PiggyBac转座酶转基因小鼠模型的建立

目的 建立表达PiggyBac转座酶转基因小鼠模型,为研究PiggyBac转座子介导基因修饰在小鼠中的应用提供工具.方法 利用Cytomegalovirus( CMV)启动子驱动PiggyBac转座酶基因的表达,经显微注射法建立C57BL/6J表达PiggyBac转座酶的转基因小鼠.PCR鉴定转基因小鼠的基因型,RT-PCR检测PiggyBac转座酶在小鼠生殖系睾丸中的表达情况.PiggyBac转座酶转基因小鼠活性的检测,是通过与转座子供体转基因小鼠杂交检测供体位置变化来确定的.结果 显微注射产生7只转基因小鼠并能传代,经RT-PCR筛选出一株在睾丸中相对高表达PiggyBac转座酶的转基因小鼠.随后与转座子供体转基因小鼠杂交,子代双阳小鼠与野生型小鼠杂交基因型分离,产生的子代转座子供体单阳性小鼠中具有转座子供体片段的转座反应.结论 成功建立了表达PiggyBac转座酶转基因小鼠动物模型,该模型为PiggyBac转座子技术在小鼠中的应用提供了有价值的工具动物.     

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.     

转座子Sleeping Beauty和PiggyBac

近10年来,得益于转座子Sleeping Beauty(SB)和PiggyBac(PB)的发现和完善,转座子作为一种遗传工程工具在脊椎动物的基因遗传研究中得到广泛应用.SB和PB宿主范围极其广泛,从单细胞生物到哺乳动物都能够发挥作用.转座过程需要转座序列和转座酶的存在,类似于"剪切"、"粘贴"的方式.转座子载体系统转座时可携带一段外源DNA序列,利用这一特性可以用于实现目的基因的转移,现已广泛用于转基因动物、基因功能研究、基因治疗等领域.当转座系统与基因捕获技术相结合,不仅可研究插入突变基因的功能,还能通过所携带的报告基因获得捕获基因的表达图谱.作为非病毒载体的SB和PB转座系统,由于具有高容量、高效率和高安全性等优势,并且PB在转座后不留任何足迹,不会造成遗传物质的不可预测改变,在动物基因工程以及基因治疗方面具有诱人的前景.     

新型二合一PiggyBac转座系统的构建及功能验证*

目的:PiggyBac(PB)转座子是一种可移动的遗传元件,采用“剪切和粘贴”机制在载体和染色体之间进行转座;通过将转座子元件和转座酶表达框整合到一个表达载体中,构建简便易用的二合一PB转座系统。方法:通过聚合酶链式反应(polymerase chain reaction,PCR)获取PiggyBac转座系统所需转座子元件和转座酶表达框,利用T4 DNA连接酶将转座酶表达框插入到pUC18载体上,再利用Gibson同源重组技术将转座子元件与重组载体结合构建二合一PB转座系统;使用该系统携带的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)以及功能性损伤抑制蛋白(damage-suppressing protein,DSUP)检测其有效性及可靠性。结果:在所有筛选获得的嘌呤霉素抗性细胞中,EGFP都是明亮可见;利用此二合一PB转座系统成功获得了可高效表达功能性损伤抑制蛋白的稳定细胞系,证明外源基因可被有效整合到基因组DNA中并表达。结论:成功构建了新型二合一PB转座系统,使稳定表达细胞系的建立更加经济简便。     

piggyBac转座子在牛基因组的整合位点及特征分析

piggyBac(PB)转座子作为一种遗传工具被广泛应用于多个物种的转基因及插入突变研究, 目前PB转座子在牛中的相关研究还较少。为了获得PB转座子在牛基因组中的整合位点, 总结其转座特征, 文章构建了PB[CMV-EGFP]和pcDNA-PBase二元转座系统, 利用细胞核电转技术共转染牛耳组织成纤维细胞, 经G-418筛选, 获得了稳定转染EGFP的转基因细胞系; 提取细胞基因组DNA, 利用基因组步移技术扩增PB转座子5′ Bac区插入位置的DNA序列; 通过与牛基因组序列进行BLAST比对, 得到PB转座子在牛基因组中的插入位点。文章共获得了8个有效的整合位点, 但仅有5个位点定位到染色体1、2、11和X染色体上。序列分析表明：在牛基因组中, PB转座子可特异性的插入到“TTAA”位置, 并整合到基因间的非调控区; 分析整合位点“TTAA”相邻一侧的5个碱基组成, 发现PB转座子5′端倾向于插入到GC(62.5%)碱基富集区。该研究表明, PB转座子可以在牛基因组中发生转座, 获得的整合位点信息为利用PB转座子在牛上开展遗传学研究提供了理论参考。     

piggyBac转座系统在哺乳动物及其细胞中的研究进展

piggyBac（PB）转座系统来源于昆虫鳞翅目,属于真核生物的第二类转座系统,主要采取＂剪切粘帖＂机制发生转座。PB系统转座效率高,宿主范围广,广泛应用于昆虫等低等生物的基因转移及突变筛选。近年来,研究发现PB系统在哺乳动物及其细胞中也具有高效的转座活性,已在动物基因组功能研究、基因转移及诱导多能干细胞等领域得到了广泛应用。本文就PB系统近年来在哺乳动物及其细胞中的研究进展、应用前景及存在问题进行了综述。     

piggyBac转座系统的功能改进及在哺乳动物中的应用

piggyBac (PB)转座系统具有转座效率高、删除精确、半随机插入和携带片段较大等优点。但是作为一种转基因实验的工具,特别是在哺乳动物个体水平的转基因方面,还需要提高其转基因效率,并降低外源基因随机插入对内源基因破坏的风险。近年来的研究结果显示,PB转座系统得到了进一步改进：采用PB转座酶与DNA特异性结合蛋白融合而构成的融合型转座酶,表现出外源片段有插入到染色体靶向位点的倾向;采用突变体筛选的方法提高了PB转座酶的活性,获得了只具有切除活性而没有插入活性的新型PB转座酶;采用PB转座系统与细菌人工染色体(Bacterial artificial chromosomes, BAC)载体联合使携带的外源片段长度提高到了207 kb。改进后的PB转座系统在基因组研究、基因治疗、诱导多能干细胞(Induced pluripotent stem cells, iPSCs)诱导及其分化方面发挥了较大的作用。文章对PB转座系统的最新研究进展和应用前景进行了综述。     

"睡美人"转座子的研究进展

“睡美人( Sleeping Beauty, SB) ”转座系统是Tc1/mariner 转座子超家族中的一员,已经失活了一千多万年。1997年,Ivics 等根据积累的系统发生数据,利用生物信息学的方法, 对其进行分子重建, 终于唤醒了其转座活性。近年来对“睡美人”转座系统的转座效率和转座机理进行的研究,已证明SB转座子在基因筛选,转基因及基因治疗等领域具有广阔的应用前景。文章重点论述了SB转座子在结构及其优化、转座机制和应用等方面的进展,同时对其研究中出现的各种问题进行了总结并提出了一些解决方案。     

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。     

piggyBac转座子AgoPLE1.1在黑腹果蝇种系 转化中的应用

【目的】通过检测黑腹果蝇Drosophila melanogaster中piggyBac (PB)转座子AgoPLE1.1的转化活性,明确AgoPLE1.1开发为昆虫转基因载体的潜力。【方法】构建AgoPLE1.1转座酶辅助质粒pAgoHsp和带有红色荧光标记的供体质粒pXLAgo-PUbDsRed,辅助质粒和供体质粒以170 ng/μL∶400 ng/μL, 90 ng/μL∶200 ng/μL和90 ng/μL∶100 ng/μL 3种不同的比例混合后分别注射新鲜的W¹¹¹⁸ 黑腹果蝇胚胎,筛选注射后代中的转基因黑腹果蝇个体;利用Southern杂交验证转基因黑腹果蝇中AgoPLE1.1转座子的插入拷贝数;利用染色体步移技术克隆AgoPLE1.1插入位点旁侧序列,明确AgoPLE1.1转座子的转座特征。【结果】AgoPLE1.1转座子在黑腹果蝇中具有转化活性,转基因频率为1.32%~1.94%。Southern杂交结果显示,AgoPLE1.1转座子在黑腹果蝇中至少有6个插入位点。染色体步移法克隆了其中4个位点,分别位于黑腹果蝇的3R, 3L, 2L和X染色体,并且AgoPLE1.1转座子在黑腹果蝇染色体中的整合带有供体质粒的骨架。【结论】PB转座子AgoPLE1.1仅可以在黑腹果蝇中以较低的频率进行非精确的剪切和转座,不具有开发为新型昆虫转基因载体的潜力。     

The <Emphasis Type="Italic">piggyBac</Emphasis> Transposon as a Tool in Genetic Engineering

Transposons are mobile genetic elements that are part of the genomic DNA of numerous organisms and belong to two classes. Unlike class I transposons, class II DNA transposons do not use the stage of RNA synthesis in their transition; they perform it by the cut-and-paste mechanism or with a replicative transposition. The integration of a DNA transposon in a new site results in the duplication of a target sequence on either side of a transposon, and its excision is, as a rule, associated with insertions and deletions. The piggyBac transposon isolated from the Trichoplusia ni moth differs from other mobile elements of its class. Due to its unique ability to leave no traces after excision from an insertion site and to perform successful transposition and transference of large DNA fragments, piggyBac is a convenient tool for the development of gene engineering approaches. The TTAA sequence serves as a target site for transposon integration: insertion in the AT-rich DNA regions is more frequent. The ability of piggyBac to be transferred to a new area independently of the cell apparatus and to restore a DNA site without error after excision lies in the mechanism of its transposition, which is discussed in detail in the present review. Along with other transposons and viruses, the piggyBac transposon is widely used in the transgenesis of various organisms; it also finds application in insertion mutagenesis and gene therapy.     

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.     

Cis requirements for transposition of Tc1-like transposons in C. elegans

The Caenorhabditis elegans transposons Tc1 and Tc3 are able to transpose in heterologous systems such as human cell lines and zebrafish. Because these transposons might be useful vectors for transgenesis and mutagenesis of diverse species, we determined the minimal cis requirements for transposition. Deletion mapping of the transposon ends shows that fewer than 100?bp are sufficient for transposition of Tc3. Unlike Tc1, Tc3 has a second, internal transposase binding site at each transposon end. We found that these binding sites play no major role in the transposition reaction, since they can be deleted without reduction of the transposition frequency. Site-directed mutagenesis was performed on the conserved terminal base pairs at the Tc3 ends. The four terminal base pairs at the ends of the Tc3 inverted repeats were shown to be required for efficient transposition. Finally, increasing the length of the transposon from 1.9?kb to 12.5 kb reduced the transposition frequency by 20-fold, both in vivo and in vitro.     

Mobilization of giant piggyBac transposons in the mouse genome

The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.     

Cis requirements for transposition of Tc1-like transposons in C. elegans

The Caenorhabditis elegans transposons Tc1 and Tc3 are able to transpose in heterologous systems such as human cell lines and zebrafish. Because these transposons might be useful vectors for transgenesis and mutagenesis of diverse species, we determined the minimal cis requirements for transposition. Deletion mapping of the transposon ends shows that fewer than 100 bp are sufficient for transposition of Tc3. Unlike Tc1, Tc3 has a second, internal transposase binding site at each transposon end. We found that these binding sites play no major role in the transposition reaction, since they can be deleted without reduction of the transposition frequency. Site-directed mutagenesis was performed on the conserved terminal base pairs at the Tc3 ends. The four terminal base pairs at the ends of the Tc3 inverted repeats were shown to be required for efficient transposition. Finally, increasing the length of the transposon from 1.9 kb to 12.5 kb reduced the transposition frequency by 20-fold, both in vivo and in vitro. Received: 21 April 1999 / Accepted: 10 June 1999