Half-site strand transfer by step-arrest mutants of yeast site-specific recombinase Flp.

The Flp recombinase of Saccharomyces cerevisae can mediate strand transfer within a half-site, between two half-sites and between a half-site and a full-site. The ability of "step-arrest" mutants of Flp to partake in half-site reactions has been examined. Arg308 variants of Flp, which show little or no strand cleavage in reactions with normal full-sites, execute significant levels of strand transfer in half-site reactions. On the other hand, His305 variants of Flp, which normally accumulate the strand cleavage product from full-sites but do not complete strand transfer, yield only minute amounts of strand transfer products from half-sites. As would be predicted, the step-arrest mutants are unable to produce "normal" or "reverse" recombinants between a half-site and a full-site. The Flp protein is able to form higher-order complexes in association with a half-site. The step-arrest mutants of Flp show specific defects in forming these complexes.     

Resolution of tethered antiparallel and parallel holliday junctions by the Flp site-specific recombinase

Members of the integrase family site-specific recombinases (also called the tyrosine family) bring about recombination in two steps by exchanging pairs of single strands at a time. The product of the first exchange reaction is a four-way DNA junction, the Holliday intermediate. The conformational dynamics by which the recombination complex "isomerizes" from the Holliday-forming to the Holliday-resolving mode are not well understood. Experiments with the lambda Int and Escherichia coli XerC/XerD systems imply that the strand configurations at the branch point of the protein-free junction dictate the resolution mode in the protein-bound junction. We have examined the question of strand bias during resolution for the Flp system by using a series of synthetic Holliday junctions that are conformationally constrained by local sequences or by strand tethering. We have not observed a strong resolution bias in favor of the strands designed to assume the "crossed" configuration within the unbound junction. The resolution patterns with antiparallel junctions in a variety of substrate contexts reveal either parity in strand choice, or only modest disparity. On the other hand, the highly biased resolutions observed in the case of tethered parallel junctions can be explained by the non-equivalence in protein occupancy of the DNA arms of these substrates and/or inefficient conversion of cleavage events to recombinants at the tethered ends.     

Marker removal from actinomycetes genome using Flp recombinase

We report here a system for the functional expression of the Flp recombinase in several actinomycetes: Streptomyces coelicolor, S. lividans, and Saccharotrix espanaensis. We have constructed a synthetic gene encoding the Flp recombinase with a GC content of 60.6% optimized for expression in high-GC bacteria. Using the synthetic flp(a) gene, we have removed an apramycin resistance gene flanked by FRT sites from the chromosome of actinomycetes with an efficiency of 40%. Sequencing the region of chromosome showed that excision of the apramycin cassette by Flp recombinase was specific.     

A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity

We have developed a dual reporter screen in Escherichia coli for identifying variants of the Flp site-specific recombinase that have acquired reactivity at an altered target site (mFRT). In one reporter, the lacZα gene segment is flanked by mFRTs in direct orientation. In the other, the red fluorescence protein (RFP) gene is flanked by the native FRTs. Hence, the color of a colony on an X-gal indicator plate indicates the recombination potential of the variant Flp protein expressed in it: blue if no recombination or only FRT recombination occurs, red if only mFRT recombination occurs and white if both FRT and mFRT recombinations occur. The scheme was validated by identification and in vivo characterization of Flp variants that show either relaxed specificity (active on FRT and mFRT) or moderately shifted specificity toward mFRT. We find that alteration of Lys-82 to Met, Thr, Arg or His enables the corresponding Flp variants to recombine FRT sites as well as altered FRT sites containing a substitution of G-C by C-G at position 1 of the Flp binding element (mFRT11). In contrast, wild-type Flp has no detectable activity on mFRT11. When Lys-82 is replaced by Tyr, the resulting Flp variant shows a small but reproducible preference for mFRT11 over FRT. However, this preference for mFRT11 is nearly lost when Tyr-82 is substituted by Phe.     

Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.     

Characterization of site-specific recombination mediated by Cre recombinase during the development of erythropoietin producing CHO cell lines

The establishment of erythropoietin (EPO) producing Chinese hamster ovary (CHO) cell lines was conducted using Cre-mediated cassette exchange. The characterization of site-specific recombination mediated by Cre-recombinase during the cell line development was also performed. A total of six parental clones, which had various green fluorescence levels ranging from high to low and containing a single copy of insertion vector (pEGFP-m2), were screened. The EPO targeting vector (pIC-m2-EPO) was targeted into the 6 parental clones by Cre-mediated cassette exchange. Correctly targeted clones were obtained from 4 out of 6 parental clones with 0∼15% of targeting efficiencies. Moreover, there was a positive relationship (R² = 0.87) between fluorescence levels of the parental clones before Cre-mediated cassette exchange and specific EPO productivities (q _EPO ) of the correctly targeted clones after Cre-mediated cassette exchange. Therefore, it was verified that the chromosomal loci’s characteristic gene expression level was not modified even after cassette exchange mediated by Cre recombinase during the development of EPO producing CHO cell lines. This finding implies that the reproducible development of CHO cell lines largely producing a desired protein is expected to be achieved by Cre-mediated cassette exchange.     

Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase.

A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.     

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine.

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.     

Spermatocyte-specific gene excision by targeted expression of Cre recombinase

Transgenic mice carrying the coding sequence of the Cre recombinase, whose expression was driven by the spermatocyte-specific Pgk-2 promoter, were generated. These mice were crossed with a reporter transgenic line, which produces beta-galactosidase depending on the occurrence of loxP-mediated DNA recombination. When DNA of the offspring was analyzed by PCR and Southern blotting, signals that appear after the recombination were detectable only in the testis. Histochemical analyses revealed that beta-galactosidase was present in spermatocytes and spermatogenic cells at later differentiation stages. However, the distribution of the protein was not uniform in all spermatocytes. Analyses of genomic DNA of the next generation indicated that recombination took place in about 70% of spermatogenic cells. From these results, we concluded that this transgenic line possessing Pgk-2-driven expression of the Cre recombinase should be useful for identifying spermatogenic genes that function at or after the spermatocyte stage.     

Retina-specific gene excision by targeted expression of Cre recombinase

The use of Cre recombinase for conditional targeting permits the controlled removal or activation of genes in specific tissues and at specific times of development. The Rho–Cre mice provide an improved tool for studying gene ablation in rod photoreceptor cells. To establish a robust expression of Rho–Cre transgenic mice that would be useful for the study of various protein functions in photoreceptor cells, a total 11,987 kb fragment (pNCHS4 Rho–NLS–cre) containing human rhodopsin promoter was cloned. The Rho–Cre plasmid was digested with EcoR1 and I Ceu-1, and the 9.316 kb fragment containing the hRho promoter and Cre recombinase gel was purified. To generate transgenic mice, the purified DNA fragment was injected into fertilized oocytes according to standard protocols. ROSA26R reported the steady expression of Rho–Cre especially in photoreceptor cells, allowing further excising proteins in rod photoreceptors across the retina. This Rho–Cre transgenic line should thus prove useful as a general deletor line for genetic analysis of diverse aspects of retinopathy.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre.

Site-specific DNA recombinases from bacteriophage and yeasts have been developed as novel tools for genome engineering both in prokaryotes and eukaryotes. The 38kDa Cre protein efficiently produces both inter- and intramolecular recombination between specific 34bp sites called loxP. We report here the in vivo use of Cre recombinase to manipulate the genome of the protozoan parasite Toxoplasma gondii. Cre catalyzes the precise removal of transgenes from T. gondii genome when flanked by two directly repeated loxP sites. The efficiency of excision has been determined using LacZ as reporter and indicates that it can easily be applied to the removal of undesired sequences such as selectable marker genes and to the determination of gene essentiality. We have also shown that the reversibility of the recombination reaction catalyzed by Cre offers the possibility to target site-specific integration of a loxP-containing vector in a chromosomally placed loxP target in the parasite. In mammalian systems, the Cre recombinase can be regulated by hormone and is used for inducible gene targeting. In T. gondii, fusions between Cre recombinase and the hormone-binding domain of steroids are constitutively active, hampering the utilization of this mode of post-translational regulation as inducible gene expression system.     

FlEx‐based transgenic reporter lines for visualization of Cre and Flp activity in live zebrafish

Site‐specific recombinases such as Cre and Flp are invaluable tools for genetic manipulations, but their usage in zebrafish has been limited. Incorporating recently developed flip‐excision (FlEx) design that allows stable inversions, we have established zebrafish reporter lines that express bright and ubiquitous EGFP, but switch to express mCherry in the presence of Cre or Flp. Here, we demonstrate the stable inversion in the reporter lines, both in somatic cells and in the germ line by Cre or Flp, and the subsequent reinversion using the other recombinase. Using the reporter lines, we characterized cardiomyocyte‐specific Cre lines and neuronal progenitor‐specific and tamoxifen‐dependent Cre lines. We also used the reporter lines for screening Cre‐ and Flp‐based enhancer trap lines. Similar to the widely used Cre reporter lines in mice, these FlEx‐based reporter lines will facilitate the use of recombinases for genetic manipulations in zebrafish. genesis 47:484–491, 2009. © 2009 Wiley‐Liss, Inc.     

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines

Background

Specific and efficient delivery of genes into targeted cells is a priority objective in non‐viral gene therapy. Polyethyleneimine‐based polyplexes have been reported to be good non‐viral transfection reagents. However, polyplex‐mediated DNA delivery occurs through a non‐specific mechanism. This article reports the construction of an immunopolyplex, a targeted non‐viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines.

Methods

Targeting elements (biotin‐labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19?), Granta 519 (CD3?/ CD19+), and J.RT3‐T3.5 (CD3?/CD19?)] using the EGFP gene as a reporter gene and anti‐CD3 and anti‐CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry.

Results

A significant selectivity of gene delivery was observed, since the anti‐CD3 immunopolyplex worked only in Jurkat cells while the anti‐CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3? cell mixture with anti‐CD3 immunopolyplexes showed up to 16‐fold more transfection in CD3+ than in CD3? cells. Several non‐specific transfection reagents showed poor or no transfection activity.

Conclusion

It is concluded that immunopolyplex is a good non‐viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) – the streptavidin–polyplex backbone remaining intact – thereby conferring high versatility. Copyright © 2002 John Wiley & Sons, Ltd.

     

A chimeric reporter gene allowing for clone selection and high-throughput screening of reporter cell lines expressing G-protein-coupled receptors

Efficient screening of ligands interacting with G-protein-coupled receptors is central for modern drug development. Here, we describe an optimized reporter vector primarily intended for use in reporter cell lines expressing such receptors. The construct consists of a synthetic enhancer containing 9x TRE (12-O-tetradecanoylphorbol-13-acetate-responsive elements) fused to a minimal CMV (cytomegalovirus) promoter. Activation of the promoter construct leads to the expression of a chimeric reporter protein based on the genes for enhanced green fluorescent protein and Photinus luciferase. The chimeric protein allows for both clonal selection by fluorescence, which facilitates the selection of optimal reporter cell lines and high-throughput screening by luminescens. In designing the vector, increasing numbers of TRE motifs were tested in front of two different minimal promoters. The reporter gene was more strongly inducible with increasing numbers of TRE motifs. The constructs were tested in two cell lines, CHO and HeLa. The latter regulated reporter gene activity stronger in response to PMA (phorbol 12-myristate 13-acetate) stimulation and were used to construct HF1 reporter cell lines. Model experiments were carried out on these reporter cells transfected with the human BLTR, human CCR5, or the rat alpha(1b) receptor. After maximal agonist stimulation reporter gene activity was increased 200-, 15-, and 50-fold, respectively.     

Conditional gene knockout using cre recombinase

Cre recombinase has become an important instrument for achieving precise genetic manipulation in mice. Many of these desired genetic manipulations rely on Cre’s ability to direct spatially and temporally specified excision of a predesignated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site. Success in achieving such conditional mutagenesis in mice depends both on the careful design of conditional alleles and on reliable detection of cre gene expression. These procedures include PCR, immunohistochemistry and the use of a recombination-proficient GFP-tagged Cre protein.     

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.