attB Site Research Articles

Characterization of the Staphylococcus aureus rRNA Methyltransferase Encoded by orfX, the Gene Containing the Staphylococcal Chromosome Cassette mec (SCCmec) Insertion Site

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/β-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 μm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/β-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.

Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by streptomyces phage phiC31 integrase

Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5′ and 3′ junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.

Multiple Mechanisms Contribute to Lateral Transfer of an Organophosphate Degradation (opd) Island in Sphingobium fuliginis ATCC 27551

The complete sequence of pPDL2 (37,317 bp), an indigenous plasmid of Sphingobium fuliginis ATCC 27551 that encodes genes for organophosphate degradation (opd), revealed the existence of a site-specific integrase (int) gene with an attachment site attP, typically seen in integrative mobilizable elements (IME). In agreement with this sequence information, site-specific recombination was observed between pPDL2 and an artificial plasmid having a temperature-sensitive replicon and a cloned attB site at the 3′ end of the seryl tRNA gene of Sphingobium japonicum. The opd gene cluster on pPDL2 was found to be part of an active catabolic transposon with mobile elements y4qE and Tn3 at its flanking ends. Besides the previously reported opd cluster, this transposon contains genes coding for protocatechuate dioxygenase and for two transport proteins from the major facilitator family that are predicted to be involved in transport and metabolism of aromatic compounds. A pPDL2 derivative, pPDL2-K, was horizontally transferred into Escherichia coli and Acinetobacter strains, suggesting that the oriT identified in pPDL2 is functional. A well-defined replicative origin (oriV), repA was identified along with a plasmid addiction module relB/relE that would support stable maintenance of pPDL2 in Sphingobium fuliginis ATCC 27551. However, if pPDL2 is laterally transferred into hosts that do not support its replication, the opd cluster appears to integrate into the host chromosome, either through transposition or through site-specific integration. The data presented in this study help to explain the existence of identical opd genes among soil bacteria.

Efficacy and Site-Specificity of Adenoviral Vector Integration Mediated by the Phage φC31 Integrase

Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage φC31 integrase (φC31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing φC31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration. Efficacy and specificity were determined by scoring the number of puromycin-resistant colonies and by sequencing integration sites. Unexpectedly, circularization of HD was unnecessary and it even reduced the integration efficacy. The maximum integration efficacy achieved was 0.5% in HeLa cells and 0.1% in C2C12 myoblasts. Up to 76% of the integration events occurred at pseudo attP sites and previously characterized hotspots were found. A small (two- to three-fold) increase in the number of γ-H2AX positive foci, accompanied by no noticeable change in γ-H2AX expression, indicated the low genotoxicity of φC31-Int. In conclusion, integration of HD mediated by φC31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity.

ΦC31 Integrase Mediates Efficient Site-Specific Integration in Sheep Fibroblasts

ΦC31 integrase, a site-specific recombinase, can effectively mediate the integration of foreign genes bearing an attB sequence into pseudo attP sites of genomes in human, mouse, and Drosophila cells. In this study, we measured ΦC31 integrase-mediated homologous recombination between attB and pseudo attP sites in sheep cells. The integration efficiency of the EGFP expression cassette with the attB sequence increased at least 2-fold in sheep fibroblasts. Three pseudo-attP sites were identified in the sheep genome, located in the intergenic regions on chromosomes 4, 13, and 7 respectively. Moreover, the transgene that was integrated at the three pseudo attP sites exhibited high levels of expression. Our study indicates that the ΦC31 integrase system provides an efficient integration tool for genetic engineering of the sheep genome.

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3' end of the tRNA(leu) gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.

Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites

Metabolic engineering of Escherichia coli using plasmids is problematic, which is addressed by developing a toolbox for genomic engineering of E. coli. This toolbox includes the attP site-based integration vectors and the attB site-based template vectors, equipped with mutant loxP sites (i.e., LE* and RE*). The former vectors facilitate integration of passenger genes into attB sites while the latter allows creation of new attB sites. Consequently, the inserted vector backbone is flanked by LE* and RE* sites and can be rescued by Cre. Based on this approach, the biosynthetic pathway of poly(3-hydroxybutyric acid) was first built in E. coli. By scoring the observable phenotype of integrants, the result revealed that the efficiency of gene integration could reach 100%. In addition, reconstruction of the n-butanol-synthesizing pathway in E. coli resulted in a plasmid-free producer strain. As a consequence, the producer strain was able to stably overproduce n-butanol (3.7g/L) from glucose (20g/L). Finally, exoglucanase was overexpressed in E. coli that carried multiple genomic copies of the celY gene. Overall, it indicates a promise of our method for cycling improvement of E. coli.

PhiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

PhiC31 integrase-mediated cassette exchange in silkworm embryos

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6 h after oviposition and recovered plasmid DNAs from the embryos 3 days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.

Streptomyces temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms)

ϕC31, ϕBT1, R4, and TG1 are temperate bacteriophages with broad host specificity for species of the genus Streptomyces. They form lysogens by integrating site-specifically into diverse attB sites located within individual structural genes that map to the conserved core region of streptomycete linear chromosomes. The target genes containing the ϕC31, ϕBT1, R4, and TG1 attB sites encode a pirin-like protein, an integral membrane protein, an acyl-CoA synthetase, and an aminotransferase, respectively. These genes are highly conserved within the genus Streptomyces, and somewhat conserved within other actinomycetes. In each case, integration is mediated by a large serine recombinase that catalyzes unidirectional recombination between the bacteriophage attP and chromosomal attB sites. The unidirectional nature of the integration mechanism has been exploited in genetic engineering to produce stable recombinants of streptomycetes, other actinomycetes, eucaryotes, and archaea. The ϕC31 attachment/integration (Att/Int) system has been the most widely used, and it has been coupled with the ϕBT1 Att/Int system to facilitate combinatorial biosynthesis of novel lipopeptide antibiotics in Streptomyces fradiae.

Site-Specific Integration with Bacteriophage ɸC31 Integrase

Few nonviral techniques exist for efficient and stable eukaryotic gene transfer and fewer still are broadly useful in both cell culture and whole-organism applications. C31 integrase, a site-specific bacteriophage recombinase, is able to catalyze chromosomal transgene insertion under a diverse range of experimental and therapeutic conditions. The enzyme recognizes and catalyzes unidirectional recombination between attachment motifs found in phage and bacterial genomes (attP and attB sites, respectively). Use of C31 integrase for gene transfer requires that an attB sequence be cloned into a transgene-bearing plasmid. When this modified plasmid is introduced into cells alongside integrase-expressing plasmid, C31 integrase is able to catalyze insertion of the transgene plasmid into one of a limited pool of sites in the target genome that show sequence similarity to wild-type attP. Efficient delivery of C31 integrase and attB donor plasmid to the tissue or cells of interest remains the most challenging aspect of the system. Unlike viral methods of genome manipulation, use of C31 integrase almost always requires an additional method of stimulating cellular DNA uptake. However, the relative simplicity of the plasmid-based system means that nearly any proven method of introducing exogenous DNA into cells can be used with C31 integrase. This protocol describes the use of C31 integrase in mammalian cell culture for the creation of clonal lines showing robust and stable expression of an experimental transgene.

Adjusting the attB Site in Donor Plasmid Improves the Efficiency of ΦC31 Integrase System

ΦC31 integrase, a site-specific recombinase, can catalyze integration of circular DNA bearing attB site into pseudo attP sites in mammalian genomes. However, the integration efficiency mediated by integrase is relatively low. Our study centered on the investigation of the impact of the position, orientation, and number of attBs in the donor plasmid on the efficiency of ΦC31 integrase system. Donor plasmids bearing various types of attBs (including forward and reverse directions, tandem, and intersperse) and reporter enhanced green fluorescent protein (EGFP) were constructed. The plasmids plus helper plasmid encoding integrase were co-transfected into HeLa cells. After G418 selection, the resistant cell colonies were counted for calculating chromosomal integration frequency. EGFP expression was detected by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay analysis. The results showed that efficiency of integration mediated by integrase accounted for 70% ± 7.1% of total integration events in the transfected HeLa cells. Compared with a forward orientation of attB in donor plasmid, a reverse direction of attB or interspersed attBs showed 1.5- or 2.8-fold increase in integration efficiency, respectively, while tandem attBs in donor plasmids caused a decreased efficiency of integration. We conclude that the adjustment of attB sites in donor plasmids may be of value for gene therapy and routine genetic engineering by using ΦC31 integrase system.

Construction of intergeneric conjugal transfer for molecular genetic studies of Streptomyces acidiscabies producing thaxtomin

Effective transformation procedure to facilitate molecular genetic studies of Streptomyces acidiscabies producing thaxtomin, which causes scab diseases in the economically important root and tuber crops was established via transconjugation from Escherichia coli ET12567 using an OC31-derived integration vector, pSET152, harboring the oriT and attP fragments. Greatest number of exconjugants was achieved on MS medium containing 50 mM MgCl2 without heat treatment of spores and E. coli cells. Additionally, the integration site, attB, of the genome of S. acidiscabies exhibited the highest degree of homology with S. avermitilis and its chromosomal location was found to exist as a single attB site within an open reading frame coding for a pirin homolog similar to those identified in other actinomycetes.

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (Φ) C31. The ΦC31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively). This recombination is stable and does not revert. A "landing pad" (LP) sequence consisting of a spectinomycin-resistance gene, aadA (SpR), and the E. coli ß-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob. The expression of the ΦC31 integrase gene (cloned from pHS62) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK7813. A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

In vivo evaluation of PhiC31 recombinase activity in transgenic mice

Genome engineering strategies employing site-specific recombinases (SSRs) such as Cre, Flp and PhiC31, have become powerful tools to analyze gene function and manipulate neural network in vertebrates. In the present study, we evaluated the ability of PhiC31 phage integrase to induce genomic recombination in transgenic mice. PhiC31 is the integrase encoded by the Streptomyces bacteriophage that promotes recombination between heterotypic attP and attB sites. We generated transgenic mice that express codon-optimized PhiC31 (PhiC31o) in neural stem/progenitor cells or tyrosine hydroxylase (TH) expressing catecholaminergic neurons. PhiC31 was functional in these cells and capable of excising a transcriptional stop cassette flanked by PhiC31-specific attP/B recognition sites. PhiC31-ERT2, a fusion protein of PhiC31o (without the nuclear localization signal) and the mutated ligand-binding domain of the human estrogen receptor, was able to induce recombination in neural stem/progenitor cells in a tamoxifen-dependent manner, but the recombination rate was less efficient than for PhiC31. Thus, PhiC31 integrase is functional in transgenic mice and is suitable for mosaic recombination in restricted cell populations.

A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome

BackgroundSingle-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration.FindingsWe have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site.ConclusionsIn this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for marking strains for in vivo studies.

Analysis of the Site-Specific Integration System of the Streptomyces aureofaciens Phage μ1/6

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

Site-specific genome integration in alphaproteobacteria mediated by TG1 integrase

The serine-type phage integrase is an enzyme that catalyzes site-specific recombination between two attachment sites of phage and host bacterial genomes (attP and attB, respectively) having relatively short but distinct sequences without host auxiliary factor(s). Previously, we have established in vivo and in vitro site-specific recombination systems based on the serine-type integrase produced by actinophage TG1 and determined the minimal sizes of attP(TG1) and attB(TG1) sites required for the in vitro TG1 integrase reaction as 43- and 39-bp, respectively. Here, DNA databases were surveyed by FASTA program with the authentic attB(TG1) sequence of Streptomyces avermitilis as a query. As a result, possible attB(TG1) sequences were extracted from genomes of bacterial strains belonging to Class Alphaproteobacteria in addition to those of Class Actinobacteria. Those sequences extracted with a high similarity score and high sequence identity (we took arbitrarily more than 80% identity) turned out to be located within a conserved region of dapC or related genes encoding aminotransferases and proved to be actually recognized as the cognate substrate of attP(TG1) site by the in vitro TG1 integrase assay. Furthermore, the possible attB(TG1) site of Rhodospirillum rubrum revealed to be used actually as a native (endogenous) attachment site for the in vivo TG1-based integration system. These features are distinct from other serine-type phage integrases and advantageous for a tool of genome technology in varied industrially important bacteria belonging to Class Alphaproteobacteria.

Bidirectional effect of MnSOD overexpression on the proliferation of esophageal cancer cells in vitro

To construct a recombinant lentiviral vector for manganese superoxide dismutase (MnSOD) gene expression, and observe its effect on the proliferation of esophageal cancer cells in vitro. Chemical methods were employed for synthesis of the MnSOD cDNA sequence sections, along with the attB sites. Target gene fragment was constructed on the pMD-18T vector, and the recombinant plasmid pDONR221 was obtained after BP recombination reaction. Sequencing was followed by LR recombination reaction between the plasmid and DEST to obtain the lentiviral vector, which worked with helper plasmid for co-transfection of human embryonic kidney epithelial cells (293T cells). Amplification was done to determine its titer, and both transfection and selection procedures were made to get two stable transfected esophageal cancer TE-1 cell lines with medium MnSOD expression (TE-1Mm cells) and high MnSOD expression (TE-1Mh cell), and empty vector cell (TE-1Mn cells). Reverse transcription polymerase chine reaction (RT-PCR), immunofluorescence, immunocytochemistry and Western blot were used to detect the target gene with respect to its expression in the TE-1 cells. Additionally, colorimetric 3-[4,5-dimethy thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, agar colony formation assay, annexin V-FITC/PI staining and flow cytometry experiments were also conducted as to observe the influence of the medium and high MnSOD overexpressions on the proliferation of esophageal cancer cells. RT-PCR indicated that the transfected TE-1 cells showed positive MnSOD expression at different levels. Immunofluorescence, immunocytochemistry and Western blot suggested that TE-1Mm cells and TE-1Mh cells had MnSOD protein expression at different levels. MTT assay indicated that TE-1Mm cells had a significantly decreased survival rate compared with that of the two control cells (TE-1 cells and TE-1Mn cells), and TE-1 Mh cells had an significantly increased survival rate (P<0.05). The colony formation ability of TE-1Mm cells was (23.0 +/- 2.7)%, and that of TE-1Mh cells was (45.3 +/- 4.5)%, significantly different form the (34.7 +/- 4.2)% in TE-1 cells and (33.7 +/- 4.7)% in TE-1Mn cells (P<0.05). Annexin V-FITC/PI double staining experiment of the stably transfected cells cultured for 48 h showed that the early apoptosis rate in TE-1Mm cells was (10.6 +/- 1.0)%, significantly higher than (2.6 +/- 0.2)% in the TE-1 cells, (2.5 +/- 0.6)% in the empty vector cells and (1.0 +/- 0.1)% in the TE-1Mh cels (P<0.05). The fluorescence index (FI) of mitochondrial apoptosis of TE-1Mm cells was 0.948 +/- 0.019, significantly lower than that of TE-1 cell (1.000 +/- 0.022) and empty vector The fluorescence index of TE-1Mn cells (0.997 +/- 0.023) and TE-1 cells (1.000 +/- 0.022) were significant different from that of 0.948 +/- 0.019 in TE-1Mm cells and 1.076 +/- 0.022 in TE-1Mh cells, indicating a significant difference of mitochondrial apoptosis between the cell groups. FCM results indicated that the ROS fluorescence index of TE-1Mm cells was 0.859 +/- 0.040, that of TE-1Mh cells was 0.763 +/- 0.039, significantly lower than that of TE-1 cells (1.000 +/- 0. 042) and empty vector cells (1.002 +/- 0.047) (P<0.05). Stably transfected cell lines with MnSOD expression have been successfully established. MnSOD overexpression shows bidirectional effect on the proliferation of esophageal cancer cells.

Progress of fC31 integrase system in site-specific integration

Integrase of phage fC31 catalyses the homologous recombination between Streptomyces attachment site attB and the phage attachment site attP. Meanwhile, this integrase can mediate integration of attB-containing donor plasmids into the pseudo attP sites in eukaryotic genomes by a site-specific manner and resulting long-term and robust expression of integrated genes. Nowadays, fC31 integrase system is becoming a potential tool for genome modification, gene therapy and transgenic research. Recent progress of fC31 integrase system in integration mode in mammalian genomes, efficiency improvement and researches concerned on transgenic safety were summarized in this review.