Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

地衣芽胞杆菌dif序列的功能鉴定

地衣芽胞杆菌是重要的工业菌株,如何为其建立一种有效的基因删除技术是对该菌株进行遗传改良的基础。枯草芽胞杆菌difBs序列已经被成功用于枯草芽胞杆菌多基因的删除。在分析地衣芽胞杆菌基因组序列并获得与枯草芽胞杆菌difBs序列十分相似的一段序列difBLi的基础上,构建了在庆大霉素抗性基因两侧具有difBLi的重组质粒pMD19-difGm和pHY-XI′::difGm,通过电击转化法将质粒pHY-XI′::difGm导入B.lichen-iformisATCC14580中,筛选获得了具有庆大霉素抗性的转化子。在转化子的传代过程中,重组质粒的庆大霉素抗性基因在体内Xer/dif位点特异性重组系统的介导下通过其侧翼的dif位点进行同源重组而被准确删除。确证了地衣芽胞杆菌中dif序列的功能,为地衣芽胞杆菌基因组中多基因的删除提供了一种新的实验途径。

XerCD/dif位点特异性重组

大约10%~15%的大肠杆菌在染色体复制过程中会形成染色体二聚体。大肠杆菌染色体编码的重组酶XerC和XerD作用于染色体复制终点区的dif序列,以同源重组的方式将染色体二聚体解离为单体,使细菌得以正常复制分裂。编码霍乱毒素的噬菌体CTXΦ以位点特异的方式整合入霍乱弧菌染色体,但其基因组中不含有任何重组酶基因,其整合过程需要细菌染色体编码的XerC和XerD重组酶,且整合位点与大肠杆菌dif序列相似。XerCD重组酶基因和dif位点在细菌染色体广泛存在,表明其可能是染色体二聚体解离,噬菌体及其他外源基因成分整合入染色体过程中一种广泛存在的途径。文章对XerCD/dif位点特异性重组在细菌染色体二聚体解离。

Establishment and application of both FLP and Cre site-specific recombination systems at the same position in the genome

Both FRT-FRT and LoxP-LoxP sites that are the target sepuences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technigue. C4LFY also contains P elements, 2 exons and 1 intron of Drosophila yellow gene, yellow promoter and enhancers, and flanking DNA. Since C4LFY made use of two pairs of FRT and LoxP sites, this vector included two site-specific recombination systems. C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation. in the presence of the FLP or Cre recombinase, either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome. Using this system, the molecular basis of yellow gene expression and regulation during development have been investigated. Results indicate that the tissue-specific expression of yellow gene is directly regulated by transcriptional enhancers. in addition, the 5’ and 3’ genomic sequences flanking the yellow gene have been

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .

Site-specific recombination in Escherichia coli mediated by actinomyces phage R4 integrase

The purpose of this study was to demonstrate that actinomyces phage R4 integrase Sre protein efficiently mediate site-specific recombination in Escherichia coll. An intramolecular recombination assay system in E. coli was constructed. The plasmid pBZP contains attB and attP sites in direct orientation flanking a lacZ gene. When pBZP was introduced into E. coli cells, in which the plasmid pSREA containing sre gene was resident, Sre protein catalyzed integration of attP into attB site, resulting in excision of the lacZ gene. This integration changed bacteria colonies from blue to white on agar plates containing X-Gal, which showed that the lacZ was removed. The integrant DNAs were identified by enzyme digestion, PCR and DNA sequencing. The minimal sizes of attB and attP were 50 bp and 47 bp for 100% recombination efficiency. The phage recombinase Sre efficiently integrated attP into attB site to create attR and attL in E. coli host environment without Streptomyces specific cofactors. This intrmolecular assay system is a simple and efficient system for Sre-mediated recombination in E. coll.

Modification and identification of a vector for making a large phage antibody library

Background The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies. Methods scFv was obtained by overlap polymerase chain reaction （PCR） amplification with the newly designed VL and VH extension primers. Ioxp511 was flanked by VL and VH and the endonuclease ACC Ⅲ encoding sequences were introduced on both sides of Ioxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated. Results The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression. Conclusions The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

Bioorthogonal Engineered Virus‑Like Nanoparticles for Efficient Gene Therapy

Gene therapy offers potentially transformative strategies for major human diseases.However,one of the key challenges in gene therapy is developing an effective strategy that could deliver genes into the specific tissue.Here,we report a novel virus-like nanoparticle,the bioorthgonal engineered viruslike recombinant biosome(reBiosome),for efficient gene therapies of cancer and inflammatory diseases.The mutant virus-like biosome(mBiosome)is first prepared by site-specific codon mutation for displaying 4-azido-L-phenylalanine on vesicular stomatitis virus glycoprotein of eBiosome at a rational site,and the reBiosome is then prepared by clicking weak acid-responsive hydrophilic polymer onto the mBiosome via bioorthogonal chemistry.The results show that the reBiosome exhibits reduced virus-like immunogenicity,prolonged blood circulation time and enhanced gene delivery efficiency to weakly acidic foci(like tumor and arthritic tissue).Furthermore,reBiosome demonstrates robust therapeutic efficacy in breast cancer and arthritis by delivering gene editing and silencing systems,respectively.In conclusion,this study develops a universal,safe and efficient platform for gene therapies for cancer and inflammatory diseases.

Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories.For some uncommon microbial hosts,such as Mycolicibacterium and Mycobacterium species,however,it is a challenge.Here,we present a multiplexed integrase-assisted site-specific recombination(miSSR)method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories.First,a single-step multi-copy integration method was established in M.neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy,the efficiencies of which were~100%for no more than three-copy integration events and decreased sharply to~20%for five-copy integration events.Second,the R4,Bxb1 andΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events.Third,a reconstructed mycolicibacterial Xer recombinases(Xer-cise)system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation.As a proof of concept,the biosynthetic pathway of ergothioneine(EGT)in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system.With six copies of the biosynthetic gene clusters(BGCs)of EGT and pentose phosphate isomerase(PRT),the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L,which was 3.77 times of that in the wild strain.The improvements indicated that the miSSR system was an effective,flexible,and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

Identification of E. coli K12 chromosomal insertion sites of bacteriophage φ297

Objective:To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods:Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which is similar to that of phage 933W to an unknown region in genomic DNA. A special adaptor is ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and subclone of PCR products, DNA sequencing and data analysis were used in this study. Results:The attL, attR and the core sequences were determined. The bacterial attachment site of phage φ297 was located in the yecE gene of E. coli K12. Conclusion:The phage φ297 integrates into the yecE gene of the E. coli K12 genome.

Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants

Targeted genome engineering refers to technologies that are used for site-specific genome modifications such as knockout, knockin and transcriptional regulation of genes of interest in organisms. Site-specific recombination system, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) technologies are the representatives of targeted genome engineering and have been widely used in crop basic and applied research. In this review, we introduce the basic information and action modes of these different genome engineering technologies, summarize the recent progresses of targeted genome engineering technologies and their applications in crop improvement, and propose perspectives for genome engineering-mediated modifications of crop plants in the future.

Recombinase-mediated Gene Stacking as a Transformation Operating System

The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site- specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.

An Open-Source System for In Planta Gene Stacking by Bxbl and Cre Recombinases

The rapid development of crops with multiple transgenic traits arouses the need for an efficient system for creating stacked cultivars. Most major crops rely on classical breeding to introgress the transgene from a laboratory variety to the numerous cultivars adapted to different growing regions. Even with vegetative propagated crops, genetic crosses are conducted during varietal improvement prior to vegetative cloning. The probability to assort the ＇x＇ number of transgenic loci into a single genome may seem trivial, （~）x for a diploid species, but given the ＇y＇ number of other nontransgenic traits that breeders also need to assemble into the same genome, the （~）~＊y probability for a ＇breeding stack＇ could quickly make the line conversion process unmanageable. Adding new transgenes onto existing transgenic varieties without creating a new segregating locus would require site-specific integration of new DNA at the existing transgenic locus. Here, we tested a recombinase-mediated gene-stacking scheme in tobacco. Sequential site-specific inte- gration was mediated by the mycobacteriophage Bxbl integrase-catalyzed recombination between attP and attB sites. Transgenic DNA no longer needed after integration was excised by Cre recombinase-mediated recombination of Iox sites. Site-specific integration occurred in -10% of the integration events, with half of those events usable as substrates for a next round of gene stacking. Among the site-specific integrants, however, a third experienced gene silencing. Overall, precise structure and reproducible expression of the sequentially added triple traits were obtained at an overall rate of -3% of the transformed clones--a workable frequency for the development of commercial cultivars. Moreover, since nei- ther the Bxbl-att nor the Cre-lox system is under patent, there is freedom to operate,

基于Red重组系统和Xer重组系统的大肠杆菌多基因删除方法

快速、高效删除大肠杆菌染色体DNA的目的基因是大肠杆菌代谢工程研究的前提和基础。利用Red重组系统结合Xer重组系统删除了野生型大肠杆菌CICIM B0013的ackA-pta基因和pps基因。实验证明了可重复应用dif位点实现大肠杆菌染色体上多基因突变的叠加,同时,在染色体上并未留下抗生素标记,借此能够高效地实现多基因缺失突变株的构建。此外,本方法重组效率高,实验步骤较简便。

产琥珀酸重组大肠杆菌的构建和发酵性能

琥珀酸是一种重要的化工产品,微生物发酵法生产琥珀酸具有广阔的应用前景.野生大肠杆菌厌氧发酵产物中琥珀酸含量低,且有大量副产物.为构建高产琥珀酸的重组大肠杆菌,阻断代谢旁路,利用Xer/dif重组酶系统对Escherichia coli CICIM B0013进行代谢途径的调控研究.结果显示,敲除了包括乙酸激酶和磷酸乙酰转移酶基因(ackA-pta)、乳酸脱氢酶基因(ldhA)、丙酮酸甲酸裂解酶基因(pflB)、乙醇脱氢酶基因(adhE)等琥珀酸合成竞争途径中关键酶基因的重组大肠杆菌菌株E.coli CICIM B0013-1040,其发酵液中琥珀酸成为主要产物得到积累;进一步对其PTS系统进行修饰并适当增强PEP羧化酶基因(ppc)表达剂量,成功获得一株具备较强琥珀酸生产能力的菌株E.coli CICIM B0013-1050(pTH-ppc),该菌株厌氧转化葡萄糖36 h,琥珀酸产量达到36.2 g/L,生产强度为1.01 g L-1 h-1,葡萄糖-琥珀酸转化率为64.3%,发酵液经高效液相色谱(HPLC)检测,无乳酸、乙酸、甲酸、乙醇生成.

Genome Editing:From Drosophila to Non-Model Insects and Beyond

Insect is the largest group of animals on land.Many insect species inflict economical and health losses to humans.Yet many more benefit us by helping to maintain balances in our ecosystem.The benefits that insects offer remain largely untapped,justifying our continuing efforts to develop tools to better understand their biology and to better manage their activities.Here we focus on reviewing the progresses made in the development of genome engineering tools for model insects.Instead of detailed descriptions of the molecular mechanisms underlying each technical advance,we focus our discussion on the logistics for implementing similar tools in non-model insects.Since none of the tools were developed specific for insects,similar approaches can be applied to other non-model organisms.

Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches： （i） AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain （GVG） chemical-inducible expression system; （ii） deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and （iii） suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element （HPT-Ds）, We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community,