVA)。機率值p <0.05被認定為具有統計上的意義。所有p 值係由雙尾測試(two-tailed test)來決定。These results are expressed as mean ± standard deviation (mean ± SE). Statistical analysis of the data was calculated by one-way ANOVA. When the main effect is obvious, the Dunnett's post-hoc test is used to identify groups that are significantly different from the control group. When compared between the two groups of treatment group, using the two-tailed student t test (two-tailed student t test) . For experiments involving more than two treatment groups, the variance analysis (ANOVA) was performed according to a post-hoc multiple range test. The probability value p < 0.05 was considered to be statistically significant. All p values are determined by a two-tailed test.

明確言之,以下說明的圖示僅供舉例說明之用而非限制本發明:圖1A-B描述內含子miRNA生體合成及其相對之基因靜默效應的機制。(圖1A)將內含子miRNA轉錄成先驅訊息RNA(pre-mRNA)之一部分,其包含蛋白質編碼的外顯子及非編碼內含子。將該等內含子從pre-mRNA中剪接出來,並將一些其二級結構進一步酶切而可誘發標的基因靜默的小型類miRNA分子內,而該等外顯子係結合在一起以形成成熟之mRNA以供標記蛋白質合成。(圖1B)將預先設計的內含子miRNA基因轉殖轉染到Tg(actin-GAL4:UAS-gfp) 品種斑馬魚的實施例顯示:在標的綠色EGFP表現上之強力的基因靜默效應(>80%之抑制,左邊第四列),然而其它非標的介子則不受影 響,包括(從第一列至第五列):不含miRNA之一空內含子(1),具對抗HIV-p24 (2)或結合蛋白integrin β1 (3)任一者之pre-miRNA介子的內含子,以及具anti-EGFP pre-miRNA介子但無功能性五端剪接位(functional 5’-splice site)的內含子(5)。將該anti-EGFP pre-miRNA插入紅色色偏螢光標記(red-shifted fluorescent marker,RGFP )基因的五端近端內含子區域(5’-proximal intron region)。北方點墨分析法(右圖)顯示成熟之miRNA家族僅由該pre-miRNA-插入RGFP 之該等剪接產物生成,而非該空RGFP (-)或該有缺陷之RGFP (△),這表示在內含子miRNA生體合成中需要RNA剪接。In other words, 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 a precursor message RNA (pre-mRNA) comprising a protein-encoded exon and a non-coding intron. The introns are spliced out of the pre-mRNA, and some of the secondary structures are further digested to induce a silent gene in a small class of miRNA molecules, and the exons are combined to form a mature The mRNA is used for labeling protein synthesis. (Fig. 1B) An example of transfection of a pre-designed intron miRNA gene into a Tg (actin-GAL4: UAS-gfp) cultivar zebrafish shows a strong gene silencing effect on the underlying green EGFP expression (> 80% inhibition, fourth column on the left), however, other non-standard mesons are unaffected, including (from the first column to the fifth column): one of the miRNAs without the miRNA (1), against HIV-p24 (2) an intron of a pre-miRNA meson of either the binding protein integrin β1 (3), and an anti- EGFP pre-miRNA meson but a functional 5'-splice site Intron (5). The anti- EGFP pre-miRNA was inserted into the 5'-proximal intron region of the red-shifted fluorescent marker ( RGFP ) gene. Northern blotting analysis (right panel) shows that the mature miRNA family is only generated by the splicing products of the pre-miRNA-inserted RGFP , rather than the empty RGFP (-) or the defective RGFP (Δ), which means RNA splicing is required in intron miRNA biosynthesis.

圖2A及2B描述使用包含SpRNAi-RGFP 轉殖基因之修飾Tet-On 載體(即:pTet-On-tTS-mir302s )的結構及策略,以表現mir-302s pre-miRNA基因群或shRNA。一種電穿孔型基因轉殖傳送方法係用來將該表現mir-302SpRNAi-RGFP 之轉殖基因轉染入標的體細胞/癌症細胞。Figures 2A and 2B depict the structure and strategy of a modified Tet-On vector (i.e., pTet-On-tTS-mir302s ) comprising a SpRNAi-RGFP transgene to express a mir-302s pre-miRNA gene population or shRNA. An electroporation-type gene transfer delivery method is used to transfect the transgenic gene expressing mir-302 SpRNAi-RGFP into a target somatic/cancer cell.

圖3A-3C顯示將人類正常頭髮毛囊hHFC細胞及癌症黑色素癌Colo細胞轉化為類ES多能性幹(mirPS)細胞,其使用預先設計之Tet-On mir-302-表現轉殖基因之電穿孔型轉染。(圖3A)在Tet-On 可誘發載體(即:pTet-On-tTS-miR302s )中之重組mir-302-表現轉殖基因(即:SpRNAi-RGFP )的結構。該SpRNAi-RGFP 轉殖基因係以370鹼基對(bp)同源區域為側翼,其用來重組插入 人類細胞基因體的標的位。(圖3B)該mir-302 pre-miRNA基因群(mir-302s)之建構,其合併為該SpRNAi-RGFP 轉殖基因之內含子的一部分。(圖3C)使用FACS流動式細胞計數儀(flow cytometry)選出陽性mir-302-轉導之mirPS細胞,該流式細胞計數儀係針對RGFP標記蛋白之抗體來分類細胞。目前本發明之轉殖基因傳送的成功率經測量約為91%-93%。Figures 3A-3C show the transformation of human normal hair follicle hHFC cells and cancer melanoma Colo cells into ES-like pluripotent stem (mirPS) cells using pre-designed Tet-On mir-302-electroporation of transgenic genes. Type transfection. (Fig. 3A) The structure of the recombinant mir- 302 -expressing transgene (i.e., SpRNAi-RGFP ) in the Tet-On inducible vector (i.e., pTet-On-tTS-miR302s ). The SpRNAi-RGFP transgenic line is flanked by a 370 base pair (bp) homology region that is used to recombine into the epitope of a human cell genome. (Fig. 3B) Construction of the mir-302 pre-miRNA gene group (mir-302s), which is incorporated as part of the intron of the SpRNAi-RGFP transgene. (FIG. 3C) Positive mir-302-transduced mirPS cells were selected using a FACS flow cytometry, which classifies cells against antibodies to RGFP-tagged proteins. At present, the success rate of the transgenic gene delivery of the present invention is measured to be about 91% to 93%.

圖4A-4B顯示在該mirPS細胞基因體之Tet-On mir-302-表現SpRNAi-RGFP 轉殖基因的併入整合,其造成在各種去氧羥(Dox)濃度的控制下之該等mir-302家族成員(mir-302s)的可誘發表現。(圖4A)由不同mirPS細胞株分離之基因體DNA的定量PCR(qPCR;左圖)分析,其顯示所有的mirPS細胞僅攜帶該轉殖基因之一或兩個伴隨拷貝序列,其中在原先之hHFC及Colo細胞中未偵測到轉殖基因(控制組)。螢光原位雜合(Fluorescentin-situ hybridization,FISH;右圖)實驗進一步地顯示該轉殖基因被插進該等人類基因體之特定位置中。此種限制基因轉殖插入暗示整體mir-302表現之濃度可影響mirPS細胞之存活。(圖4B)對應於該Dox濃度之該可誘發mir-302s表現的北方點墨分析及柱形圖展示。Figures 4A-4B show the integration of the Tet-On mir-302-expressing SpRNAi-RGFP transgene in the miRNA of the mirPS cell, which results in these mir- under the control of various deoxygenated (Dox) concentrations. Inducible performance of members of the 302 family (mir-302s). (Fig. 4A) Quantitative PCR (qPCR; left panel) analysis of genomic DNA isolated from different mirPS cell lines, showing that all mirPS cells carry only one or two accompanying copy sequences of the transgene, where No transgenes were detected in hHFC and Colo cells (control group). Fluorescent in-situ hybridization (FISH; right panel) experiments further showed that the transgenic genes were inserted into specific positions of the human genome. Such restriction gene insertion insertions suggest that the concentration of the overall mir-302 expression can affect the survival of mirPS cells. (Fig. 4B) Northern blot analysis and histogram display corresponding to the Dox concentration which induces mir-302s performance.

圖5A-5C顯示將人類正常頭髮毛囊(hHFC)及癌症黑色素癌Colo細胞轉化為類ES多能性幹(mirPS)細胞的變化。(圖5A)在該等mir-302-轉導細胞(即:mirPS-hHFC及mirPS-Colo細胞) 中之形態的改變及細胞增殖率。在具去氧羥(Dox)誘發之mir-302-轉導細胞中發現類ES圓形細胞形狀及極慢之細胞再生率。(圖5B)源自該等mirPS細胞之類胚胎體(EB)的形成以及經導引分化成具陽性Tuj1及/或ABCA2標記之神經元前驅細胞。(圖5C)在限數稀釋法(limiting dilution)之後,由單一mirPS細胞形成EB的時程變化圖。該等細胞循環估計約為20-24小時。Figures 5A-5C show changes in the conversion of human normal hair follicles (hHFC) and cancer melanoma Colo cells to ES-like pluripotent stem (mirPS) cells. (Fig. 5A) in these mir-302-transduced cells (ie: mirPS-hHFC and mirPS-Colo cells) Morphological changes and cell proliferation rates. The shape of ES-like round cells and the very slow rate of cell regeneration were found in hydroxy-302-transduced cells with deoxygenated hydroxyl (Dox) induction. (Fig. 5B) Formation of embryonic bodies (EB) derived from such mirPS cells and guided differentiation into neuronal precursor cells with positive Tuj1 and/or ABCA2 markers. (Fig. 5C) A time course change map of EB formed from a single mirPS cell after limiting dilution. These cell cycles are estimated to be approximately 20-24 hours.

圖6A-6B顯示mir-302s轉染及ES標記表現間的相關性。(圖6A)整體miRNA表現之微陣列分析,其顯示全部之mir-302家族成員(mir-302s)係高度表現在具Dox誘發之mirPS細胞中,而非在該等原始之體細胞中(n=3,p<0.01)。(圖6B)北方點墨及西方點墨分析法,其顯示具Dox誘發之該等mirPS細胞表現了豐富的ES細胞標記,包括:Oct3/4(Oct4)、SSEA-3、SSEA-4、Sox2及Nanog,但表現較少的Klf4(n=4,p<0.01),這與在人類ES WA01-H1及WA09-H9細胞中觀察到的細胞標記極為相似。Figures 6A-6B show the correlation between mir-302s transfection and ES marker expression. (Fig. 6A) Microarray analysis of overall miRNA expression, showing that all mir-302 family members (mir-302s) are highly expressed in Dox-induced mirPS cells, rather than in such primitive somatic cells (n = 3, p < 0.01). (Fig. 6B) Northern blotting and western dot blot analysis, which showed that Dox-induced mirPS cells exhibited abundant ES cell markers, including: Oct3/4 (Oct4), SSEA-3, SSEA-4, Sox2 And Nanog, but with less performance of Klf4 (n=4, p<0.01), which is very similar to the cell markers observed in human ES WA01-H1 and WA09-H9 cells.

圖7A-7D顯示在各種mirPS細胞株中之基因體DNA去甲基化的型式。(圖7A)HpaII酶切顯示在mirPS細胞中泛基因體規模下之整體CpG甲基化的降低。(圖7B)將未甲基化之ACGT經二亞硫酸鹽修飾為Oct3/4啟動子之9,400鹼基對調節區域中的AUGT位,其顯示在所有mirPS細胞中未甲基化之ACTG(或 AUCT)位的顯著增加。(圖7C)二亞硫酸鹽DNA序列,其顯示在Oct3/4啟動子之起始位側面的詳細甲基化映像圖。黑色圓圈及白色圓圈分別表示該等甲基化及未甲基化之胞嘧啶位。(圖7D)與mirPS-PC3細胞之起始轉移性癌症PC3細胞相較,mirPS-PC3細胞中之遷移能力的喪失。Figures 7A-7D show the pattern of de-methylation of genetic DNA in various mirPS cell lines. (FIG. 7A) HpaII digestion revealed a decrease in overall CpG methylation at ubiquitin scale in mirPS cells. (FIG. 7B) The unmethylated ACGT was modified with disulfite to the AUGT position in the 9,400 base pair regulatory region of the Oct3/4 promoter, which shows unmethylated ACTG in all mirPS cells (or A significant increase in the AUCT) position. (Fig. 7C) Disulfite DNA sequence showing a detailed methylation map on the side of the start of the Oct3/4 promoter. Black circles and white circles indicate the methylated 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.

圖8A-8B顯示在Colo、mirPS-Colo及人類ES WA01-H1(H1)與WA09-H9(H9)細胞間之泛基因體基因表現分析。(圖8A)使用人類基因體基因晶片(Human genome GeneChip)U133A&B及plus 2.0陣列(Affymetrix)之改變基因表現樣式的比較,其顯示在mirPS-Colo與H1(89%)以及H9(86%)間的高相似度,但與癌症型Colo(53%)細胞較不相似。白點表示與該穩定表現基因(綠點)相較之高度可變基因。(圖8B)微陣列識別分化表現基因之功能性基因群集,其證明在mirPS細胞中偵測到:ES細胞標記之顯著增加與黑色素癌致癌基因、發育訊息以及mir-302-標的細胞增殖與DNA甲基化基因之顯著減少,其極為類似H1及H9細胞中所偵測到的現象(n=4,p<0.01)。圖8C顯示北方點墨及西方點墨分析,其確認在mirPS-Colo細胞中mir-302s、人類ES細胞標記及預測mir-302標的基因之表現樣式間的相關性,這與人類ES H1及H9細胞中之表現樣式類似,除了Klf4之表現以外(n=3,p<0.01)。Figures 8A-8B show ubiquitin gene expression analysis between Colo, mirPS-Colo and human ES WA01-H1 (H1) and WA09-H9 (H9) cells. (Fig. 8A) Comparison of altered gene expression patterns using Human genome GeneChip U133A&B and plus 2.0 array (Affymetrix), shown between mirPS-Colo and H1 (89%) and H9 (86%) High similarity, 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 a functional gene cluster of differentiation-expressing genes, which demonstrates detection in mirPS cells: a significant increase in ES cell markers and melanoma oncogenes, developmental messages, and mir-302-target cell proliferation and DNA Significantly reduced methylation genes, which are very similar to those detected in H1 and H9 cells (n=4, p<0.01). 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-302 genes in mirPS-Colo cells, which is related to human ES H1 and H9. The pattern of expression in the cells was similar except for the expression of Klf4 (n=3, p<0.01).

圖9A-9B顯示在hHFC、mirPS-hHFC及人類ES WA01-H1(H1)與WA09-H9(H9)細胞間之泛基因體基因表現 分析。(圖9A)使用人類基因體基因晶片U133 plus 2.0陣列(Affymetrix)之改變基因表現樣式的比較,其顯示在mirPS-hHFC與H1(96%)以及H9(91%)間的高相似度,但與hHFC(47%-56%)體細胞較不相似。(圖9B)西方點墨分析,其確認在mirPS-hHFC細胞中mir-302s、人類ES細胞標記及預測mir-302標的基因之該等表現樣式間的相關性,這與人類ES H1及H9細胞中之該等表現樣式類似,除了Klf4及Klf5之表現以外。所列出之mir-302標的基因包括:十七個轉錄調節子、一個組織蛋白去乙醯酶(HDA4)、兩個甲基CpG-結合蛋白質(MECP1-p66及MECP2),以及三個細胞循環檢驗點蛋白質(CDK2、細胞週期素cyclin D1及D2)。Figures 9A-9B show pan-gene expression of genes in hHFC, mirPS-hHFC, and human ES WA01-H1 (H1) and WA09-H9 (H9) cells. analysis. (Fig. 9A) Comparison of altered gene expression patterns using the human genome gene chip U133 plus 2.0 array (Affymetrix) showing high similarity between mirPS-hHFC and H1 (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-302s, human ES cell markers and predicted mir-302 genes in mirPS-hHFC cells, which are related to human ES H1 and H9 cells. These performance styles are similar, except for the performance of Klf4 and Klf5. The listed mir-302 genes include: seventeen transcriptional regulators, one tissue protein deacetylase (HDA4), two methyl CpG-binding proteins (MECP1-p66 and MECP2), and three cell cycles. Checkpoint proteins (CDK2, cyclin D1 and D2).

圖10顯示源自在雌性假性懷孕免疫功能不足SCID-beige小鼠之子宮或腹腔中之mirPS移植物的類畸胎瘤原生組織(n/total=6/6)。該等已分化組織包括所有三個胚胎生殖層:外胚層、中胚層及內胚層,此係在蘇木精(hematoxylin)及曙紅(eosin)(H & E)染色後以其截然不同的細胞形態來判定。顯微鏡照片係以Nikon TE2000系統在200x放大倍率下拍攝。Figure 10 shows teratomas native tissue derived from mirPS grafts in the uterus or peritoneal cavity of female pseudopregnant immunodeficiency SCID-beige mice (n/total = 6/6). These differentiated tissues include all three embryonic germ layers: ectoderm, mesoderm and endoderm, which are distinct cells after hematoxylin and eosin (H & E) staining. Form to determine. Microscope photographs were taken at 200x magnification using a Nikon TE2000 system.

圖11A-11O顯示mirPS細胞之導引多能性。分別由上至下處理DHT、TGF-ß1及BMP4,將該mirPS細胞誘發分化成類精原細胞(圖11A-E)、纖維組織母細胞(圖11F-J)及軟骨細胞(chondrocyte)(圖11K-O)組織細胞,其係在免疫功能不足之小 鼠中離體處理。該免疫功能不足之裸鼠係用於體內環境模擬移植治療中。由左至右顯示之顯微照片指出:使用微分干涉差之蘇木精染色(圖11A、F、K)、以轉殖基因mir-302標記RGFP標示之明視野(紅色)(圖11B、G、L)、以4,6-脒基-2-苯基吲哚(4,6-diamidino-2-phenylindole)標示之第一組織標記的免疫染色法(藍色DAPI)(圖11C、H、M)、以螢光素標示之第二組織標記的免疫染色法(綠色EGFP)(圖11D、I、N),以及所有三個螢光標記之合併(圖11E、J、O)。在該等RGFP-明視野中之小窗口中顯示在高放大倍率(600x)下之分化mirPS細胞的形態。Figures 11A-11O show the guided pluripotency of mirPS cells. DHT, TGF-ß1 and BMP4 were treated from top to bottom, respectively, and the mirPS cells were induced to differentiate into spermatogonia (Fig. 11A-E), fibroblasts (Fig. 11F-J) and chondrocyte (Fig. 11K-O) tissue cells, which are small in immune function Ex vivo treatment in rats. This immunocompromised nude mouse is used in in vivo environmental mimic transplantation therapy. The micrographs shown from left to right indicate: hematoxylin staining using differential interference (Fig. 11A, F, K), and clear field of view (red) labeled with the transgenic gene mir-302 (Fig. 11B, G) , L), immunostaining method (blue DAPI) of the first tissue marker indicated by 4,6-diamidino-2-phenylindole (Fig. 11C, H, M), immunostaining of the second tissue marker labeled with luciferin (green EGFP) (Fig. 11D, I, N), and the combination of all three fluorescent markers (Fig. 11E, J, O). The morphology of differentiated mirPS cells at high magnification (600x) is shown in a small window in these RGFP-bright fields.

<110> Lin,Shi-Lung 林希龍 Wu,David TS 吳堂熙<110> Lin, Shi-Lung Lin Xilong Wu, David TS Wu Tangxi

<120> 使用可誘導之重組核醣核酸生成不具癌細胞特性之類胚胎幹細胞/Generation of Tumor-Free Embryonic Stem-Like Pluripotent Cells Using Inducible Recombinant RNAAgents<120> Using 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 RNAAgents

<150> 12/149725<150> 12/149725

<151> 2008-05-07<151> 2008-05-07

<150> 61/191327<150> 61/191327

<151> 2008-09-08<151> 2008-09-08

<150> 61/193438<150> 61/193438

<151> 2008-11-28<151> 2008-11-28

<150> PCT/US09/30432<150> PCT/US09/30432

<151> 2009-01-08<151> 2009-01-08

<150> 12/318806<150> 12/318806

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<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 6 <400> 6

<210> 7<210> 7

<211> 17<211> 17

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 7 <400> 7

<210> 8<210> 8

<211> 19<211> 19

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<220><220>

<221> misc_feature<221> misc_feature

<222> (15)..(15)<222> (15)..(15)

<223> n is a,c,g,or t<223> n is a,c,g,or t

<400> 8 <400> 8

<210> 9<210> 9

<211> 23<211> 23

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 9 <400> 9

<210> 10<210> 10

<211> 23<211> 23

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 10 <400> 10

<210> 11<210> 11

<211> 23<211> 23

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 11 <400> 11

<210> 12<210> 12

<211> 23<211> 23

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 12 <400> 12

<210> 13<210> 13

<211> 23<211> 23

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 13 <400> 13

<210> 14<210> 14

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 14 <400> 14

<210> 15<210> 15

<211> 46<211> 46

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 15 <400> 15

<210> 16<210> 16

<211> 42<211> 42

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 16 <400> 16

<210> 17<210> 17

<211> 46<211> 46

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 17 <400> 17

<210> 18<210> 18

<211> 70<211> 70

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 18 <400> 18

<210> 19<210> 19

<211> 74<211> 74

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 19 <400> 19

<210> 20<210> 20

<211> 47<211> 47

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 20 <400> 20

<210> 21<210> 21

<211> 45<211> 45

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 21 <400> 21

<210> 22<210> 22

<211> 689<211> 689

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 藉由從海葵(Heteractis crispa )衍生來之HcRed1彩色蛋白質基因(chromoprotein gene)於第69個胺基酸插入天門冬胺酸(aspartate(Asp))而生成之突變紅色螢光蛋白基因<223> A mutant red fluorescent protein gene produced by insertion of aspartate (Asp) into the 69th amino acid by an HcRed1 chromoprotein gene derived from an anemone ( Heteractis crispa )

<400> 22 <400> 22

<210> 23<210> 23

<211> 27<211> 27

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 23 <400> 23

<210> 24<210> 24

<211> 27<211> 27

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 24 <400> 24

<210> 25<210> 25

<211> 89<211> 89

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 25 <400> 25

<210> 26<210> 26

<211> 89<211> 89

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 26 <400> 26

<210> 27<210> 27

<211> 82<211> 82

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 27 <400> 27

<210> 28<210> 28

<211> 82<211> 82

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 28 <400> 28

<210> 29<210> 29

<211> 91<211> 91

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 29 <400> 29

<210> 30<210> 30

<211> 91<211> 91

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 30 <400> 30

<210> 31<210> 31

<211> 95<211> 95

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 31 <400> 31

<210> 32<210> 32

<211> 95<211> 95

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 32 <400> 32

<210> 33<210> 33

<211> 90<211> 90

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 33 <400> 33

<210> 34<210> 34

<211> 90<211> 90

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 34 <400> 34

<210> 35<210> 35

<211> 82<211> 82

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 35 <400> 35

<210> 36<210> 36

<211> 82<211> 82

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 36 <400> 36

<210> 37<210> 37

<211> 10<211> 10

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 37 <400> 37

<210> 38<210> 38

<211> 6<211> 6

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 38 <400> 38

<210> 39<210> 39

<211> 8<211> 8

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 39 <400> 39

<210> 40<210> 40

<211> 7<211> 7

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 40 <400> 40

<210> 41<210> 41

<211> 6<211> 6

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 41 <400> 41

<210> 42<210> 42

<211> 12<211> 12

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 42 <400> 42

<210> 43<210> 43

<211> 11<211> 11

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> 化學合成寡核苷酸<223> Chemically synthesized oligonucleotide

<400> 43 <400> 43

<210> 44<210> 44

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> primer<223> primer

<400> 44 <400> 44

<210> 45<210> 45

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> primer<223> primer

<400> 45 <400> 45

<210> 46<210> 46

<211> 21<211> 21

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> primer<223> primer

<400> 46 <400> 46

<210> 47<210> 47

<211> 27<211> 27

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> primer<223> primer

<400> 47 <400> 47

<210> 48<210> 48

<211> 27<211> 27

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> primer<223> primer

<400> 48 <400> 48

<210> 49<210> 49

<211> 63<211> 63

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> primer<223> primer

<400> 49 <400> 49

<210> 50<210> 50

<211> 69<211> 69

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> intron<223> intron

<400> 50 <400> 50

<210> 51<210> 51

<211> 73<211> 73

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> intron<223> intron

<400> 51 <400> 51

<210> 52<210> 52

<211> 68<211> 68

<212> RNA<212> RNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> intron<223> intron

<400> 52 <400> 52

<210> 53<210> 53

<211> 62<211> 62

<212> DNA<212> DNA

<213> 人造序列<213> Artificial sequence

<220><220>

<223> intron<223> intron

<400> 53 <400> 53

Claims (55)

一種生成至少一不具癌細胞特性且不具有腫瘤形成之傾向的多能性幹細胞之方法,其包含以下步驟:(a)建構一重組核酸組成物,其能夠被傳送、轉錄及處理成至少一個基因靜默效應子,該基因靜默效應子包含一SEQ.ID.NO.3所示序列,與mir-302分享超過91%同源性,且抑制複數個由mir-302所標的之細胞基因的表現;其中該基因靜默效應子的表現量高於人類胚胎幹細胞中mir-302的表現量,且該些基因之一為CDK2基因;(b)以該重組核酸組成物來處理一細胞基質。 A method of producing at least one pluripotent stem cell having no cancer cell characteristics and having no tendency to form a tumor, comprising the steps of: (a) constructing a recombinant nucleic acid composition capable of being transmitted, transcribed, and processed into at least one gene The silent effector, the gene silencing effector comprises a sequence of SEQ.ID.NO.3, shares more than 91% homology with mir-302, and inhibits the expression of a plurality of cellular genes designated by mir-302; Wherein the expression level of the gene silencing effector is higher than the expression level of mir-302 in human embryonic stem cells, and one of the genes is a CDK2 gene; (b) treating the cell matrix with the recombinant nucleic acid composition. 如請求項1之方法,其中該細胞係選自一哺乳動物細胞、一人類細胞、一正常體細胞、一患病體細胞、一腫瘤細胞、一癌症細胞、一人類頭髮毛囊細胞、一人類皮膚細胞、及其組合細胞。 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 body cell, a tumor cell, a cancer cell, a human hair follicle cell, and a human skin. Cells, and combinations thereof. 如請求項1之方法,其中該細胞基質表現複數個由mir-302所標的之細胞基因。 The method of claim 1, wherein the cell matrix exhibits a plurality of cellular genes designated by mir-302. 如請求項1之方法,其中處理該細胞基質之該步驟係在由mir-302所標的之該等細胞基因受到抑制的一條件下進行。 The method of claim 1, wherein the step of treating the cell matrix is carried out under conditions in which the cellular genes identified by mir-302 are inhibited. 如請求項1之方法,其中該重組核酸組成物包括至少一個重組內含子,其編碼與mir-302分享超過91%同源性之一序列。 The method of claim 1, wherein the recombinant nucleic acid composition comprises at least one recombinant intron encoding a sequence that shares more than 91% homology with mir-302. 如請求項1之方法,其中該重組核酸組成物包括一載體,其係選自質體、病毒載體、反轉位子、及其組合。 The method of claim 1, wherein the recombinant nucleic acid composition comprises a vector selected from the group consisting of a plastid, a viral vector, an inverted position, and combinations thereof. 如請求項1之方法,其中該重組核酸組成物包括一藥物可誘發之基因表現啟動子。 The method of claim 1, wherein the recombinant nucleic acid composition comprises a drug-inducible gene expression promoter. 如請求項7之方法,其中該藥物可誘發之基因表現啟動子係以一四環黴素衍生物或同等物來控制。 The method of claim 7, wherein the drug-inducible gene expression promoter is controlled by a tetracycline derivative or equivalent. 如請求項8之方法’其中該四環黴素衍生物或同等物係選自G418、四環黴素、去氧羥、新黴素、安比西林、康黴素、其等衍生物、及其組合。 The method of claim 8 wherein the tetracycline derivative or equivalent is selected from the group consisting of G418, tetracycline, deoxyhydroxyl, neomycin, ampicillin, ketomycin, derivatives thereof, and the like combination. 如請求項1之方法,其中該重組核酸組成物包括一Tet-OnTet-Off 基因表現系統。The method of claim 1, wherein the recombinant nucleic acid composition comprises a Tet-On or Tet-Off gene expression system. 如請求項1之方法,其中該重組核酸組成物包括:一五端供體剪接位、一內含子插入位、一分支點基序、一多嘧啶段、及一三端受體剪接位。 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 polypyrimidine segment, and a tri-terminal acceptor splice site. 如請求項11之方法,其中該重組核酸組成物係由一程序形成,該程序係選自化學合成、核苷酸重組、基因工程、及其組合。 The method of claim 11, wherein the recombinant nucleic acid composition is formed by a program selected from the group consisting of chemical synthesis, nucleotide recombination, genetic engineering, and combinations thereof. 如請求項12之方法,其中該五端供體剪接位包括一SEQ.ID.NO.4序列。 The method of claim 12, wherein the five-terminal donor splice site comprises a sequence of SEQ.ID.NO.4. 如請求項12之方法,其中該五端供體剪接位係包括5’-GTAAG-3’。 The method of claim 12, wherein the five-terminal donor splice site comprises 5'-GTAAG-3'. 如請求項12之方法,其中該分支點基序包括一SEQ.ID.NO.6序列。 The method of claim 12, wherein the branch point motif comprises a sequence of SEQ.ID.NO.6. 如請求項12之方法,其中該分支點基序包括5’-TACTAAC-3’。 The method of claim 12, wherein the branch point motif comprises 5'-TACTAAC-3'. 如請求項12之方法,其中該多嘧啶段包括一SEQ.ID.NO.7序列或一SEQ.ID.NO.8序列。 The method of claim 12, wherein the polypyrimidine segment comprises a SEQ.ID.NO.7 sequence or a SEQ.ID.NO.8 sequence. 如請求項12之方法,其中該三端受體剪接位包括一SEQ.ID.NO.5序列。 The method of claim 12, wherein the tripartite receptor splice site comprises a SEQ.ID.NO.5 sequence. 如請求項12之方法,其中該三端受體剪接位係包括5’-CTGCAG-3’。 The method of claim 12, wherein the tripartite receptor splice site comprises 5'-CTGCAG-3'. 如請求項11之方法,其中該內含子插入位包括與mir-302分享超過91%同源性之該基因靜默效應子。 The method of claim 11, wherein the intron insertion site comprises a gene silencing effector that shares more than 91% homology with mir-302. 如請求項1之方法,其中該重組核酸組成物進一步包括複數個外顯子,該等外顯子係選自螢光蛋白質標記基因、螢光酵素基因、lac-Z乳糖表現基因、胚胎幹細胞標記基因、病毒基因、細菌基因、細胞標記基因、跳躍基因、轉位子、及其組合。 The method of claim 1, wherein the recombinant nucleic acid composition 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, and an embryonic stem cell marker. Genes, viral genes, bacterial genes, cell marker genes, skip genes, transposons, and combinations thereof. 如請求項1之方法,其中該基因靜默效應子包含一SEQ.ID.NO.1序列或一SEQ.ID.NO.2序列。 The method of claim 1, wherein the gene silencing effector comprises a SEQ.ID.NO.1 sequence or a SEQ.ID.NO.2 sequence. 如請求項1之方法,其中該基因靜默效應子係一重組類髮夾型RNA,其包括一SEQ.ID.NO.9序列。 The method of claim 1, wherein the gene silencing effector is a recombinant hairpin RNA comprising a SEQ.ID.NO.9 sequence. 如請求項1之方法,其中該基因靜默效應子係一重組核酸序列,其係以一SEQ.ID.NO.27序列及一SEQ.ID.NO.28序列之雜合物形成。 The method of claim 1, wherein the gene silencing effector is a recombinant nucleic acid sequence formed by a SEQ.ID.NO.27 sequence and a SEQ.ID.NO.28 sequence hybrid. 如請求項1之方法,其中該基因靜默效應子包括一重組RNA,其序列包含選自於一SEQ.ID.NO.9序列、一SEQ.ID.NO.10序列、一SEQ.ID.NO.11序列、一SEQ.ID.NO.12序列、以及一SEQ.ID.NO.13序列所組成之群組。 The method of claim 1, wherein the gene silencing effector comprises a recombinant RNA, the sequence comprising a sequence selected from the group consisting of a SEQ.ID.NO.9, a sequence of SEQ.ID.NO.10, and a SEQ.ID.NO A group consisting of a .11 sequence, a SEQ.ID.NO.12 sequence, and a SEQ.ID.NO.13 sequence. 如請求項1之方法,其中該基因靜默效應子係一重組核酸序列,該重組核酸序列係以選自一SEQ.ID.NO.29序列及一SEQ.ID.NO.30序列之雜合物、一SEQ.ID.NO.31序列及一SEQ.ID.NO.32序列之雜合物、一SEQ.ID.NO.33序列及一SEQ.ID.NO.34序列之雜合物、以及一SEQ.ID.NO.35序列及一SEQ.ID.NO.36序列之雜合物所組成之群組接合鏈結形成。 The method of claim 1, wherein the gene silencing effector is a recombinant nucleic acid sequence which is a hybrid selected from the group consisting of a sequence of SEQ.ID.NO.29 and a sequence of SEQ.ID.NO.30. a SEQ.ID.NO.31 sequence and a SEQ.ID.NO.32 sequence hybrid, a SEQ.ID.NO.33 sequence and a SEQ.ID.NO.34 sequence hybrid, and A group junction linkage consisting of a SEQ.ID.NO.35 sequence and a SEQ.ID.NO.36 sequence hybrid is formed. 如請求項1之方法,其中該重組核酸組成物係選自一四環黴素反應元素、一病毒或一第二型RNA聚合酶啟動子(Pol-II)、或以上兩者、一Kozak一致轉譯起始位、聚腺苷酸化訊息、複數個限制/選殖位、及其組合。 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 Kozak consistent Translation initiation sites, polyadenylation messages, multiple restriction/selection sites, and combinations thereof. 如請求項1之方法,其中該重組核酸組成物係選自一pUC複製來源(origin of replication)、用來表現在複製勝任原核生物 細胞中之至少一個抗生素抗藥性基因的一SV40早熟啟動子、用來在哺乳動物細胞中一可選擇的SV40複製來源、及其組合。 The method of claim 1, wherein the recombinant nucleic acid composition is selected from a pUC origin of replication for expression in a competent prokaryotes An SV40 precocious promoter of at least one antibiotic resistance gene in a cell, a selectable source of SV40 replication in mammalian cells, and combinations thereof. 如請求項1之方法,其中該重組核酸組成物係以一基因傳送方法引介至該細胞中,該基因傳送方法係選自微脂體轉染、化學轉染法、基因轉殖DNA重組、病毒感染、轉位子插入、跳躍基因插入、顯微注射、電穿孔法、基因槍穿透、及其組合。 The method of claim 1, wherein the recombinant nucleic acid composition is introduced into the cell by a gene delivery method selected from the group consisting of liposome transfection, chemical transfection, gene transfer DNA recombination, virus Infection, transposition insertion, skip gene insertion, microinjection, electroporation, gene gun penetration, and combinations thereof. 如請求項1之方法,其中該靜默效應子係以一細胞內機制釋放,該機制係選自RNA剪接、外體處理、無義介導降解、及其組合。 The method of claim 1, wherein the silent effector is released by an intracellular mechanism selected from the group consisting of RNA splicing, exosome treatment, nonsense mediated degradation, and combinations thereof. 如請求項1之方法,其中該多能性幹細胞表現一胚胎幹細胞標記Oct3/4。 The method of claim 1, wherein the pluripotent stem cell exhibits an embryonic stem cell marker Oct3/4. 如請求項1之方法,其中該多能性幹細胞係在一無哺乳細胞之條件下培養。 The method of claim 1, wherein the pluripotent stem cell line is cultured in the absence of a mammalian cell. 如請求項1之方法,其中該多能性幹細胞分化成類生殖細胞系細胞。 The method of claim 1, wherein the pluripotent stem cells differentiate into germ cell line-like cells. 如請求項1之方法,其中該多能性幹細胞分化成一類精原細胞。 The method of claim 1, wherein the pluripotent stem cells differentiate into a type of spermatogonia. 如請求項1之方法,其中該多能性幹細胞分化成一正常體細胞。 The method of claim 1, wherein the pluripotent stem cells differentiate into a normal somatic cell. 如請求項1之方法,其中該多能性幹細胞分化成一纖維組織母細胞。 The method of claim 1, wherein the pluripotent stem cells differentiate into a fibrous tissue mother cell. 如請求項1之方法,其中該多能性幹細胞分化成一軟骨細胞。 The method of claim 1, wherein the pluripotent stem cells differentiate into a chondrocyte. 如請求項1之方法,其中該多能性幹細胞係使用mir-302微型核醣核酸及Oct3/4作為標記來選擇性地分離。 The method of claim 1, wherein the pluripotent stem cell line is selectively isolated using mir-302 miniribonucleic acid and Oct3/4 as a label. 一種用來生成至少一不具癌細胞特性且不具有腫瘤形成之傾向的多能性幹細胞之重組核酸組成物,其包含:至少一個內含子,其編碼一基因靜默效應子,該基因靜默效應子包含一SEQ.ID.NO.3所示序列,與mir-302分享超過91%同源性;其中該內含子在該細胞中被剪接出來以誘發mir-302-介導基因靜默效應,抑制複數個細胞基因的表現;其中該基因靜默效應子的表現量高於人類胚胎幹細胞中mir-302的表現量,且該些基因之一為CDK2基因。 A recombinant nucleic acid composition for producing at least one pluripotent stem cell having no cancer cell characteristics and having no tendency to form tumors, comprising: at least one intron encoding a gene silencing effector, the gene silencing effector Containing a sequence of SEQ.ID.NO.3, sharing more than 91% homology with mir-302; wherein the intron is spliced out in the cell to induce mir-302-mediated gene silencing effect, inhibition The expression of a plurality of cellular genes; wherein the expression of the silencing effector of the gene is higher than the expression of mir-302 in human embryonic stem cells, and one of the genes is the CDK2 gene. 如請求項39之組成物,其中該重組核酸組成物係以一程序形成,該程序係選自化學合成、核苷酸重組、基因工程、及其組合。 The composition of claim 39, wherein the recombinant nucleic acid composition is formed by a process selected from the group consisting of chemical synthesis, nucleotide recombination, genetic engineering, and combinations thereof. 如請求項39之組成物,其中該內含子包含:(a)一內含子插入位,其編碼與mir-302分享超過91%同源性之該基因靜默效應子; (b)一五端供體剪接位及一三端受體剪接位;(c)一分支點基序;及(d)一多嘧啶段。 The composition of claim 39, wherein the intron comprises: (a) an intron insertion site encoding a gene silencing effector that shares more than 91% homology with mir-302; (b) a five-terminal donor splice site and a three-terminal acceptor splice site; (c) a branch point motif; and (d) a polypyrimidine segment. 如請求項41之組成物,其中該內含子插入位包括一類髮夾型核酸序列,其具有一包含一SEQ.ID.NO.1序列或一SEQ.ID.NO.2序列的一幹環結構。 The composition of claim 41, wherein the intron insertion site comprises a hairpin-type nucleic acid sequence having a dry loop comprising a sequence of SEQ.ID.NO.1 or a sequence of SEQ.ID.NO.2 structure. 如請求項41之組成物,其中該內含子插入位包括一類髮夾型先驅微型核醣核酸(pre-miRNA)序列,其含有選自於一SEQ.ID.NO.9序列、一SEQ.ID.NO.10序列、一SEQ.ID.NO.11序列、一SEQ.ID.NO.12序列以及一SEQ.ID.NO.13序列所組成之群組。 The composition of claim 41, 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.NO.9, a SEQ.ID A group consisting of a .NO.10 sequence, a SEQ.ID.NO.11 sequence, a SEQ.ID.NO.12 sequence, and a SEQ.ID.NO.13 sequence. 如請求項41之組成物,其中該分支點基序包括一腺核苷(A)核苷酸,其位在包含一SEQ.ID.NO.6序列的一核酸序列內。 The composition of claim 41, wherein the branch point motif comprises a nucleotide of adenosine (A) which is located within a nucleic acid sequence comprising a sequence of SEQ. ID. NO. 如請求項41之組成物,其中該分支點基序包括一腺核苷(A)核苷酸,其位在包含至少一個5’-TACTAAC-3’之寡核苷酸基序的一核酸序列內。 The composition of claim 41, wherein the branch point motif comprises a nucleotide of adenosine (A), which is located in a nucleic acid sequence comprising an oligonucleotide motif of at least one 5'-TACTAAC-3' Inside. 如請求項41之組成物,其中該多嘧啶段包括一高T或C含量之核酸序列,其包含一SEQ.ID.NO.7序列或一SEQ.ID.NO.8序列。 The composition of claim 41, wherein the polypyrimidine segment comprises a high T or C content nucleic acid sequence comprising a SEQ.ID.NO.7 sequence or a SEQ.ID.NO.8 sequence. 如請求項41之組成物,其中該五端供體剪接位為一核酸序列,其包含一SEQ.ID.NO.4序列。 The composition of claim 41, wherein the five-terminal donor splice site is a nucleic acid sequence comprising a sequence of SEQ.ID.NO.4. 如請求項41之組成物,其中該五端供體剪接位為一核酸序列,其包含5’-GTAAG-3’。 The composition of claim 41, wherein the five-terminal donor splice site is a nucleic acid sequence comprising 5'-GTAAG-3'. 如請求項41之組成物,其中該三端受體剪接位為一核酸序列,其包含一SEQ.ID.NO.5序列。 The composition of claim 41, wherein the triad receptor splice site is a nucleic acid sequence comprising a sequence of SEQ.ID.NO.5. 如請求項41之組成物,其中該三端受體剪接位為一核酸序列,其包含5’-CTGCAG-3’。 The composition of claim 41, wherein the triad receptor splice site is a nucleic acid sequence comprising 5'-CTGCAG-3'. 如請求項39之組成物,其中該基因靜默效應子包括一核酸序列,且含有一SEQ.ID.NO.3序列。 The composition of claim 39, wherein the gene silencing effector comprises a nucleic acid sequence and comprises a sequence of SEQ.ID.NO.3. 如請求項39之組成物,其中該基因靜默效應子包括一核酸序列,其包含選自於一SEQ.ID.NO.10序列、一SEQ.ID.NO.11序列、一SEQ.ID.NO.12序列,及一SEQ.ID.NO.13序列所組成之群組。 The composition of claim 39, wherein the gene silencing effector comprises a nucleic acid sequence comprising a sequence selected from the group consisting of a SEQ.ID.NO.10, a sequence of SEQ.ID.NO.11, and a SEQ.ID.NO The .12 sequence, and a group of SEQ.ID.NO.13 sequences. 如請求項39之組成物,其中該基因靜默效應子包括一重組核酸序列,該重組核酸序列係以選自一SEQ.ID.NO.29序列及一SEQ.ID.NO.30序列之雜合物、一SEQ.ID.NO.31序列及一SEQ.ID.NO.32序列之雜合物、一SEQ.ID.NO.33序列及一SEQ.ID.NO.34序列之雜合物、以及一SEQ.ID.NO.35序列及一SEQ.ID.NO.36序列之雜合物所組成之群組接合鏈結形成。 The composition of claim 39, wherein the gene silencing effector comprises a recombinant nucleic acid sequence which is heterozygous for a sequence selected from the group consisting of a SEQ.ID.NO.29 sequence and a SEQ.ID.NO.30 sequence. a SEQ.ID.NO.31 sequence and a SEQ.ID.NO.32 sequence hybrid, a SEQ.ID.NO.33 sequence and a SEQ.ID.NO.34 sequence hybrid, And a group junction linkage formed by a SEQ.ID.NO.35 sequence and a SEQ.ID.NO.36 sequence hybrid. 如請求項39之組成物,其中該基因靜默效應子包括一核酸序列,其包含一SEQ.ID.NO.9序列。 The composition of claim 39, wherein the gene silencing effector comprises a nucleic acid sequence comprising a sequence of SEQ.ID.NO.9. 如請求項39之組成物,其中該基因靜默效應子包括一重組核酸序列,其以一SEQ.ID.NO.27序列或一SEQ.ID.NO.28序列之一雜合物來形成。 The composition of claim 39, wherein the gene silencing effector comprises a recombinant nucleic acid sequence formed as a SEQ.ID.NO.27 sequence or a SEQ.ID.NO.28 sequence hybrid.
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