Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Genetic Elements Required for Integration

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP x attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.     

Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile “transgene maintenance and control” strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.     

Plant development inhibitory genes in binary vector backbone improve quality event efficiency in soybean transformation

Conventional Agrobacterium-mediated plant transformation often produces a significant frequency of transgenic events containing vector backbone sequence, which is generally undesirable for biotechnology applications. We tested methods to reduce the frequency of transgenic plants containing vector backbone by incorporating genes into the backbone that inhibit the development of transgenic plants. Four backbone frequency reduction genes, bacterial levansucrase (sacB), maize cytokinin oxidase (CKX), Phaseolus GA 2-oxidase (GA 2-ox), and bacterial phytoene synthase (crtB), each expressed by the enhanced CaMV 35S promoter, were placed individually in a binary vector backbone near the left border (LB) of binary vectors. In transformed soybean plants, the lowest frequency of backbone presence was observed when the constitutively expressed CKX gene was used, followed by crtB. Higher backbone frequencies were found among the plants transformed with the GA 2-oxidase and sacB vectors. In some events, transfer of short backbone fragments appeared to be caused by LB readthrough and termination within the backbone reduction gene. To determine the effect of the backbone genes on transformation frequency, the crtB and CKX vectors were then compared to a control vector in soybean transformation experiments. The results revealed that there was no significant transformation frequency difference between the crtB and control vectors, but the CKX vector showed a significant transformation frequency decrease. Molecular analysis revealed that the frequency of transgenic plants containing one or two copies of the transgene and free of backbone was significantly increased by both the CKX and crtB backbone reduction vectors, indicating that there may be a correlation between transgene copy number and backbone frequency.     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.     

The Determination of Importance of Sequences Neighboring the Psi Sequence in Lentiviral Vector Transduction and Packaging Efficiency

A number of lentiviral vector systems have been developed for gene delivery and therapy by eliminating and/or modifying viral genetic elements. However, all lentiviral vector systems derived from HIV-1 must have a viral packaging signal sequence, Psi (Ψ), which is placed downstream of 5′ long terminal repeat in a transgene plasmid to effectively package and deliver transgene mRNA. In this study, we examined feasible regions or sequences around Psi that could be manipulated to further modify the packaging sequence. Surprisingly, we found that the sequences immediately upstream of the Psi are highly refractory to any modification and resulted in transgene vectors with very poor gene transduction efficiency. Analysis around the Psi region revealed that there are a few sites that can be used for manipulation of the Psi sequence without disturbing the virus production as well as the efficiency of transgene RNA packaging and gene transduction. By exploiting this new vector system, we investigated the requirement of each of four individual stem-loops of the Psi sequence by deletion mapping analysis and found that all stem-loops, including the SL4 region, are needed for efficient transgene RNA packaging and gene delivery. These results suggest a possible frame of the lentiviral vector that might be useful for further modifying the region/sequence around the packaging sequence as well as directly on the Psi sequence without destroying transduction efficiency.     

Locus control region of the human CD2 gene in a lentivirus vector confers position-independent transgene expression

Vectors derived from murine leukemia virus (MLV) have been used in many human gene therapy clinical trials. However, insertion of the locus control regions (LCRs) derived from the beta-globin gene locus or the CD2 gene into MLV vectors frequently led to vector rearrangement. Since the human immunodeficiency virus (HIV) sequence diverges significantly from the MLV sequence, we tested whether the LCR sequence is more stable in the context of an HIV vector. Clones derived from human fibrosarcoma line HT1080 cells transduced with an HIV vector containing the T-cell-specific CD2 LCR exhibit the same wide range of transgene expression as clones lacking the LCR. In contrast, Jurkat and primary T-cell clones derived from the transduction of the LCR-containing vector show, on average, a three- to fourfold increase in transgene expression relative to that of the control vector. This is consistent with previous observations that the CD2 LCR contains a T-cell-specific enhancer. In addition, the clones derived from the LCR-containing vector have a much lower clonal variation in transgene expression than those derived from the control vector. We also demonstrate that the level of transgene expression is proportional to the vector copy number. These results suggest that the human CD2 LCR sequence is compatible with HIV vector sequences and confers enhanced integration site-independent and copy number-dependent expression of the transgene. Thus, HIV vectors may represent the ideal vehicle to deliver genes controlled by various cis-acting elements such as LCRs.     

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.     

Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted.

Although adenovirus vectors offer many advantages, it would be desirable to develop vectors with improved expression and decreased toxicity. Toward this objective, an adenovirus vector system with deletion of both the El and E2a regions was developed. A 5.9-kb fragment of the adenovirus type 5 (Ad5) genome containing the E2a gene and its early and late promoters was transfected into 293 cells. A complementing cell line, designated 293-C2, expressed the E2a mRNA and protein and was found to complement the defect in Ad5 viruses with temperature-sensitive or deletion mutations in E2a. A deletion of 1.3 kb removing codons 40 to 471 of the 529 amino acids of E2a was introduced into plasmids for preparation of viruses and vectors. An Ad5 virus with disruption of the El gene and deletion of E2a grew on 293-C2 cells but not on 293 cells. Vectors with E1 and E2a deleted expressing Escherichia coli beta-galactosidase or human alpha1-antitrypsin were prepared and expressed the reporter genes after intravenous injection into mice. This vector system retains sequences in common between the complementing cell line and the vectors, including 3.4 kb upstream and 1.1 kb downstream of the deletion. These vectors have potential advantages of increased capacity for insertion of transgene sequences, elimination of expression of E2a, and possibly reduction in expression of other viral proteins. Although the titers of the vectors with deleted are about 10- to 30-fold below those of vectors with E2a wild-type regions, the former vectors are suitable for detailed studies with animals to evaluate the effects on host immune responses, on duration of expression, and on safety.     

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.     

Understanding and improving transgene stability and expression in insects for SIT and conditional lethal release programs

Genetically transformed insect pests provide significant opportunities to create strains for improved sterile insect technique and new strategies based on conditional lethality. A major concern for programs that rely on the release of transgenic insects is the stability of the transgene, and maintenance of consistent expression of genes of interest within the transgene. Transgene instability would influence the integrity of the transformant strain upon which the effectiveness of the biological control program depends. Loss or intra-genomic transgene movement would result in strain attributes important to the program being lost or diminished, and the mass-release of such insects could significantly exacerbate the insect pest problem. Instability resulting in intra-genomic movement may also be a prelude to inter-genomic transgene movement between species resulting in ecological risks. This is less of a concern for short-term releases, where transgenic insects are not expected to survive in the environment beyond two or three generations. Transgene movement may occur, however, into infectious agents during mass-rearing, and the potential for movement after release is a possibility for programs using many millions of insects. The primary methods of addressing potential transgene instability relate to an understanding of the vector system used for gene transfer, the potential for its mobilization by the same or a related vector system, and methods required to identify transformants and determine if unexpected transgene movement has occurred. Methods also exist for preventing transposon-mediated mobilization, by deleting or rearranging vector sequences required for transposition using recombination systems. Stability of transgene expression is also a critical concern, especially in terms of potential epigenetic interactions with host genomes resulting in gene silencing that have been observed in plants and fungi, and it must be determined if this or related phenomena can occur in insects.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

Employing site-specific recombination for conditional genetic analysis in Sinorhizobium meliloti

The ability to remove a genetic function from an organism with good temporal resolution is crucial for characterizing essential genes or genes that act in complex developmental programs. The rhizobium-legume symbiosis involves an elaborate two-organism interaction requiring multiple levels of signal exchange. As an important step toward probing rhizobium genetic functions with temporal resolution, we present the development of a conditional gene deletion system in Sinorhizobium meliloti that employs Cre/loxP site-specific recombination. This system enables chemically inducible and irreversible gene deletion or gene upregulation. Recombinase-mediated excision events can be positively or negatively selected or monitored by a colorimetric assay. The system may be adaptable to various bacterial species, in which recombinase activity may be placed under the control of diverse user-defined promoters. This system also shows promise for uses in promoter trapping and biosensing applications.     

Construction of an integration-proficient vector based on the site-specific recombination mechanism of enterococcal temperate phage phiFC1

The genome of temperate phage phiFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage phiFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage phiFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 x 10(3) transformants/microg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage phiFC1 can be used for genetic engineering in E. faecalis and in other hosts.     

The long road to recombinase‐mediated plant transformation

The use of site‐specific recombinases to manipulate eukaryotic genomes began nearly three decades ago. Although seemingly parallel efforts were being made in animal and plant systems, the motivation for its development in plants was unique to, at least at the time, crop bioengineering issues. The impetus behind site‐specific deletion in plants was to remove antibiotic resistance genes used during transformation but unnecessary in commercial products. Site‐specific integration in plants was more than academic curiosity of position effects on gene expression, but a necessary step towards developing the serial stacking of DNA to the same chromosome locus – to insure that bioengineered crops can be improved over time through transgene additions without inflating the number of segregating loci. This article is not a review of the literature on site‐specific recombination, but a first person account of the series of events leading to the development of a gene stacking transformation system in plants.     

A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector

The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.     

Marker-Free Transgenic Chrysanthemum Obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation with Twin T-DNA Binary Vectors

In this study, a superbinary vector was constructed to evaluate the potential of a twin T-DNA system for generating selectable marker-free transgenic chrysanthemum plants. The first T-DNA of the pCAMBIA 1300 vector contained the hygromycin phosphotransferase (hpt) selectable marker gene, while the second T-DNA carried the β-glucuronidase gene (uidA) and featuring the gene of interest. The two T-DNA regions were placed adjacent to each other with no intervening region. This vector was then used to transform transversal thin cell layers (1–2 mm thick) of internodal stem segments of chrysanthemum via Agrobacterium-mediated transfer. Putative transgenic plants were obtained and analyzed for presence and integration of the transgene using polymerase chain reaction amplification and Southern blotting. The primary cotransformation frequency was calculated at 38.4%. A total of 17 hpt-resistant/gus-positive T0 plants were evaluated for segregation in the next generation (T1), and among those approximately 15.7% carried the transgene. Overall, the two T-DNA system appeared to be a useful approach to generate marker-free transgenic chrysanthemum plants, thereby eliminating public concerns regarding proliferation of antibiotic and herbicide resistance genes into the environment.     

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.