Tol1转座子研究进展

Tol1和Tol2是在青鳉基因组中发现的具有自主活性的DNA转座子,而Tol1转座子的自主活性是新近才发现的,因此对它的报道较少。较之Tol2,Tol1可以携带更大片段的DNA进行转座,且Tol1的转座不受转座酶"过量表达抑制"的影响。研究已证实,Tol1转座子在秀丽线虫、斑马鱼、爪蟾和人等多种生物中具有转座活性。因此,在动物转基因和基因功能研究等方面有重要的应用前景。从Tol1转座子的结构特征、转座机制和作为基因转移载体的优点,以及应用研究等方面进行了简要的综述。     

Tol2转座子系统在转基因动物中的应用

Tol2是在青鳉鱼基因组中发现的一种具有自主性的转座子元件.它编码转座酶,催化Tol2转座子结构中5’端200 bp和3’端150 bp序列发生转座反应.Tol2的多种特性,如可携带大片段外源DNA、单拷贝整合效率高、转座子活性强等,使得以Tol2特座子系统为载体的转基因技术在多种生物中得到应用.综述了Tol2转座子系统的结构、特性以及近年来在多种动物转基因中的应用.     

金鱼hAT家族转座子Tgf2的克隆及其结构

hAT家族转座子以果蝇hobo、玉米Ac和金鱼草(Ceratophyllum demersum L.)Tam3为代表,以"剪切-粘帖"方式进行DNA转座。1996年,日本学者首次在白化青鳉(Oryzias latipes)中发现具有天然活性的脊椎动物hAT家族转座子,即青鳉Tol2转座子,该转座子已在模式生物斑马鱼转基因、基因和启动子捕获方面进行了广泛应用。文章根据玉米Ac与青鳉Tol2转座子序列保守区设计一对引物,在19种不同鱼类物种或品系中进行PCR筛选,最后发现此类hAT家族转座子在我国不同品系金鱼中存在,命名为金鱼Tgf2转座子。金鱼Tgf2转座子全长4720bp,由4个阅读框组成,与青鳉Tol2转座子的相似度为97%。金鱼Tgf2与青鳉Tol2转座子在末端倒位重复和亚末端重复上存在一定差异,此外,金鱼Tgf2转座子的中间反向重复序列(1453bp到2091bp)可形成一种"十"字结构,明显有别于青鳉Tol2转座子形成的茎环结构,这些区域与转座活性密切相关。文章预示金鱼Tgf2转座子可能具有更高的天然转座活性,构建高效金鱼Tgf2转基因元件可供鱼类转基因和基因捕获研究。     

利用Tol2转座子构建转基因草金鱼初探

青鳉Tol2转座子是脊椎动物中发现的第一例天然具有活性的转座子,目前已经被成功应用于多种模式动物的转基因研究中。采用PCR的方法以质粒pCAgcGH为模板亚克隆出一段含有草鱼生长激素(GH)5个外显子、鲤鱼β-actin启动子及多个酶切位点的序列,并将这段序列与经双酶切处理的质粒pTol2-MCS-EGFP进行重组连接,得到重组质粒pTol2-GH-EGFP。采用显微注射的方法将重组质粒pTol2-GH-EGFP与体外合成的Tol2转座酶mRNA一起注入草金鱼受精卵中。通过观察绿色荧光和PCR检验绿色荧光蛋白和GH基因的表达,筛选出成功转入外源基因的草金鱼。阳性表达检出率为17.3%。利用Tol2转座子构建转基因草金鱼,不仅丰富了Tol2转座子的应用范围,而且为进一步利用Tol2转座元件进行观赏鱼转基因及基因表达研究奠定了基础。     

斑马鱼sat1.a突变体的鉴定

目的在我们的前期研究工作中,通过Tol2转座子介导的插入突变,筛选到了一批组织特异性表达绿色荧光蛋白GFP的斑马鱼品系。其中一个品系Tol2:20141221t的GFP在神经系统中表达,但还没有鉴定到Tol2转座子插入到基因组什么位置,造成了哪个基因的突变。本文的主要研究目标就是对这一由Tol2转座子插入诱导的斑马鱼突变品系进行鉴定。方法使用交错式热不对称PCR(thermal asymmetric interlaced PCR,TAIL-PCR)鉴定Tol2转座子插入的基因组位置;利用原位杂交检测被突变的基因的时空表达是否与该品系GFP表达具有一致性;筛选鉴定纯合体突变体,并进一步分析该基因突变造成的发育缺陷。结果在该品系中,Tol2转座子插入到了亚精胺/精胺N1-乙酰基转移酶1a(spermidine/spermine N1-acetyltransferase 1a,sat1.a)的第八个内含子区域,致使sat1.a基因转录提前终止。我们筛选到了sat1.a纯合突变体,但是没有检测到明显的发育缺陷。结论筛选鉴定的Tol2转座子介导的斑马鱼sat1.a突变体没有明显的发育缺陷,但可以作为研究神经系统发育的有力工具。     

一种包含转座子Sleeping Beauty和PiggyBac的新型多功能载体的构建

DNA转座子作为一种遗传学工具对脊椎动物的转基因、突变体产生、癌基因发现和基因治疗方面都有巨大的贡献. 目前,哺乳动物中应用最为广泛、活性最高的DNA转座子为重构于鲑鱼的Sleeping Beauty (SB)转座子和来源于甘蓝蠖度尺蛾 (cabbage looper moth Trichoplusia ni)的PiggyBac (PB)转座子. 本研究中,我们成功构建了包含PB和SB两种转座子的杂合转座载体,命名为PBSBD. 在杂合转座载体中融入了基因捕获框及loxp/Frt元件,用以实现转座过程中的基因捕获和条件性敲除. 在HepG2细胞中通过检测报告基因的表达情况及阳性克隆的定位,对构建的杂合转座载体PBSBD进行了活性的初步验证. 结果表明,PBSBD能够有效被2种转座酶识别,并能检测到报告基因的表达. 本研究所构建的杂合转座载体PBSBD结合2种转座酶,可以应用于大规模筛选突变基因和研究基因功能. 并且该杂合转座载体还可以利用SB转座酶的邻近转座特性,结合载体内所包含的loxp/Frt元件用以邻近区域DNA片段的条件性敲除,研究大片段DNA在生物体中的作用.     

酵母Ty1和Ty3转座调控机制研究进展

LTR(Long Terminal Repetition, LTR)反转录转座子广泛存在于真核生物界,是逆转录病毒的进化祖先。LTR反转录转座子有两个古老的家族,Ty1/Copia和Ty3/Gypsy。目前关于LTR反转录转座子转座机制及调控机制研究最透彻的是来源于酵母的两个活性转座子Ty1和Ty3。全面综述了Ty1和Ty3的分子生物学机制相关的最新研究进展。系统总结了Ty1和Ty3的结构特征及转座特性,归纳了Ty1和Ty3与宿主共生的调控机制,为进一步了解酵母LTR反转录转座子相关转座调控机制提供参考。     

动物遗传工程

920了38利用转基因植物了解转位机理并开发转座子标签技术[英〕/Haring,M.A.…1 Plant Mol.Biol一1991,16(3)。一449~461[译自DBA,1991,10(12),91一06900〕 综述了植物可转座因子Ac(活化子)、Tam3、En/Spm(增强子/抑制子一突变子)和Mu(突变子)在异源植物种及其原始宿主中的活性‘讨论了下列问题:1.基因克隆中可转座因子的应用一转座子标签,11.自主植物可转座因子在新的遗传背景中的活性;111。用2一因子系统控制转座子标签,iv.烟草中可转座因子A。的突变分析,v.烟草中En/Spm抑制子功能分析,vi.异源诱动DNA因子在转座子标签中的应用前…     

根瘤菌共生固氮的遗传学研究(二)

2.在根瘤菌研究中成功地运用了转座子诱变技术。转座子(Transposon)是一种特殊的DNA短片段,它带有抗药性基因,并具有在DNA复制子之间转座插入的能力,转座的发生并不需要recA基因产物,一些转座子象Tn 5的转座插入位点的分布是相当随机的,但另一些象Tn 10,它的转座插入似乎具有“热点”(Hot spot),转座子插入到一个新位点时,被插入位点原基因的连续性受到阻断,因而该基因的功     

9种硬骨鱼类转座组注释

通过生物信息学手段对9种硬骨鱼转座组进行注释。结果表明9种硬骨鱼类转座组大小和构成差异显著,其转座组含量从高到低分别为斑马鱼、矛尾鱼、青鳉鱼、罗非鱼、花斑剑尾鱼、大西洋鳕鱼、三刺鱼、金娃娃和红鳍东方鲀,转座子含量和基因组大小呈正相关。DNA转座子在硬骨鱼类中具有多样性高和含量差异大的特点(0.50%–38.37%),是硬骨鱼类转座组差异的主要决定因素,其中h AT和Tc/Mariner超家族是硬骨鱼类主要的DNA转座子。RNA转座子在硬骨鱼类中也具有多样性高的特点,其中LINE转座子占硬骨鱼类基因组的0.53%–5.75%,共检测到14个超家族分布,其中L1、L2、RTE和Rex转座子扩增较为明显,LTR转座子除了在斑马鱼和三刺鱼中含量达到5.58%和2.51%,在大多硬骨鱼类基因组中的含量低于2%,在硬骨鱼类中共检测到6个LTR转座子(Copia、DIRS、ERV、Gypsy、Ngaro和Pao)超家族分布,其中扩增最为明显的是Gypsy。而SINE转座子在硬骨鱼类中扩增最弱,仅在斑马鱼和矛尾鱼中分别达到3.28%和5.64%,在其他7个物种中低于1%。SINE中t RNA、5S和MIR三个超家族在部分硬骨鱼类中有一定程度扩增。本研究表明硬骨鱼类转座组具有多样性丰富、差异大的特点,转座组差异与硬骨鱼基因组大小有很强的相关性,转座组是决定硬骨鱼基因组大小的重要因素。     

Genome-wide target profiling of <Emphasis Type="Italic">piggyBac</Emphasis> and <Emphasis Type="Italic">Tol2</Emphasis>in HEK 293: pros and cons for gene discovery and gene therapy

Background  

DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery.     

Harnessing a high cargo-capacity transposon for genetic applications in vertebrates

Viruses and transposons are efficient tools for permanently delivering foreign DNA into vertebrate genomes but exhibit diminished activity when cargo exceeds 8 kilobases (kb). This size restriction limits their molecular genetic and biotechnological utility, such as numerous therapeutically relevant genes that exceed 8 kb in size. Furthermore, a greater payload capacity vector would accommodate more sophisticated cis cargo designs to modulate the expression and mutagenic risk of these molecular therapeutics. We show that the Tol2 transposon can efficiently integrate DNA sequences larger than 10 kb into human cells. We characterize minimal sequences necessary for transposition (miniTol2) in vivo in zebrafish and in vitro in human cells. Both the 8.5-kb Tol2 transposon and 5.8-kb miniTol2 engineered elements readily function to revert the deficiency of fumarylacetoacetate hydrolase in an animal model of hereditary tyrosinemia type 1. Together, Tol2 provides a novel nonviral vector for the delivery of large genetic payloads for gene therapy and other transgenic applications.     

<Emphasis Type="Italic">Tol2</Emphasis>: a versatile gene transfer vector in vertebrates

The medaka fish Tol2 element is an autonomous transposon that encodes a fully functional transposase. The transposase protein can catalyze transposition of a transposon construct that has 200 and 150 base pairs of DNA from the left and right ends of the Tol2 sequence, respectively. These sequences contain essential terminal inverted repeats and subterminal sequences. DNA inserts of fairly large sizes (as large as 11 kilobases) can be cloned between these sequences without reducing transpositional activity. The Tol2 transposon system has been shown to be active in all vertebrate cells tested thus far, including zebrafish, Xenopus, chicken, mouse, and human. In this review I describe and discuss how the Tol2 transposon is being applied to transgenic studies in these vertebrates, and possible future applications.     

Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster

The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila.     

Excision of the Tol2 transposable element of the medaka fish Oryzias latipes in Xenopus laevis and Xenopus tropicalis

The Tol2 transposable element from the medaka fish belong to the hAT family of transposons. In the previous studies, we have identified an autonomous member of this element, which encodes a fully functional transposase, and have shown that it can catalyze transposition in the zebrafish germ lineage. To date, the Tol2 element is the only natural transposon in vertebrates from which an autonomous member has been identified. We report here transposase-dependent excision of the Tol2 element in Xenopus laevis and Xenopus (Silurana) tropicalis embryos. We coinjected a plasmid DNA containing a nonautonomous Tol2 element and the transposase mRNA synthesized in vitro into two-cell-stage embryos, and analyzed DNA extracted from the injected embryos by polymerase chain reaction (PCR). We demonstrated that the Tol2 element could be excised from the plasmid DNA in both X. laevis and X. tropicalis only when it was coinjected with the transposase mRNA. In most cases, a complete loss of the Tol2 sequence was accompanied by addition of a short DNA sequence to the target sequence, indicating that transposase-dependent excision occurred. While these footprints were characteristic to those created upon excision of transposons of the hAT family, the additional bases found in Xenopus were longer and their structures were more complicated than those detected upon excision in zebrafish. This may reflect differences in the activities of host factors involved in either transposition, repair, or both between fish and frog. Our present study suggests that the Tol2 transposon system should be used as a novel genetic tool to develop transgenesis and mutagenesis methods in Xenopus.     

Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.     

转座子PiggyBac在哺乳动物中的应用

DNA转座子作为一种遗传工程工具已广泛应用于多物种的转基因及产生插入突变等研究。目前,在哺乳动物中有转座活性的转座子可分为三类：1）hAT样转座子;2）Tcl样转座子包括Sleeping Beauty和FrogPrince;3）PiggyBac转座子家族。其中甘蓝蠖度尺蛾（Cabbage looper moth Trichoplusia ni）来源的PiggyBac转座子是目前在哺乳动物中活性最高的转座子,并且可以携带十几kb的外源基因转座而不影响其效率,使其在哺乳动物的转基因、癌基因的发现、基因治疗研究方面具有巨大的应用潜力。此外,PB的无痕迹转座对于无转基因、无遗传物质改变的诱导多潜能干细胞（iPS）研究也具有非常重要的意义。本文主要对针对PB在哺乳动物中的应用现状及前景作一介绍。     

Retargeting transposon insertions by the adeno-associated virus Rep protein

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties of the transposon systems by taking advantage of the locus-specific integration system of adeno-associated virus (AAV). The AAV Rep protein binds to Rep recognition sequences (RRSs) in the human genome, and mediates viral integration into nearby sites. A series of fusion constructs consisting of the N-terminal DNA-binding domain of Rep and the transposases or the N57 domain of SB were generated. A plasmid-based transposition assay showed that Rep/SB yielded a 15-fold enrichment of transposition at a particular site near a targeted RRS. Genome-wide insertion site analysis indicated that an approach based on interactions between the SB transposase and Rep/N57 enriched transgene insertions at RRSs. We also provide evidence of biased insertion of the PB and Tol2 transposons. This study provides a comparative insight into target site selection properties of transposons, as well as proof-of-principle for targeted chromosomal transposition by composite protein-protein and protein-DNA interactions.     

Phylogenetic and functional characterization of the hAT transposon superfamily

Transposons are found in virtually all organisms and play fundamental roles in genome evolution. They can also acquire new functions in the host organism and some have been developed as incisive genetic tools for transformation and mutagenesis. The hAT transposon superfamily contains members from the plant and animal kingdoms, some of which are active when introduced into new host organisms. We have identified two new active hAT transposons, AeBuster1, from the mosquito Aedes aegypti and TcBuster from the red flour beetle Tribolium castaneum. Activity of both transposons is illustrated by excision and transposition assays performed in Drosophila melanogaster and Ae. aegypti and by in vitro strand transfer assays. These two active insect transposons are more closely related to the Buster sequences identified in humans than they are to the previously identified active hAT transposons, Ac, Tam3, Tol2, hobo, and Hermes. We therefore reexamined the structural and functional relationships of hAT and hAT-like transposase sequences extracted from genome databases and found that the hAT superfamily is divided into at least two families. This division is supported by a difference in target-site selections generated by active transposons of each family. We name these families the Ac and Buster families after the first identified transposon or transposon-like sequence in each. We find that the recently discovered SPIN transposons of mammals are located within the family of Buster elements.