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Comparison of Transformation Efficiency of piggybac Transposon among Three Different Silkworm Bombyx mori Strains

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Acta Biochimica et Biophysica Sinica 2007年第2期39卷 117-122页

作者： Boxiong ZHONG~1 Jianying LI~1 Jin'e CHEN~(1,2) Jian YE~1 and Songdong YU~1 ~1College of Animal Sciences,Zhejiang University,Hangzhou 310029,China ~2Institute of Sericultural Research,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China College of Animal Sciences Zhejiang University Hangzhou 310029 China Institute of Sericultural Research Zhejiang Academy of Agricultural Sciences Hangzhou 310021 China

The transformation rate of three different strains of silkworm Bombyx mori was comparedafter the introduction of enhanced green fluorescence protein （EGFP）-encoding genes into the silkwormeggs by microinjection of a mixture of piggybac vector and helper plasmid containing a transposase-encodingsequence.Although there were no significant differences among the three strains in the percentages offertile moths in microinjected eggs （P=0.1258）,the percentages of G0transformed moths in fertile mothsand injected eggs were both significantly different （P=0.01368 and P=0.02398, respectively）.Thetransformation rate of the Nistari strain （Indian strain） was significantly higher than that of the other twostrains,Golden-yellow-cocoon （Vietnamese strain） and Jiaqiu （Chinese strain）,which had similar rate. Theseresults indicate that the transformation efficiency of the piggybac-based system might vary with silkwormstrains with different genetic backgrounds.The presence of endogenous piggybac-like elements might bean important factor influencing the transformation efficiency of introduced piggybac-derived vectors,andthe diverse amount and activation in different silkworm strains might account for the significant differences.

关键词： Bombyx mori piggybac transposon transgenesis transformation efficiency

Using chimeric piggybac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2010年第9期11卷 728-734页

作者： Na WANG Cai-ying JIANG Ming-xing JIANG Chuan-xi ZHANG Jia-an CHENG Institute of lnsect Sciences Zhejiang University Hangzhou 310029 China Institute of Biochemistry Zhejiang Sci-Tech University Hangzhou 310018 China

The piggybac transposon has been long used to integrate foreign DNA into insect genomes.However,undesirable transgene expression can result from random insertions into the genome.In this study,the efficiency of chimeric Gal4-piggybac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2(S2) cells.The Gal4-piggybac transposase has a Gal4 DNA-binding domain(DBD),and the target plasmid has upstream activating sequences(UAS) to which the Gal4 DBD can bind with high affinity.The results indicate that,in the Bm-12 and S2 cells,transpositional activity of Gal4-piggybac transposase was increased by 4.0 and 7.5 times,respectively,compared to controls,where Gal4-UAS interaction was absent.Moreover,the Gal4-piggybac transposase had the ability of directing piggybac element integration to certain sites of the target plasmid,although the target-directing specificity was not as high as expected.The chimeric piggybac transposase has the potential for use in site-directed transgenesis and gene function research in B.mori.

关键词：给词 Bombyx mori 调音 piggybac Gal4 在上游的激活序列(UAS ) 调换试金 Transgenesis

Discovering genes responsible for silk synthesis in Bombyx moriby piggybac-based random insertional mutagenesis

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Insect Science 2019年第5期26卷 821-830页

作者： Xing-Bao Feng Zi-Wen Zheng Xian Zhang Jun Gu Qi-Li Feng Li-Hua Huang Guangzhou Key Laboratory of Insect Development Regulation and Applied Research Institute of Insect Science and TechnologySchool of Life SciencesSouth China Normal UniversityGuangzhouChina

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. piggybac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggybac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggybac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.

关键词： aquaporin Bombyx mori development mutant piggybac silk gland

Rapid construction of a whole-genome mutant library by combining haploid stem cells and inducible self-inactivating piggybac transposon

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Protein & Cell 2020年第6期11卷 452-457页

作者： Junjie Mao Kai Xu Jiabao Han Guihai Feng Ying Zhang Wei Li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Institute for Stem Cell and Regeneration Chinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Dear Editor,Genome-scale screening is a powerful method used to explore phenotypes that are of interest,and numerous screening systems have been built for functional sites identification.Classical genetic screening in eukaryotes has been performed using chemical mutagens(Chen et al.,2000),transposon mediated gene trapping(Dupuy et al.,2005),and CRISPR-Cas9 system(Wang et al.,2014).

关键词： piggybac inducible inactivating

piggybac转座子在牛基因组的整合位点及特征分析

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遗传 2013年第6期35卷 771-777页

作者：杜新华高雪张路培高会江李俊雅许尚忠中国农业科学院北京畜牧兽医研究所中国农业科学院肉牛研究中心农业部畜禽遗传资源与利用重点开放实验室北京100193

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,... 详细信息

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。

关键词： piggybac 转基因牛基因组整合位点

piggybac转座子在绒山羊基因组中的整合位点及其特征分析

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中国农业科学 2012年第5期45卷 958-965页

作者：白丁平方堃杨明明屈雷陈玉林西北农林科技大学动物科技学院新疆农垦科学院畜牧兽医研究所榆林学院陕西省陕北绒山羊工程技术研究中心

【目的】构建piggybac(PB)转座子表达载体系统,研究PB转座子在绒山羊基因组中的整合位点。【方法】构建pB-CMV-EGFP-Neo转座载体和pcDNA-Trans辅助载体,利用lipofectamine 2000介导共转染绒山羊胎儿成纤维细胞,经G418筛选获得稳定转染... 详细信息

【目的】构建piggybac(PB)转座子表达载体系统,研究PB转座子在绒山羊基因组中的整合位点。【方法】构建pB-CMV-EGFP-Neo转座载体和pcDNA-Trans辅助载体,利用lipofectamine 2000介导共转染绒山羊胎儿成纤维细胞,经G418筛选获得稳定转染的转基因细胞系。提取转基因细胞基因组DNA,利用反向PCR检测piggybac转座子整合位点。【结果】构建了转座系统并成功介导外源基因在绒山羊成纤维细胞染色体中整合,获得了转基因细胞系;反向PCR检测显示在绒山羊基因组中有21个PB转座子整合位点;整合位点TTAA毗邻一侧的5个碱基组成中,PB转座子3′倾向于插入到富含AT(58.35%)碱基的区域,PB转座子5′倾向于插入到富含GC(57.8%)。【结论】PB转座子可在绒山羊基因组中发生高效转座,获得的整合位点信息可为利用PB转座子开展绒山羊研究提供理论参考。

关键词： piggybac 绒山羊基因组整合位点

利用CRISPR/Cas9和piggybac实现果蝇基因组无缝编辑

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遗传 2019年第5期41卷 422-429,451-461页

作者：王珏黄娟许蕊南京医科大学基础医学院医学遗传学系南京211166

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9)是第三代基因组编辑技术。在sgRNA引导下,Cas9核酸内切酶作用于特定基因组序列,产生DNA双链断裂(double-strandedbreaks,DSBs),利用同源定向修复(homology-directedrepair,HDR)可实现对靶基因的特异性基因敲除(knock-out)或敲入(knock-in)。传统的技术方案将CRISPR/Cas9技术与Cre/loxP或FLP/FRT系统联用,可实现高效的基因打靶,也易于移除打靶过程中引入的筛选标记。然而,筛选标记移除过程中会在基因组中残留34个碱基的标签序列。因此,对基因组进行精确编辑的同时不引入无关序列仍有一定难度。在人工诱导多能干细胞(induced pluripotent stem cells, iPSCs)的基因组编辑中,CRISPR/Cas9技术和piggybac转座酶联用的两步法策略能够实现这一目标:首先运用CRISPR/Cas9技术,利用同源定向修复原理引入基因突变及筛选标记,然后利用piggybac转座酶将筛选标记精确移除。借鉴该方法的技术原理,本研究对果蝇(Drosophila melanogaster)CG4894基因进行了无缝编辑(seamless genome editing),成功将该基因第18外显子上第21位的酪氨酸(tyrosine,Y)突变为半胱氨酸(cysteine,C),且测序结果显示基因组中除设计位点之外并无其他外源序列残留。CRISPR/Cas9技术和piggybac转座酶联用策略为果蝇基因组的精确编辑提供了更多选择。

关键词：果蝇无缝基因组编辑 CRISPR/Cas9 piggybac

piggybac转座系统在哺乳动物及其细胞中的研究进展

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生命科学 2011年第3期23卷 255-260页

作者：谢飞蒋世忠马晴雯上海市儿童医院医学遗传研究所上海200040 上海交通大学医学院上海200025

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... 详细信息

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。

关键词： piggybac 转座哺乳动物转基因

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piggybac在转基因小鼠制备中的应用

中国细胞生物学学报 2014年第7期36卷 934-940页

作者：刘金烨庄华孙瑞林费俭同济大学生命科学与技术学院上海200092 上海南方模式生物研究中心上海201203

受精卵雄原核裸DNA注射是目前制备转基因小鼠的主要技术,而转基因表达成功率低是这种技术的主要缺点。piggybac转座子系统已被报道用于制备转基因小鼠,但这一方法是否能够提高转基因的表达成功率尚不清楚。为此,我们利用毛色基因agouti... 详细信息

受精卵雄原核裸DNA注射是目前制备转基因小鼠的主要技术,而转基因表达成功率低是这种技术的主要缺点。piggybac转座子系统已被报道用于制备转基因小鼠,但这一方法是否能够提高转基因的表达成功率尚不清楚。为此,我们利用毛色基因agouti为报告基因,采用piggybac转座子系统以C57/BL6小鼠为背景进行转基因小鼠的制备。结果表明,将piggybac转座酶cRNA和转基因载体进行受精卵雄原核共注射后,转基因阳性率为18.4%,转基因表达率为88.89%,显著高于单独进行转基因载体DNA受精卵雄原核注射法。同时,利用agouti基因作为报告基因,可根据毛色变化直接对表达阳性的转基因小鼠进行初步筛选,提高了筛选效率。

关键词：转基因小鼠 piggybac agouti基因基因表达受精卵雄原核共注射