Integration specificity of phage phiC31 integrase in the human genome

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.     

PhiC31 integrase facilitates genetic approaches combining multiple recombinases

Homologous and site-specific DNA recombination has revolutionized genetic engineering. The reliability of recombinases such as Cre and FLP has allowed scientists to design complex strategies to study gene function in mammals. However, the retention of recombination sites in the genome limits the use of Cre and FLP recombinases in subsequent modifications. Access to additional recombinases in the ES cell toolbox would enormously widen the number of possibilities to manipulate the genome. In the method presented here, we combine the use of PhiC31, a site-specific integrase, with FLP to obtain site-specific insertion and replacement in pre-inserted docking sites in the genome of mouse ES cells. This method allows for the integration of any sequence of interest in a pre-defined locus, leaving Cre recombinase available for downstream applications. The selection strategy is based on a silent selection marker activated by a plasmid-delivered promoter, making the integration system highly reliable and reducing the need for extensive molecular screens. This article describes how to create "dockable" mouse embryonic stem (ES) cell lines, integrate incoming vectors, and analyze the resulting clones. Current applications of this technology are also discussed.     

无标记转Fat-1基因真核表达载体的构建及转基因绵羊细胞系的建立

为了建立无筛选标记基因的转Fat-1基因绵羊细胞系,本研究将PCR克隆得到的Fat-1基因,合成的attB、Loxp序列并克隆入pN1-EGFP框架载体,得到可删除筛选标记基因的pEGFP-N1-Fat-1真核表达载体。体外转录合成phiC31整合酶mRNA并与线性化的pEGFP-N1-Fat-1载体共转染绵羊胎儿皮肤成纤维细胞,G418筛选得到表达绿色荧光的单克隆,再利用pET-28a-His-NLS-TAT-Cre质粒诱导Cre重组蛋白表达,将纯化后的Cre穿膜肽转导表达绿色荧光的单克隆细胞,将荧光淬灭的细胞系扩繁,提取基因组DNA,进行PCR及测序鉴定,得到无标记转Fat-1基因绵羊胎儿皮肤成纤维细胞系,为生产无筛选标记基因的转基因绵羊奠定基础。     

Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery

BACKGROUND: Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phiC31 integrase in murine lungs. METHODS: Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI-DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described 'hotspot' were confirmed by polymerase chain reaction (PCR). RESULTS: phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in MLE12 cells. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long-term expression of luciferase after intravenous injection of PEI-DNA complexes could be observed only in the lungs of mice which were co-injected with the integrase-encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on 'day 1' post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described 'hot spot' from liver. CONCLUSIONS: These results provide evidence for the activity of phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.     

A novel approach to plastid transformation utilizes the phiC31 phage integrase

Thus far plastid transformation in higher plants has been based on incorporation of foreign DNA in the plastid genome by the plastid's homologous recombination machinery. We report here an alternative approach that relies on integration of foreign DNA by the phiC31 phage site-specific integrase (INT) mediating recombination between bacterial and phage attachment sites (attB and attP, respectively). Plastid transformation by the new approach depends on the availability of a recipient line in which an attB site has been incorporated in the plastid genome by homologous recombination. Plastid transformation involves insertion of an attP vector into the attB site by INT and selection of transplastomic clones by selection for antibiotic resistance carried in the attP plastid vector. INT function was provided by either expression from a nuclear gene, which encoded a plastid-targeted INT, or expressing INT transiently from a non-integrating plasmid in plastids. Transformation was successful with both approaches using attP vectors with kanamycin resistance or spectinomycin resistance as the selective marker. Transformation efficiency in some of the stable nuclear INT lines was as high as 17 independently transformed lines per bombarded sample. As this system does not rely on the plastid's homologous recombination machinery, we expect that INT-based vectors will make plastid transformation a routine in species in which homologous recombination rarely yields transplastomic clones.     

Identification of pseudo attP sites for phage phiC31 integrase in bovine genome

Streptomyces phage phiC31 integrase was found to mediate site-specific integration of foreign genes at pseudo attP sites of genomes in human, mouse, rat, and Drosophila. This paper reports that phiC31 integrase can also mediate homologous recombination between attB and pseudo attP sites in bovine cells and foreign gene integration was increased at least 2-fold in bovine fibroblasts or Madin-Darby bovine kidney (MDBK) cells. Two intrinsic pseudo attP sites named BpsF1 and BpsM1 located in the inter-gene regions on chromosome 28 and 19, respectively, were identified in bovine genome. These pseudo attP sites shared similar characteristics with those from other species as previously described. Our study demonstrated that the phiC31 integrase system provides a new potential for genetic engineering of the bovine genome and might be beneficial for the research on ruminant.     

家蚕miRNA Novel-31~*上调溶血素基因的表达

【目的】Novel-31*是在家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,Bm CPV)感染的家蚕中发现的一个差异表达miRNA。本研究旨在验证Novel-31*对其靶基因表达的调控作用,以便进一步研究miRNA及其靶基因在昆虫免疫调节中的作用。【方法】用生物信息学方法预测Novel-31*的靶基因,荧光定量PCR分析Novel-31*及其靶基因在家蚕感染Bm CPV后不同时间点的表达变化;构建miRNA慢病毒表达载体和靶基因慢病毒表达载体,转染293T细胞,同时合成Novel-31*mimics转染家蚕培养细胞Bm N,使用荧光定量PCR检测Novel-31*对靶基因表达的调控作用。【结果】生物信息学方法预测发现,溶血素基因是Novel-31*的靶基因,其结合位点位于溶血素基因的5'UTR区域。荧光定量PCR分析表明,Novel-31*及溶血素基因在感染Bm CPV的家蚕血淋巴细胞中呈现明显的上调表达。荧光定量PCR检测表明,在Novel-31*慢病毒表达载体和溶血素基因5'UTR慢病毒表达载体转染的293T细胞中和在转染Novel-31*mimics的家蚕Bm N细胞中,溶血素基因都上调表达。【结论】溶血素基因是miRNA Novel-31*的靶基因,Novel-31*与溶血素基因5'UTR结合,上调溶血素基因的表达。     

Transgene excision in zebrafish using the phiC31 integrase

Genome engineering strategies employing site‐specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase‐estrogen receptor hormone‐binding domain variant fusion protein catalyzes attB‐attP recombination in zebrafish embryos in a 4‐hydroxytamoxifen‐dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish. genesis 48:137–143, 2010. © 2010 Wiley‐Liss, Inc.     

Efficient reversal of phiC31 integrase recombination in mammalian cells

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR. To date, use of phiC31 integrase has been limited to attP × attB recombination. The factor needed for the reverse reaction – the excisionase or recombination directionality factor (RDF) – was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL–attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.     

利用家蚕杆状病毒表达家蚕肌质网型钙离子ATP酶蛋白

肌质网型钙离子ATP酶(Sacro/endoplasmic reticulum Ca2+-ATPase,Serca)负责将细胞中多余的Ca2+转运并存储于内质网中,从而维持细胞内适宜的Ca2+环境。家蚕Serca创造的细胞内及细胞外Ca2+平衡对家蚕正常生命活动的维持具有重要作用。由于Serca分子量较大并具有10次跨膜结构,很难在大肠杆菌表达系统中表达。为了获得具有生物学活性的重组Serca蛋白,利用p Fast Bac Dual载体构建了用于表达egfp和serca的双元杆状病毒表达载体,转染细胞后获得重组病毒,将重组病毒感染细胞后,成功地在细胞中表达了EGFP和Serca。通过荧光观察及Western blotting分析表明,感染后细胞中Serca和EGFP表达模式一致,从感染后48 h开始表达,在感染后96 h表达量最大。对获得的重组蛋白进行酶活分析,发现感染后48 h至120 h的细胞Serca酶活显著提高。表明具有生物学活性的Serca在此系统中成功获得表达,为深入研究Serca蛋白的功能奠定了基础。     

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.     

家蚕V型ATP酶B亚基的克隆及表达特征

V型ATP酶(Vacuolar-type ATPase)是一种定位于细胞膜和细胞器膜上的氢离子转运酶。它利用ATP水解的能量将氢离子转运到液泡、囊泡或者胞外,从而维持细胞内正常的酸碱环境。V型ATP酶B亚基(V-ATPase B)作为ATP的催化位点,也有着非常重要的作用。为了探讨家蚕V-ATPase B(Bm V-ATPase B)的功能,首先从家蚕五龄幼虫的中肠c DNA中克隆了Bm V-ATPase B基因并构建原核表达载体进行原核表达,获得了重组蛋白,经质谱鉴定正确后,通过镍柱亲和层析的方法纯化了该蛋白并制备了多克隆抗体;最后分析了该蛋白在家蚕丝腺中的表达特征并利用免疫荧光对其在丝腺中的表达位置进行了定位。结果显示Bm V-ATPase B基因序列全长1 473 bp,预测蛋白分子量55 k Da,预测等电点5.3。通过Western blotting对家蚕5龄第3天和上蔟第1天幼虫丝腺的不同区段进行Bm V-ATPase B蛋白的表达特征分析,发现在两个时期该蛋白均在前部丝腺高量表达,而在中部丝腺和后部丝腺表达量相对较低。进一步对两个时期丝腺的不同区段进行免疫荧光定位,发现该蛋白在两个时期的前部丝腺、中部丝腺和后部丝腺均定位于细胞层。利用激光共聚焦显微镜对该蛋白进行进一步的定位,发现该蛋白主要在丝腺的细胞膜表达。研究结果明确了该蛋白在丝腺中的表达模式,为深入研究该蛋白在蚕丝纤维形成中的作用奠定了基础。     

High-efficient expression of porcine IL-2 with recombinant baculovirus infected silkworm,Bombyx mori

Biologically active porcine Interleukin-2(poIL-2) was produced fromin vitro andin vivo baculovirus expression systems, namely theAutographa californica nuclear polyhedrosis virus (AcNPV)-cell culture system and the Hybrid nuclear polyhedrosis virus (HyNPV)— silkworm larva system. The concentration of the recombinant poIL-2(rpoIL-2) in the larvae hemolymph was 1 to 3 mg/mL, which was about 7 to 20 times those of the cell culture systems. The level of this expression efficiency is equal to that with transgenic livestock, secretion products in milk.     

A new inducible adenoviral expression system that responds to inflammatory stimuli in vivo

BACKGROUND: Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. METHODS: A luciferase reporter vector with a hybrid promoter containing the human IL-1beta enhancer region (-3690 to - 2720) and the human CIITA promoter IV (-399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1beta/CIITApIV promoter (Ad-IL1beta/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1beta/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1beta/CIITApIV promoter. RESULTS: The IL1beta/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1beta/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. CONCLUSIONS: Using the IL1beta/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity.     

蜕皮激素对家蚕卵黄原蛋白基因表达的调控

蜕皮激素（20-hydroxy ecdysone,20E）是由前胸腺分泌的能调节节肢动物昆虫纲、甲壳纲等动物蜕皮的激素. 家蚕属于完全变态昆虫,一生经历卵、幼虫、蛹、成虫四个发育时期. 20E在家蚕发育过程中有不可替代的作用. 本研究通过酶联免疫吸附反应（ELISA）测定家蚕变态期前后血淋巴中20E滴度,发现在卵黄原蛋白基因 (Bombyx mori vitellogenin, BmVg) 高量表达前的雌雄血淋巴中20E含量没有明显差异,但含量变化趋势与BmVg表达趋势相一致. 用20E注射上蔟后60 h的家蚕和添食5龄家蚕, 能诱导雌性和雄性脂肪体中BmVg表达和蛋白合成. 调查处理后的家蚕所产卵中的卵黄磷蛋白（BmVn）含量及重量,发现蚕卵中BmVn含量及蚕卵重量都明显增加|本研究还通过脂肪体体外培养,证明了20E是直接作用于脂肪体来诱导BmVg的表达. 本研究结果表明,20E是通过诱导脂肪体中BmVg的转录来增加蚕卵中BmVn积累,从而最终使得蚕卵重量也相应增加.     

家蚕组织蛋白酶基因家族的鉴定及表达特征分析

家蚕是鳞翅目完全变态昆虫,在其变态过程中伴随着巨大的形态变化,包括旧组织的解离和新组织的形成,在这过程中有多种组织蛋白酶参与。组织蛋白酶是一类细胞内蛋白酶,广泛存在于各个物种中,包括组织蛋白酶B、H、L等几个亚家族。对家蚕组织蛋白酶的研究将有利于阐明家蚕变态发育的详细过程。通过对家蚕基因组数据库进行筛选,共在家蚕中鉴定到13种组织蛋白酶,并对这13种组织蛋白酶的基本信息和表达模式进行了分析。另外,利用家蚕基因芯片数据和荧光定量PCR分析,鉴定编号为BGIBMGA004622的基因为卵巢特异表达的组织蛋白酶L亚家族基因。该基因全长1 209 bp,编码402个氨基酸。经过序列分析,该酶与其他物种的组织蛋白酶L具有较高的同源性,其活性位点高度保守,且与鳞翅目的组织蛋白酶L在进化上聚为一支。同时,对该基因进行克隆并原核表达,结果显示重组蛋白以包涵体的形式表达。定量PCR结果显示,该酶在蛹发育初期表达量逐渐升高,至蛹3 d达到最高值,推测其可能参与卵巢与卵母细胞的发育过程。     

Generating Customized Transgene Landing Sites and Multi-Transgene Arrays in Drosophila Using phiC31 Integrase

Transgenesis in numerous eukaryotes has been facilitated by the use of site-specific integrases to stably insert transgenes at predefined genomic positions (landing sites). However, the utility of integrase-mediated transgenesis in any system is constrained by the limited number and variable expression properties of available landing sites. By exploiting the nonstandard recombination activity exhibited by a phiC31 integrase mutant, we developed a rapid and inexpensive method for isolating landing sites that exhibit desired expression properties. Additionally, we devised a simple technique for constructing arrays of transgenes at a single landing site, thereby extending the utility of previously characterized landing sites. Using the fruit fly Drosophila melanogaster, we demonstrate the feasibility of these approaches by isolating new landing sites optimized to express transgenes in the nervous system and by building fluorescent reporter arrays at several landing sites. Because these strategies require the activity of only a single exogenous protein, we anticipate that they will be portable to species such as nonmodel organisms, in which genetic manipulation is more challenging, expediting the development of genetic resources in these systems.     

A bipartite system for the constitutive and inducible expression of high levels of foreign proteins in plants

We have developed combined transgene/virus vector systems for the expression of heterologous proteins in plants. The systems are based on the bipartite RNA plant virus, cowpea mosaic virus (CPMV), and involve the amplification of integrated copies of either full-length or deleted versions of RNA-2 carrying a foreign gene. In the case of plants transgenic for full-length versions of RNA-2 carrying the green fluorescent protein (GFP), amplification can be achieved by supplying RNA-1 either exogenously or by crossing. This allows either inducible or constitutive expression of the foreign gene and results in an infection that can be passaged to further plants. Replication of deleted versions of RNA-2 harbouring GFP requires the presence of both RNA-1 and a suppressor of gene silencing, a function which we show can be supplied by HcPro from potato virus Y. Replication of the deleted versions of RNA-2 can be achieved by supplying the suppressor and RNA-1 either exogenously or by crossing, showing that this system can also be used in an inducible and constitutive format. The use of deleted forms of RNA-2 has the advantage that no infectious virus is produced, providing an effective method of biocontainment. The CPMV-based systems have advantages over existing plant expression systems in terms of the expression levels obtainable and the simplicity and flexibility of use, and should be of great practical benefit in the development of plants as bioreactors.