Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.     

Flexible pseudotyping of retrovirus using recombinase-mediated cassette exchange

Objective

Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.

Results

Dual-FLEX cells (dFLEX) contain two independent recombinase-mediated cassette exchange (RMCE) systems which confer flexibility to the expression of different transgene and envelope combinations. The flexible envelope expression in dFLEX cells was validated by pseudotyping retrovirus particles with three different viral envelope proteins—GaLV, 4070A and VSV-G. Our results show that dFLEX cells are able to provide high titers of infectious retroviral particles with a single-copy integration of the envelope constructs after RMCE. The integrated CRE/Lox tagging cassette was amenable to express envelope proteins both using constitutive (i.e. CMV) and inducible (i.e. Tet-on) promoters.

Conclusions

dFLEX cell line provides predictable productivities of recombinant retrovirus pseudotyped with different envelope proteins broadening the tropism of particles that can be generated and thus accelerating the research and development of retrovirus-based products.

     

Gene tagging and gene replacement using recombinase-mediated cassette exchange in Schizosaccharomyces pombe

Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.     

Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange

Elk-1 is a member of the TCF subfamily of Ets proteins. TCFs interact with SRF at serum response elements (SREs) of immediate early genes (IEGs), such as c-fos and Egr-1, thereby mediating IEG induction upon extracellular stimulation. We previously generated an Elk-1 null allele (Elk1-137) in murine embryonic stem (ES) cells by homologous recombination. In Elk1-137, the Elk-1 gene was replaced by a Hygromycin B phosphotransferase - Thymidine Kinase (HygTk) fusion gene, flanked by two nonidentical Flp recombinase recognition (FRT) sites (Cesari et al., [2004] Mol Cell Biol, in press) to allow for the subsequent generation of alternative alleles of interest by recombinase-mediated cassette exchange (RMCE). Elk1-deficient mice derived from Elk-1((137/0)) ES cells are viable and do not reveal strong phenotypical abnormalities, apart from male sterility. However, the Elk-1 locus contains the Tk cassette, which has previously been related to this defect. Therefore, in our first experiment involving the technique of Flp RMCE we chose to remove the HygTk cassette in Elk-1((137/0)) ES cells and to generate Elk-1((RMCE16/0)) and Elk-1((RMCE16/RMCE16)) mice. In so doing, we provide evidence that the sterility of Elk1((137/0)) mice was not due to the absence of Elk-1 but rather the presence of HygTk. This is the first report of mice derived from ES cells which were subjected to Flp RMCE and thus proves that RMCE is a powerful tool for the genetic engineering of previously tagged loci in the mouse genome.     

High efficiency of a sequential recombinase-mediated cassette exchange reaction in Escherichia coli

A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.     

Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.     

Stable transformation of Pinus radiata embryogenic tissue by Agrobacterium tumefaciens

Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation.     

Genotype-independent transformation of lettuce using Agrobacterium tumefaciens

The genetic manipulation of lettuce (Lactuca sativa) necessitatesa reliable and efficient, genotype-independent method of transformation.Thirteen lettuce cultivars have been assessed for their amenabilityto Agrobacterrum-mediated gene transfer linked to their tissueculture responsiveness, including callus induction and shootregeneration. A reliable protocol has been developed for theroutine production of transgenic plants for all 13 cultivarsinvestigated. Key words: Agrobacterium-mediated transformation, Lactuca sativa genotypes, lettuce     

Transformation of forage legumes using Agrobacterium tumefaciens

Summary Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines.     

Transformation of Liquidambar styraciflua using Agrobacterium tumefaciens

Summary We describe the molecular transformation of Liquidambar styraciflua using Agrobacterium tumefaciens. A binary TI-plasmid vector containing a chimeric neomycin phosphotransferagene which confers resistance to kanamycin and either a chimeric Bacillus thuringiensis toxin gene, a chimeric E. coli -glucuronida(GUS), or a chimeric tobacco anionic peroxidase gene was introduced into sweetgum by co-cultivation with Agrobacterium tumefaciens. Sweetgum shoots regenerated in the presence of kanamycin were confirmed to be transformed by genomic DNA blots or the presence of GUS activity. The optimization of the transformation protocol and the incorporation of molecular transformation into a rapid germplasm improvement protocol are discussed.     

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l^–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS ß-glucuronidase - MS Murashige-Skoog medium - BA 6-benzylaminopurine - 2ip 6-(, -dimethylallylamino)purine - IAA indole-3-acetic acid - GA₃ gibberellic acid - npt II neomycin phosphotransferase II - NOS nopaline synthase - MUG 4-methyl umbelliferyl glucuronide - MU 7-hydroxy-4-methylcoumarin - X-gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid     

Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.     

A simple polymerase chain reaction-based method for the construction of recombinase-mediated cassette exchange donor vectors

Here we describe a simple method for generating donor vectors suitable for targeted transgenesis via recombinase-mediated cassette exchange (RMCE) using the ΦC31 integrase. This PCR-based strategy employs small attB “tails” on the primers used to amplify a sequence of interest, permitting the rapid creation of transgenes for in vivo analysis.     

Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are less susceptible to Agrobacterium transformation

Agrobacterium tumefaciens causes crown gall disease on many plant species and can result in considerable economic losses. Here we report a new strategy to control crown gall disease by over-expressing Agrobacterium tumefaciens VirD2 protein in plants. Transgenic Arabidopsis plants over-expressing virD2 from constitutive or wound-inducible promoters are less susceptible to Agrobacterium -mediated transformation. Additionally, the transient introduction of an A. tumefaciens virD2 gene in tobacco BY-2 cells reduces subsequent Agrobacterium -mediated transformation.     

Transfection in Agrobacterium tumefaciens

Intact cells of Agrobacterium tumefaciens were examined for ability to take up biologically active LR-4 phage deoxyribonucleic acid (DNA) from the surrounding medium. DNA incorporation as measured by subsequent plaque formation (transfection) failed to occur when the bacteria were grown in defined minimal salts media, and was restricted to a 4-hr period in the early log phase of growth in enriched media. In the latter case, maximal transfection frequencies were obtained after a 25- to 30-min incubation with 22.5 mug of phage DNA/ml. Higher DNA concentrations or longer incubation times were inhibitory. Transfection was completely inhibited by deoxyribonuclease but not by ribonuclease, trypsin, or phage-specific antisera.