PIWIL1基因启动子调控的特异表达Cre重组酶的转基因猪的构建
|
摘要
非编码小RNA (non-coding RNA, ncRNA)，主要包括siRNA (smallinterfering RNA)、miRNA(microRNA)和piRNA (PIWI-interacting RNA)三类，其中piRNA是科学家们持续关注的热点研究区域。2006年，piRNA被发现于动物（果蝇、小鼠、大鼠、人等）生殖细胞中，它是一类内源的新小分子非编码RNA。piRNA基因在整个基因组上几乎都有分布，但高度不连续，其长度集中在26～32nt，因其特异地与PIWI家族蛋白结合作用，所以piRNA才被称为PIWI-interacting RNA[1-4]。piRNA基因簇则主要分布于转座子、重复序列等区域，在染色体上的位置具有物种间保守性。piRNA主要表达于生殖细胞系，代表了一个独特小RNA通路，它能使生殖细胞转座子沉默。可能通过表观遗传调控及转录后调控等方式，参与生殖干细胞的自我维持和分化命运决定、减数分裂、精子形成等生殖相关过程。对维持生殖系DNA完整、抑制转座子转录、抑制翻译、参与异染色质的形成、执行表观遗传调控和生殖细胞发生等均有重要作用[5-7]。
Argonaute（AGO）蛋白家族主要分为两大亚家族，即类似于植物拟南芥Argonautel的AGO蛋白亚家族和类似果蝇PIWI的PIWI蛋白亚家族。人类AGO蛋白家族由8名成员组成，通过序列比较，它们分别是eIF2C/AGO亚家族的4个成员(EIF2C1/hAGO1，EIF2C2/hAGO2，EIF2C3/hAGO3和EIF2C4/hAGO4)，以及PIWI亚家族的4个成员(PIWIL1/HIWI，PIWIL2/HILI，PIWIL3和PIWIL4/HIWI2)[8]。eIF2C/AGO亚家族的4个成员在成人各个组织中包括睾丸、心脏、肾、胸腺等都有表达，而PIWI亚家族的4个成员主要在睾丸的精原细胞或精母细胞中表达。AGO蛋白亚族和PIWI蛋白亚族均能与非编码小RNA结合，在转录、转录后甚至翻译水平上对靶基因及蛋白的调节发挥关键作用[9-10]。Argonaute蛋白家族是RNA沉默途径的关键因子[11]，而PIWI成员则伴随着piRNA的生物发生和生物学功能行使等一系列过程[5]，包括参与沉默转录基因过程、维持生殖系和干细胞功能以及调节翻译和mRNA的稳定性等。
虽然科学家们已经对于piRNA展开非常广泛的研究，并且已经取得了阶段性的成果，但仍然存在许多问题亟待我们进一步探索，如piRNA和PIWI蛋白相互作用的机制及调控作用等。有研究报道，在非生殖器官中PIWI蛋白也有高表达，提示piRNA在配子发育的过程中起重要作用外，在其他组织中可能也具有重要的调控作用。
本实验的目的是建立PIWIL1基因启动子调控的特异性表达Cre重组酶的转基因猪，为后续在猪体内实现时空特异性缺失PIWI家族蛋白同一时空表达的基因，为研究piRNA的功能、揭示piRNA与PIWI蛋白的相互作用机制提供可靠的动物平台奠定基础。
本实验以上述研究成果为背景，将PIWIL1启动子连接于猪源Cre重组酶的表达载体pET28a-MHC-Cre-BGHpo1yA-FRT2neor上，获得PIWIL1特异性启动Cre重组酶表达的pPIWIL1-Cre载体。转染小型猪胎儿成纤维细胞，药物筛选获得细胞阳性克隆后进行核移植和胚胎移植，最终获得PIWIL1特异性表达Cre重组酶的转基因猪，为研究piRNA的功能、揭示PIWI与PIWI蛋白的相互作用机制提供可靠的动物平台奠定基础。
Non-coding RNA mainly including siRNA (small interfering RNA)、miRNA(microRNA) and piRNA (PIWI-interacting RNA), piRNA is the hot researcharea which scientists continue pay attention to. piRNA was found ingerm cells ofanimals (Drosophila melanogaster, rats, mice and human), it is a kind of novel smallmolecules of endogenous non-coding RNA. piRNAs are found in clusters throughoutthe genome,but it is highly discontinuous, the length of a piRNA is, by definition,between26and32nucleotides, because of its specifical interaction with pIWIproteins, so piRNA is also known as PIWI-interacting RNA.piRNA gene clusters aremainly distributed in the region of transposons and repeat sequences， thechromosomal location of species is conservatism. piRNA mainly expressed in thegerm cell line represents a unique small RNA pathway, which enables the germ cellstransposon silencing. They may be involved in germline stem cell self-sustaining anddifferentiation fate decision, meiosis, sperm formation and other reproductive processthrough epigenetic and post-transcriptional regulation.They play an important role inMaintaining germline DNA integrity, inhibiting the transcription of the transposon,inhibiting translation, involving in heterochromatin formation, implementing ofepigenetic regulation and germ cells cytogenesis.
The Argonaute (AGO) protein family is divided into two major subfamilies,Theyare the AGO subfamily similar to Arabidopsis thaliana Argonautel and PIWI proteinsubfamily similar to Drosophila PIWI. There are eight members in Human AGOprotein family by sequence comparing, they are4eIF2C/AGO subfamily members(EIF2C1/hAGO1, EIF2C2/hAGO2EIF2C3/hAGO3and EIF2C4/hAGO4), as well asthe four members of PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, andPIWIL4/HIWI2)[8]. The four members of eIF2C/AGO subfamily are expressed invarious tissues in the adult，including testis, heart, kidney, thymus.and four membersof the PIWI subfamily are expressed in the testis of spermatogonia or spermatocytes.
AGO protein subfamily and PIWI protein subfamily can bind with non-coding small RNA, in transcription, and play a key role in regulating target genes andproteins in transcription or translation level [9-10]. The Argonaute protein family arethe key factors of the RNA silencing pathway [11], PIWI members are along with thepiRNA in the biogenesis and the exercise of the biological functions of the processand silence transcription of genes, including participation in the process ofmaintaining the germ system and stem cell function and regulation of translation andmRNA stability.
Although there are extensive research for the piRNA and we have achievedinitial results, there are still many problems to make further exploring, such as piRNAand PIWI protein interaction mechanism and regulation. Some studies have reportedthat PIWI protein has a high expression in non-reproductive organs, suggesting thatpiRNA may also have an important role in the regulation in other tissues besides playan important role in the process of gamete development.
In this experiment, the background is the above-mentioned researchresults.Amplify the promoter of pig PIWIL1gene by PCR. Replace the MHCpromoter of pig specific Cre recombinase expression plasmid (pET28a-MHC-Cre-BGHpo1yA-FRT2neor) with PIWIL1promoter and specific Crerecombinase plasmid(pPIWIL1-Cre) was abtained. Transfect the plasmid tomini-pig fetal fibroblast cells and screen for positive clones with drug and donuclear transfer. We generate transgenic pigs that specific express Crerecombinase successfully, lay a foundation for researching the function ofpiRNA, revealing the piRNA and PIWI protein interaction mechanism whichproviding a reliable animal platform.
Argonaute（AGO）蛋白家族主要分为两大亚家族，即类似于植物拟南芥Argonautel的AGO蛋白亚家族和类似果蝇PIWI的PIWI蛋白亚家族。人类AGO蛋白家族由8名成员组成，通过序列比较，它们分别是eIF2C/AGO亚家族的4个成员(EIF2C1/hAGO1，EIF2C2/hAGO2，EIF2C3/hAGO3和EIF2C4/hAGO4)，以及PIWI亚家族的4个成员(PIWIL1/HIWI，PIWIL2/HILI，PIWIL3和PIWIL4/HIWI2)[8]。eIF2C/AGO亚家族的4个成员在成人各个组织中包括睾丸、心脏、肾、胸腺等都有表达，而PIWI亚家族的4个成员主要在睾丸的精原细胞或精母细胞中表达。AGO蛋白亚族和PIWI蛋白亚族均能与非编码小RNA结合，在转录、转录后甚至翻译水平上对靶基因及蛋白的调节发挥关键作用[9-10]。Argonaute蛋白家族是RNA沉默途径的关键因子[11]，而PIWI成员则伴随着piRNA的生物发生和生物学功能行使等一系列过程[5]，包括参与沉默转录基因过程、维持生殖系和干细胞功能以及调节翻译和mRNA的稳定性等。
虽然科学家们已经对于piRNA展开非常广泛的研究，并且已经取得了阶段性的成果，但仍然存在许多问题亟待我们进一步探索，如piRNA和PIWI蛋白相互作用的机制及调控作用等。有研究报道，在非生殖器官中PIWI蛋白也有高表达，提示piRNA在配子发育的过程中起重要作用外，在其他组织中可能也具有重要的调控作用。
本实验的目的是建立PIWIL1基因启动子调控的特异性表达Cre重组酶的转基因猪，为后续在猪体内实现时空特异性缺失PIWI家族蛋白同一时空表达的基因，为研究piRNA的功能、揭示piRNA与PIWI蛋白的相互作用机制提供可靠的动物平台奠定基础。
本实验以上述研究成果为背景，将PIWIL1启动子连接于猪源Cre重组酶的表达载体pET28a-MHC-Cre-BGHpo1yA-FRT2neor上，获得PIWIL1特异性启动Cre重组酶表达的pPIWIL1-Cre载体。转染小型猪胎儿成纤维细胞，药物筛选获得细胞阳性克隆后进行核移植和胚胎移植，最终获得PIWIL1特异性表达Cre重组酶的转基因猪，为研究piRNA的功能、揭示PIWI与PIWI蛋白的相互作用机制提供可靠的动物平台奠定基础。
Non-coding RNA mainly including siRNA (small interfering RNA)、miRNA(microRNA) and piRNA (PIWI-interacting RNA), piRNA is the hot researcharea which scientists continue pay attention to. piRNA was found ingerm cells ofanimals (Drosophila melanogaster, rats, mice and human), it is a kind of novel smallmolecules of endogenous non-coding RNA. piRNAs are found in clusters throughoutthe genome,but it is highly discontinuous, the length of a piRNA is, by definition,between26and32nucleotides, because of its specifical interaction with pIWIproteins, so piRNA is also known as PIWI-interacting RNA.piRNA gene clusters aremainly distributed in the region of transposons and repeat sequences， thechromosomal location of species is conservatism. piRNA mainly expressed in thegerm cell line represents a unique small RNA pathway, which enables the germ cellstransposon silencing. They may be involved in germline stem cell self-sustaining anddifferentiation fate decision, meiosis, sperm formation and other reproductive processthrough epigenetic and post-transcriptional regulation.They play an important role inMaintaining germline DNA integrity, inhibiting the transcription of the transposon,inhibiting translation, involving in heterochromatin formation, implementing ofepigenetic regulation and germ cells cytogenesis.
The Argonaute (AGO) protein family is divided into two major subfamilies,Theyare the AGO subfamily similar to Arabidopsis thaliana Argonautel and PIWI proteinsubfamily similar to Drosophila PIWI. There are eight members in Human AGOprotein family by sequence comparing, they are4eIF2C/AGO subfamily members(EIF2C1/hAGO1, EIF2C2/hAGO2EIF2C3/hAGO3and EIF2C4/hAGO4), as well asthe four members of PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, andPIWIL4/HIWI2)[8]. The four members of eIF2C/AGO subfamily are expressed invarious tissues in the adult，including testis, heart, kidney, thymus.and four membersof the PIWI subfamily are expressed in the testis of spermatogonia or spermatocytes.
AGO protein subfamily and PIWI protein subfamily can bind with non-coding small RNA, in transcription, and play a key role in regulating target genes andproteins in transcription or translation level [9-10]. The Argonaute protein family arethe key factors of the RNA silencing pathway [11], PIWI members are along with thepiRNA in the biogenesis and the exercise of the biological functions of the processand silence transcription of genes, including participation in the process ofmaintaining the germ system and stem cell function and regulation of translation andmRNA stability.
Although there are extensive research for the piRNA and we have achievedinitial results, there are still many problems to make further exploring, such as piRNAand PIWI protein interaction mechanism and regulation. Some studies have reportedthat PIWI protein has a high expression in non-reproductive organs, suggesting thatpiRNA may also have an important role in the regulation in other tissues besides playan important role in the process of gamete development.
In this experiment, the background is the above-mentioned researchresults.Amplify the promoter of pig PIWIL1gene by PCR. Replace the MHCpromoter of pig specific Cre recombinase expression plasmid (pET28a-MHC-Cre-BGHpo1yA-FRT2neor) with PIWIL1promoter and specific Crerecombinase plasmid(pPIWIL1-Cre) was abtained. Transfect the plasmid tomini-pig fetal fibroblast cells and screen for positive clones with drug and donuclear transfer. We generate transgenic pigs that specific express Crerecombinase successfully, lay a foundation for researching the function ofpiRNA, revealing the piRNA and PIWI protein interaction mechanism whichproviding a reliable animal platform.
引文
[1] GIRARD A, SACHIDANANDAM R, HANNON G J, et al. A germline-specificclass of small RNAs binds mammalian PIWI proteins [J]. Nature,2006,442(7099):199-202.
[2] ARAVIN A, GAIDATZIS D, PFEFFER S, et al. A novel class of small RNAs bindto MILI protein in mouse testes [J]. Nature,2006,442(7099):203-207.
[3] GRIVNA S T, BEYRET E, WANG Z, et al. A novel class of small RNAs in mousespermatogenic cells [J]. Genes Dev,2006,20(13):1709-1714.
[4] LAU N C, SETO A G, KIM J, et al. Characterization of the piRNA complex fromrat testes [J]. Science,2006,313(5785):363-367.
[5] BROWER-TOLAND B, FINDLEY S D, JIANG L, et al. Drosophila PIWIassociates with chromatin and interacts directly with HP1a [J]. Genes Dev,2007,21(18):2300-2311.
[6] ARAVIN A A, SACHIDANANDAM R, GIRARD A, et al. Developmentallyregulated piRNA clusters implicate MILI in transposon control [J]. Science,2007,316(5825):744-747.
[7] NISHIDA K M, SAITO K, MORI T, et al. Gene silencing mechanisms mediatedby Aubergine piRNA complexes in Drosophila male gonad [J]. RNA,2007,13(11):1911-1922.
[8] SASAKI T, SHIOHAMA A, MINOSHIMA S, et al. Identification of eightmembers of the Argonaute family in the human genome small star, filled [J].Genomics,2003,82(3):323-330.
[9] BANDYOPADHYAY S, MITRA R, MAULIK U, et al. Development of thehuman cancer microRNA network [J]. Silence,2010,1(1):6.
[10] BARANWAL S, ALAHARI S K. miRNA control of tumor cell invasion andmetastasis [J]. Int J Cancer,2010,126(6):1283-1290.
[11] KIM V N. Small RNAs just got bigger: PIWI-interacting RNAs (piRNAs) inmammalian testes [J]. Genes Dev,2006,20(15):1993-1997.
[12] CARMELL M A, XUAN Z, ZHANG M Q, et al. The Argonaute family: tentaclesthat reach into RNAi, developmental control, stem cell maintenance, andtumorigenesis [J]. Genes Dev,2002,16(21):2733-2742.
[13] RIVAS F V, TOLIA N H, SONG J J, et al. Purified Argonaute2and an siRNAform recombinant human RISC [J]. Nat Struct Mol Biol,2005,12(4):340-349.
[14] SAITO K, NISHIDA K M, MORI T, et al. Specific association of PIWI withrasiRNAs derived from retrotransposon and heterochromatic regions in theDrosophila genome [J]. Genes Dev,2006,20(16):2214-2222.
[15] COX D N, CHAO A, BAKER J, et al. A novel class of evolutionarily conservedgenes defined by pIWI are essential for stem cell self-renewal [J]. Genes Dev,1998,12(23):3715-3727.
[16] WATANABE T, TAKEDA A, TSUKIYAMA T, et al. Identification andcharacterization of two novel classes of small RNAs in the mouse germline:retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes [J].Genes Dev,2006,20(13):1732-1743.
[17] THOMSON T, LIN H. The biogenesis and function of PIWI proteins andpiRNAs: progress and prospect [J]. Annu Rev Cell Dev Biol,2009,25355-376.
[18] BOHMERT K, CAMUS I, BELLINI C, et al. AGO1defines a novel locus ofArabidopsis controlling leaf development [J]. EMBO J,1998,17(1):170-180.
[19] MEISTER G, TUSCHL T. Mechanisms of gene silencing by double-strandedRNA [J]. Nature,2004,431(7006):343-349.
[20] PETERS L, MEISTER G. Argonaute proteins: mediators of RNA silencing [J].Mol Cell,2007,26(5):611-623.
[21] LINGEL A, SIMON B, IZAURRALDE E, et al. Structure and nucleic-acidbinding of the Drosophila Argonaute2PAZ domain [J]. Nature,2003,426(6965):465-469.
[22] LINGEL A, SIMON B, IZAURRALDE E, et al. Nucleic acid3'-end recognitionby the Argonaute2PAZ domain [J]. Nat Struct Mol Biol,2004,11(6):576-577.
[23] MA J B, YE K, PATEL D J. Structural basis for overhang-specific smallinterfering RNA recognition by the PAZ domain [J]. Nature,2004,429(6989):318-322.
[24] SONG J J, LIU J, TOLIA N H, et al. The crystal structure of the Argonaute2PAZdomain reveals an RNA binding motif in RNAi effector complexes [J]. Nat StructBiol,2003,10(12):1026-1032.
[25] YAN K S, YAN S, FAROOQ A, et al. Structure and conserved RNA binding ofthe PAZ domain [J]. Nature,2003,426(6965):468-474.
[26] SONG J J, SMITH S K, HANNON G J, et al. Crystal structure of Argonaute andits implications for RISC slicer activity [J]. Science,2004,305(5689):1434-1437.
[27] YUAN Y R, PEI Y, MA J B, et al. Crystal structure of A. aeolicus argonaute, asite-specific DNA-guided endoribonuclease, provides insights intoRISC-mediated mRNA cleavage [J]. Mol Cell,2005,19(3):405-419.
[28] PARKER J S, ROE S M, BARFORD D. Crystal structure of a PIWI proteinsuggests mechanisms for siRNA recognition and slicer activity [J]. EMBO J,2004,23(24):4727-4737.
[29] LIU J, CARMELL M A, RIVAS F V, et al. Argonaute2is the catalytic engine ofmammalian RNAi [J]. Science,2004,305(5689):1437-1441.
[30] MEISTER G, LANDTHALER M, PATKANIOWSKA A, et al. HumanArgonaute2mediates RNA cleavage targeted by miRNAs and siRNAs [J]. MolCell,2004,15(2):185-197.
[31] MA J B, YUAN Y R, MEISTER G, et al. Structural basis for5'-end-specificrecognition of guide RNA by the A. fulgidus PIWI protein [J]. Nature,2005,434(7033):666-670.
[32] KIRIAKIDOU M, TAN G S, LAMPRINAKI S, et al. An mRNA m7G capbinding-like motif within human Ago2represses translation [J]. Cell,2007,129(6):1141-1151.
[33] HOCK J, MEISTER G. The Argonaute protein family [J]. Genome Biol,2008,9(2):210.
[34] JAKYMIW A, LIAN S, EYSTATHIOY T, et al. Disruption of GW bodiesimpairs mammalian RNA interference [J]. Nat Cell Biol,2005,7(12):1267-1274.
[35] EULALIO A, BEHM-ANSMANT I, IZAURRALDE E. P bodies: at thecrossroads of post-transcriptional pathways [J]. Nat Rev Mol Cell Biol,2007,8(1):9-22.
[36] EULALIO A, BEHM-ANSMANT I, SCHWEIZER D, et al. P-body formation isa consequence, not the cause, of RNA-mediated gene silencing [J]. Mol Cell Biol,2007,27(11):3970-3981.
[37] LEUNG A K, CALABRESE J M, SHARP P A. Quantitative analysis ofArgonaute protein reveals microRNA-dependent localization to stress granules[J]. Proc Natl Acad Sci U S A,2006,103(48):18125-18130.
[38] KOTAJA N, SASSONE-CORSI P. The chromatoid body: a germ-cell-specificRNA-processing centre [J]. Nat Rev Mol Cell Biol,2007,8(1):85-90.
[39] ZILBERMAN D, CAO X, JOHANSEN L K, et al. Role of ArabidopsisARGONAUTE4in RNA-directed DNA methylation triggered by inverted repeats[J]. Curr Biol,2004,14(13):1214-1220.
[40] CHEN P Y, MEISTER G. microRNA-guided posttranscriptional gene regulation[J]. Biol Chem,2005,386(12):1205-1218.
[41] NOTTROTT S, SIMARD M J, RICHTER J D. Human let-7a miRNA blocksprotein production on actively translating polyribosomes [J]. Nat Struct Mol Biol,2006,13(12):1108-1114.
[42] VASUDEVAN S, TONG Y, STEITZ J A. Switching from repression to activation:microRNAs can up-regulate translation [J]. Science,2007,318(5858):1931-1934.
[43] BEHM-ANSMANT I, REHWINKEL J, DOERKS T, et al. mRNA degradationby miRNAs and GW182requires both CCR4:NOT deadenylase and DCP1:DCP2decapping complexes [J]. Genes Dev,2006,20(14):1885-1898.
[44] DING L, SPENCER A, MORITA K, et al. The developmental timing regulatorAIN-1interacts with miRISCs and may target the argonaute protein ALG-1tocytoplasmic P bodies in C. elegans [J]. Mol Cell,2005,19(4):437-447.
[45] LIPPMAN Z, MARTIENSSEN R. The role of RNA interference inheterochromatic silencing [J]. Nature,2004,431(7006):364-370.
[46] PAL-BHADRA M, LEIBOVITCH B A, GANDHI S G, et al. Heterochromaticsilencing and HP1localization in Drosophila are dependent on the RNAimachinery [J]. Science,2004,303(5658):669-672.
[47] ARAVIN A A, KLENOV M S, VAGIN V V, et al. Dissection of a natural RNAsilencing process in the Drosophila melanogaster germ line [J]. Mol Cell Biol,2004,24(15):6742-6750.
[48] YIN H, LIN H. An epigenetic activation role of PIWI and a PIWI-associatedpiRNA in Drosophila melanogaster [J]. Nature,2007,450(7167):304-308.
[49] KURAMOCHI-MIYAGAWA S, KIMURA T, IJIRI T W, et al. Mili, amammalian member of pIWI family gene, is essential for spermatogenesis [J].Development,2004,131(4):839-849.
[50] HOCK J, WEINMANN L, ENDER C, et al. Proteomic and functional analysis ofArgonaute-containing mRNA-protein complexes in human cells [J]. EMBO Rep,2007,8(11):1052-1060.
[51] TAUBERT H, GREITHER T, KAUSHAL D, et al. Expression of the stem cellself-renewal gene HIWI and risk of tumour-related death in patients withsoft-tissue sarcoma [J]. Oncogene,2007,26(7):1098-1100.
[52] JIN P, ZARNESCU D C, CEMAN S, et al. Biochemical and genetic interactionbetween the fragile X mental retardation protein and the microRNA pathway [J].Nat Neurosci,2004,7(2):113-117.
[53] ILLMENSEE K, MAHOWALD A P. Transplantation of posterior polar plasm inDrosophila. Induction of germ cells at the anterior pole of the egg [J]. Proc NatlAcad Sci U S A,1974,71(4):1016-1020.
[54] MAHOWALD A P. Assembly of the Drosophila germ plasm [J]. Int Rev Cytol,2001,203187-213.
[55] SAFFMAN E E, LASKO P. Germline development in vertebrates andinvertebrates [J]. Cell Mol Life Sci,1999,55(8-9):1141-1163.
[56] MEGOSH H B, COX D N, CAMPBELL C, et al. The role of PIWI and themiRNA machinery in Drosophila germline determination [J]. Curr Biol,2006,16(19):1884-1894.
[57] BERNSTEIN E, CAUDY A A, HAMMOND S M, et al. Role for a bidentateribonuclease in the initiation step of RNA interference [J]. Nature,2001,409(6818):363-366.
[58] LIN H, SPRADLING A C. A novel group of pumilio mutations affects theasymmetric division of germline stem cells in the Drosophila ovary [J].Development,1997,124(12):2463-2476.
[59] HOUWING S, KAMMINGA L M, BEREZIKOV E, et al. A role for PIWI andpiRNAs in germ cell maintenance and transposon silencing in Zebrafish [J]. Cell,2007,129(1):69-82.
[60] CARMELL M A, GIRARD A, VAN DE KANT H J, et al. MIWI2is essential forspermatogenesis and repression of transposons in the mouse male germline [J].Dev Cell,2007,12(4):503-514.
[61] CHEN Y, PANE A, SCHUPBACH T. Cutoff and aubergine mutations result inretrotransposon upregulation and checkpoint activation in Drosophila [J]. CurrBiol,2007,17(7):637-642.
[62] DENG W, LIN H. mIWI, a murine homolog of pIWI, encodes a cytoplasmicprotein essential for spermatogenesis [J]. Dev Cell,2002,2(6):819-830.
[63] BATISTA P J, RUBY J G, CLAYCOMB J M, et al. PRG-1and21U-RNAsinteract to form the piRNA complex required for fertility in C. elegans [J]. MolCell,2008,31(1):67-78.
[64] NGUYEN T T, SAXENA A, BEVERIDGE T J. Effect of surfacelipopolysaccharide on the nature of membrane vesicles liberated from theGram-negative bacterium Pseudomonas aeruginosa [J]. J Electron Microsc(Tokyo),2003,52(5):465-469.
[65] GUNAWARDANE L S, SAITO K, NISHIDA K M, et al. A slicer-mediatedmechanism for repeat-associated siRNA5' end formation in Drosophila [J].Science,2007,315(5818):1587-1590.
[66] RO S, PARK C, SONG R, et al. Cloning and expression profiling oftestis-expressed piRNA-like RNAs [J]. RNA,2007,13(10):1693-1702.
[67] BRENNECKE J, ARAVIN A A, STARK A, et al. Discrete small RNA-generatingloci as master regulators of transposon activity in Drosophila [J]. Cell,2007,128(6):1089-1103.
[68] FANTI L, DORER D R, BERLOCO M, et al. Heterochromatin protein1bindstransgene arrays [J]. Chromosoma,1998,107(5):286-292.
[69] ARONICA L, BEDNENKO J, NOTO T, et al. Study of an RNA helicaseimplicates small RNA-noncoding RNA interactions in programmed DNAelimination in Tetrahymena [J]. Genes Dev,2008,22(16):2228-2241.
[70] GIRARD A, HANNON G J. Conserved themes in small-RNA-mediatedtransposon control [J]. Trends Cell Biol,2008,18(3):136-148.
[71] SAROT E, PAYEN-GROSCHENE G, BUCHETON A, et al. Evidence for apIWI-dependent RNA silencing of the gypsy endogenous retrovirus by theDrosophila melanogaster flamenco gene [J]. Genetics,2004,166(3):1313-1321.
[72] SHPIZ S, KWON D, UNEVA A, et al. Characterization of Drosophila telomericretroelement TAHRE: transcription, transpositions, and RNAi-based regulation ofexpression [J]. Mol Biol Evol,2007,24(11):2535-2545.
[73] GRIVNA S T, PYHTILA B, LIN H. MIWI associates with translationalmachinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis[J]. Proc Natl Acad Sci U S A,2006,103(36):13415-13420.
[74] SAUER B. Inducible gene targeting in mice using the Cre/lox system [J].Methods,1998,14:381-392.
[75] GU H, MARTH J D, ORBAN P C, et al. Deletion of a DNA polymerase betagene segment in T cells using cell type-specific gene targeting [J]. Science,1994,265:103-106.
[76] MICHEL G, YU Y, CHANG T, et al. Site-specific gene insertion mediated by aCre-loxP-carrying lentiviral vector [J]. Mol Ther,2010,18:1814-1821.
[77] SUEHIRO Y, KINOSHITA M, OKUYAMA T, et al. Transient and permanentgene transfer into the brain of the teleost fish medaka (Oryzias latipes) usinghuman adenovirus and the Cre-loxP system [J]. FEBS Lett,2010,584:3545-3549.
[78] LI L, PANG D, CHEN L, et al. Establishment of a transgenic pig fetal fibroblastreporter cell line for monitoring Cre recombinase activity [J]. DNA Cell Biol,2009,28:303-308.
[79] LI L, PANG D, WANG T, et al. Production of a reporter transgenic pig formonitoring Cre recombinase activity [J]. Biochem Biophys Res Commun,2009,382:232-235.
[80] CHEN L, LI L, PANG D, et al. Construction of transgenic swine with inducedexpressionof Cre recombinase [J]. Animal,2009,00:1-5.
[81] Lai, L.X., PRATHER, R.S. Production of cloned pigs by using somatic cells asdonors[J]. Cloning Stem Cells,2003(5):233–241.
[82] Liu Y, ROBERT F W. Thermal asymmetric interlaced PCR: automatableamplification and sequencing of insert end fragment from P1and YAC clones forchromosome walking. Genomics,1995(25):674681.
[2] ARAVIN A, GAIDATZIS D, PFEFFER S, et al. A novel class of small RNAs bindto MILI protein in mouse testes [J]. Nature,2006,442(7099):203-207.
[3] GRIVNA S T, BEYRET E, WANG Z, et al. A novel class of small RNAs in mousespermatogenic cells [J]. Genes Dev,2006,20(13):1709-1714.
[4] LAU N C, SETO A G, KIM J, et al. Characterization of the piRNA complex fromrat testes [J]. Science,2006,313(5785):363-367.
[5] BROWER-TOLAND B, FINDLEY S D, JIANG L, et al. Drosophila PIWIassociates with chromatin and interacts directly with HP1a [J]. Genes Dev,2007,21(18):2300-2311.
[6] ARAVIN A A, SACHIDANANDAM R, GIRARD A, et al. Developmentallyregulated piRNA clusters implicate MILI in transposon control [J]. Science,2007,316(5825):744-747.
[7] NISHIDA K M, SAITO K, MORI T, et al. Gene silencing mechanisms mediatedby Aubergine piRNA complexes in Drosophila male gonad [J]. RNA,2007,13(11):1911-1922.
[8] SASAKI T, SHIOHAMA A, MINOSHIMA S, et al. Identification of eightmembers of the Argonaute family in the human genome small star, filled [J].Genomics,2003,82(3):323-330.
[9] BANDYOPADHYAY S, MITRA R, MAULIK U, et al. Development of thehuman cancer microRNA network [J]. Silence,2010,1(1):6.
[10] BARANWAL S, ALAHARI S K. miRNA control of tumor cell invasion andmetastasis [J]. Int J Cancer,2010,126(6):1283-1290.
[11] KIM V N. Small RNAs just got bigger: PIWI-interacting RNAs (piRNAs) inmammalian testes [J]. Genes Dev,2006,20(15):1993-1997.
[12] CARMELL M A, XUAN Z, ZHANG M Q, et al. The Argonaute family: tentaclesthat reach into RNAi, developmental control, stem cell maintenance, andtumorigenesis [J]. Genes Dev,2002,16(21):2733-2742.
[13] RIVAS F V, TOLIA N H, SONG J J, et al. Purified Argonaute2and an siRNAform recombinant human RISC [J]. Nat Struct Mol Biol,2005,12(4):340-349.
[14] SAITO K, NISHIDA K M, MORI T, et al. Specific association of PIWI withrasiRNAs derived from retrotransposon and heterochromatic regions in theDrosophila genome [J]. Genes Dev,2006,20(16):2214-2222.
[15] COX D N, CHAO A, BAKER J, et al. A novel class of evolutionarily conservedgenes defined by pIWI are essential for stem cell self-renewal [J]. Genes Dev,1998,12(23):3715-3727.
[16] WATANABE T, TAKEDA A, TSUKIYAMA T, et al. Identification andcharacterization of two novel classes of small RNAs in the mouse germline:retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes [J].Genes Dev,2006,20(13):1732-1743.
[17] THOMSON T, LIN H. The biogenesis and function of PIWI proteins andpiRNAs: progress and prospect [J]. Annu Rev Cell Dev Biol,2009,25355-376.
[18] BOHMERT K, CAMUS I, BELLINI C, et al. AGO1defines a novel locus ofArabidopsis controlling leaf development [J]. EMBO J,1998,17(1):170-180.
[19] MEISTER G, TUSCHL T. Mechanisms of gene silencing by double-strandedRNA [J]. Nature,2004,431(7006):343-349.
[20] PETERS L, MEISTER G. Argonaute proteins: mediators of RNA silencing [J].Mol Cell,2007,26(5):611-623.
[21] LINGEL A, SIMON B, IZAURRALDE E, et al. Structure and nucleic-acidbinding of the Drosophila Argonaute2PAZ domain [J]. Nature,2003,426(6965):465-469.
[22] LINGEL A, SIMON B, IZAURRALDE E, et al. Nucleic acid3'-end recognitionby the Argonaute2PAZ domain [J]. Nat Struct Mol Biol,2004,11(6):576-577.
[23] MA J B, YE K, PATEL D J. Structural basis for overhang-specific smallinterfering RNA recognition by the PAZ domain [J]. Nature,2004,429(6989):318-322.
[24] SONG J J, LIU J, TOLIA N H, et al. The crystal structure of the Argonaute2PAZdomain reveals an RNA binding motif in RNAi effector complexes [J]. Nat StructBiol,2003,10(12):1026-1032.
[25] YAN K S, YAN S, FAROOQ A, et al. Structure and conserved RNA binding ofthe PAZ domain [J]. Nature,2003,426(6965):468-474.
[26] SONG J J, SMITH S K, HANNON G J, et al. Crystal structure of Argonaute andits implications for RISC slicer activity [J]. Science,2004,305(5689):1434-1437.
[27] YUAN Y R, PEI Y, MA J B, et al. Crystal structure of A. aeolicus argonaute, asite-specific DNA-guided endoribonuclease, provides insights intoRISC-mediated mRNA cleavage [J]. Mol Cell,2005,19(3):405-419.
[28] PARKER J S, ROE S M, BARFORD D. Crystal structure of a PIWI proteinsuggests mechanisms for siRNA recognition and slicer activity [J]. EMBO J,2004,23(24):4727-4737.
[29] LIU J, CARMELL M A, RIVAS F V, et al. Argonaute2is the catalytic engine ofmammalian RNAi [J]. Science,2004,305(5689):1437-1441.
[30] MEISTER G, LANDTHALER M, PATKANIOWSKA A, et al. HumanArgonaute2mediates RNA cleavage targeted by miRNAs and siRNAs [J]. MolCell,2004,15(2):185-197.
[31] MA J B, YUAN Y R, MEISTER G, et al. Structural basis for5'-end-specificrecognition of guide RNA by the A. fulgidus PIWI protein [J]. Nature,2005,434(7033):666-670.
[32] KIRIAKIDOU M, TAN G S, LAMPRINAKI S, et al. An mRNA m7G capbinding-like motif within human Ago2represses translation [J]. Cell,2007,129(6):1141-1151.
[33] HOCK J, MEISTER G. The Argonaute protein family [J]. Genome Biol,2008,9(2):210.
[34] JAKYMIW A, LIAN S, EYSTATHIOY T, et al. Disruption of GW bodiesimpairs mammalian RNA interference [J]. Nat Cell Biol,2005,7(12):1267-1274.
[35] EULALIO A, BEHM-ANSMANT I, IZAURRALDE E. P bodies: at thecrossroads of post-transcriptional pathways [J]. Nat Rev Mol Cell Biol,2007,8(1):9-22.
[36] EULALIO A, BEHM-ANSMANT I, SCHWEIZER D, et al. P-body formation isa consequence, not the cause, of RNA-mediated gene silencing [J]. Mol Cell Biol,2007,27(11):3970-3981.
[37] LEUNG A K, CALABRESE J M, SHARP P A. Quantitative analysis ofArgonaute protein reveals microRNA-dependent localization to stress granules[J]. Proc Natl Acad Sci U S A,2006,103(48):18125-18130.
[38] KOTAJA N, SASSONE-CORSI P. The chromatoid body: a germ-cell-specificRNA-processing centre [J]. Nat Rev Mol Cell Biol,2007,8(1):85-90.
[39] ZILBERMAN D, CAO X, JOHANSEN L K, et al. Role of ArabidopsisARGONAUTE4in RNA-directed DNA methylation triggered by inverted repeats[J]. Curr Biol,2004,14(13):1214-1220.
[40] CHEN P Y, MEISTER G. microRNA-guided posttranscriptional gene regulation[J]. Biol Chem,2005,386(12):1205-1218.
[41] NOTTROTT S, SIMARD M J, RICHTER J D. Human let-7a miRNA blocksprotein production on actively translating polyribosomes [J]. Nat Struct Mol Biol,2006,13(12):1108-1114.
[42] VASUDEVAN S, TONG Y, STEITZ J A. Switching from repression to activation:microRNAs can up-regulate translation [J]. Science,2007,318(5858):1931-1934.
[43] BEHM-ANSMANT I, REHWINKEL J, DOERKS T, et al. mRNA degradationby miRNAs and GW182requires both CCR4:NOT deadenylase and DCP1:DCP2decapping complexes [J]. Genes Dev,2006,20(14):1885-1898.
[44] DING L, SPENCER A, MORITA K, et al. The developmental timing regulatorAIN-1interacts with miRISCs and may target the argonaute protein ALG-1tocytoplasmic P bodies in C. elegans [J]. Mol Cell,2005,19(4):437-447.
[45] LIPPMAN Z, MARTIENSSEN R. The role of RNA interference inheterochromatic silencing [J]. Nature,2004,431(7006):364-370.
[46] PAL-BHADRA M, LEIBOVITCH B A, GANDHI S G, et al. Heterochromaticsilencing and HP1localization in Drosophila are dependent on the RNAimachinery [J]. Science,2004,303(5658):669-672.
[47] ARAVIN A A, KLENOV M S, VAGIN V V, et al. Dissection of a natural RNAsilencing process in the Drosophila melanogaster germ line [J]. Mol Cell Biol,2004,24(15):6742-6750.
[48] YIN H, LIN H. An epigenetic activation role of PIWI and a PIWI-associatedpiRNA in Drosophila melanogaster [J]. Nature,2007,450(7167):304-308.
[49] KURAMOCHI-MIYAGAWA S, KIMURA T, IJIRI T W, et al. Mili, amammalian member of pIWI family gene, is essential for spermatogenesis [J].Development,2004,131(4):839-849.
[50] HOCK J, WEINMANN L, ENDER C, et al. Proteomic and functional analysis ofArgonaute-containing mRNA-protein complexes in human cells [J]. EMBO Rep,2007,8(11):1052-1060.
[51] TAUBERT H, GREITHER T, KAUSHAL D, et al. Expression of the stem cellself-renewal gene HIWI and risk of tumour-related death in patients withsoft-tissue sarcoma [J]. Oncogene,2007,26(7):1098-1100.
[52] JIN P, ZARNESCU D C, CEMAN S, et al. Biochemical and genetic interactionbetween the fragile X mental retardation protein and the microRNA pathway [J].Nat Neurosci,2004,7(2):113-117.
[53] ILLMENSEE K, MAHOWALD A P. Transplantation of posterior polar plasm inDrosophila. Induction of germ cells at the anterior pole of the egg [J]. Proc NatlAcad Sci U S A,1974,71(4):1016-1020.
[54] MAHOWALD A P. Assembly of the Drosophila germ plasm [J]. Int Rev Cytol,2001,203187-213.
[55] SAFFMAN E E, LASKO P. Germline development in vertebrates andinvertebrates [J]. Cell Mol Life Sci,1999,55(8-9):1141-1163.
[56] MEGOSH H B, COX D N, CAMPBELL C, et al. The role of PIWI and themiRNA machinery in Drosophila germline determination [J]. Curr Biol,2006,16(19):1884-1894.
[57] BERNSTEIN E, CAUDY A A, HAMMOND S M, et al. Role for a bidentateribonuclease in the initiation step of RNA interference [J]. Nature,2001,409(6818):363-366.
[58] LIN H, SPRADLING A C. A novel group of pumilio mutations affects theasymmetric division of germline stem cells in the Drosophila ovary [J].Development,1997,124(12):2463-2476.
[59] HOUWING S, KAMMINGA L M, BEREZIKOV E, et al. A role for PIWI andpiRNAs in germ cell maintenance and transposon silencing in Zebrafish [J]. Cell,2007,129(1):69-82.
[60] CARMELL M A, GIRARD A, VAN DE KANT H J, et al. MIWI2is essential forspermatogenesis and repression of transposons in the mouse male germline [J].Dev Cell,2007,12(4):503-514.
[61] CHEN Y, PANE A, SCHUPBACH T. Cutoff and aubergine mutations result inretrotransposon upregulation and checkpoint activation in Drosophila [J]. CurrBiol,2007,17(7):637-642.
[62] DENG W, LIN H. mIWI, a murine homolog of pIWI, encodes a cytoplasmicprotein essential for spermatogenesis [J]. Dev Cell,2002,2(6):819-830.
[63] BATISTA P J, RUBY J G, CLAYCOMB J M, et al. PRG-1and21U-RNAsinteract to form the piRNA complex required for fertility in C. elegans [J]. MolCell,2008,31(1):67-78.
[64] NGUYEN T T, SAXENA A, BEVERIDGE T J. Effect of surfacelipopolysaccharide on the nature of membrane vesicles liberated from theGram-negative bacterium Pseudomonas aeruginosa [J]. J Electron Microsc(Tokyo),2003,52(5):465-469.
[65] GUNAWARDANE L S, SAITO K, NISHIDA K M, et al. A slicer-mediatedmechanism for repeat-associated siRNA5' end formation in Drosophila [J].Science,2007,315(5818):1587-1590.
[66] RO S, PARK C, SONG R, et al. Cloning and expression profiling oftestis-expressed piRNA-like RNAs [J]. RNA,2007,13(10):1693-1702.
[67] BRENNECKE J, ARAVIN A A, STARK A, et al. Discrete small RNA-generatingloci as master regulators of transposon activity in Drosophila [J]. Cell,2007,128(6):1089-1103.
[68] FANTI L, DORER D R, BERLOCO M, et al. Heterochromatin protein1bindstransgene arrays [J]. Chromosoma,1998,107(5):286-292.
[69] ARONICA L, BEDNENKO J, NOTO T, et al. Study of an RNA helicaseimplicates small RNA-noncoding RNA interactions in programmed DNAelimination in Tetrahymena [J]. Genes Dev,2008,22(16):2228-2241.
[70] GIRARD A, HANNON G J. Conserved themes in small-RNA-mediatedtransposon control [J]. Trends Cell Biol,2008,18(3):136-148.
[71] SAROT E, PAYEN-GROSCHENE G, BUCHETON A, et al. Evidence for apIWI-dependent RNA silencing of the gypsy endogenous retrovirus by theDrosophila melanogaster flamenco gene [J]. Genetics,2004,166(3):1313-1321.
[72] SHPIZ S, KWON D, UNEVA A, et al. Characterization of Drosophila telomericretroelement TAHRE: transcription, transpositions, and RNAi-based regulation ofexpression [J]. Mol Biol Evol,2007,24(11):2535-2545.
[73] GRIVNA S T, PYHTILA B, LIN H. MIWI associates with translationalmachinery and PIWI-interacting RNAs (piRNAs) in regulating spermatogenesis[J]. Proc Natl Acad Sci U S A,2006,103(36):13415-13420.
[74] SAUER B. Inducible gene targeting in mice using the Cre/lox system [J].Methods,1998,14:381-392.
[75] GU H, MARTH J D, ORBAN P C, et al. Deletion of a DNA polymerase betagene segment in T cells using cell type-specific gene targeting [J]. Science,1994,265:103-106.
[76] MICHEL G, YU Y, CHANG T, et al. Site-specific gene insertion mediated by aCre-loxP-carrying lentiviral vector [J]. Mol Ther,2010,18:1814-1821.
[77] SUEHIRO Y, KINOSHITA M, OKUYAMA T, et al. Transient and permanentgene transfer into the brain of the teleost fish medaka (Oryzias latipes) usinghuman adenovirus and the Cre-loxP system [J]. FEBS Lett,2010,584:3545-3549.
[78] LI L, PANG D, CHEN L, et al. Establishment of a transgenic pig fetal fibroblastreporter cell line for monitoring Cre recombinase activity [J]. DNA Cell Biol,2009,28:303-308.
[79] LI L, PANG D, WANG T, et al. Production of a reporter transgenic pig formonitoring Cre recombinase activity [J]. Biochem Biophys Res Commun,2009,382:232-235.
[80] CHEN L, LI L, PANG D, et al. Construction of transgenic swine with inducedexpressionof Cre recombinase [J]. Animal,2009,00:1-5.
[81] Lai, L.X., PRATHER, R.S. Production of cloned pigs by using somatic cells asdonors[J]. Cloning Stem Cells,2003(5):233–241.
[82] Liu Y, ROBERT F W. Thermal asymmetric interlaced PCR: automatableamplification and sequencing of insert end fragment from P1and YAC clones forchromosome walking. Genomics,1995(25):674681.