A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation

Safe Production of a Product of Interest in Hybrid Seed

Publication Number: WO/2004/108934 International Application No.: PCT/EP2004/006069 Publication Date: 16.12.2004 International Filing Date: 04.06.2004process of the production of a product of interest in an F1 seed obtained by a hybridization of a first and a second transgenic parental plant, said hybridization generating a genetic endowment in said F1 seed for said production by combining in said F1 seed first and second partial genetic endowments of said first and second transgenic parental plants, followed by isolating said product of interest from said F1 seed or a seedling thereo

In planta engineering of viral RNA replicons: Efficient assembly by recombination of DNA modules delivered by Agrobacterium

We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon. The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols. The process described here is very fast (expression requires 3–4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using in vivo manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods

γ\gamma spectroscopy of the 96Y^{96}\mathrm{Y} isotope: Searching for the onset of shape coexistence before N=60N=60

International audienceMedium and high spin states of the Y96 nucleus, located in the shape-coexistence region near Z=40 and N=60, were populated in thermal-neutron-induced fission of U233 and U235 targets, diluted in a scintillator. γ rays were measured with the FIssion Product Prompt γ-ray Spectrometer (FIPPS) high-purity germanium (HPGe) detector array, using double and triple γ-ray coincidence techniques and taking advantage of the efficient fission tag provided by the scintillating target material. A complex level scheme, extending up to 5.2 MeV and including excitations above the 8+β-decaying isomer, was investigated, and firm spin and parity assignments were given to a number of states, on the basis of angular correlation analysis and considerations on the γ-decay patterns. While the structures built on the 0− ground state and the 8+ isomer show irregular patterns typical for spherical shapes, the (6+) isomeric state at 1655 keV [with half-life of 181(9) ns], and the rotational band built on it [with spin-parity values between (6+) and (9+)], can be explained by Hartree-Fock-Bogoliubov calculations, if an oblate deformation is assumed. This is the first observation of a deformed structure in an N=57 isotone, lying three neutrons away from the N=60 line. An important finding is also the 115-keV transition which connects the (6+) 181(9)-ns isomer to the β-decaying 8+ spherical isomer, allowing us to firmly place the latter at 1541 keV excitation energy. This may be relevant for calculations of electron and antineutrino spectra from fission of actinides, for which Y96 is a prominent product

Improved sterile neutrino constraints from the STEREO experiment with 179 days of reactor-on data

International audienceThe STEREO experiment is a very short baseline reactor antineutrino experiment. It is designed to test the hypothesis of light sterile neutrinos being the cause of a deficit of the observed antineutrino interaction rate at short baselines with respect to the predicted rate, known as the reactor antineutrino anomaly. The STEREO experiment measures the antineutrino energy spectrum in six identical detector cells covering baselines between 9 and 11 m from the compact core of the ILL research reactor. In this article, results from 179 days of reactor turned on and 235 days of reactor turned off are reported at a high degree of detail. The current results include improvements in the modelling of detector optical properties and the γ-cascade after neutron captures by gadolinium, the treatment of backgrounds, and the statistical method of the oscillation analysis. Using a direct comparison between antineutrino spectra of all cells, largely independent of any flux prediction, we find the data compatible with the null oscillation hypothesis. The best-fit point of the reactor antineutrino anomaly is rejected at more than 99.9% C.L