Sec22b-dependent antigen cross-presentation is a significant contributor of T cell priming during infection with the parasite Trypanosoma cruzi.

Antigen cross-presentation is a vital mechanism of dendritic cells and other antigen presenting cells to orchestrate the priming of cytotoxic responses towards killing of infected or cancer cells. In this process, exogenous antigens are internalized by dendritic cells, processed, loaded onto MHC class I molecules and presented to CD8+ T cells to activate them. Sec22b is an ER-Golgi Intermediate Compartment resident SNARE protein that, in partnership with sintaxin4, coordinates the recruitment of the transporter associated with antigen processing protein and the peptide loading complex to phagosomes, where antigenic peptides that have been proteolyzed in the cytosol are loaded in MHC class I molecules and transported to the cell membrane. The silencing of Sec22b in dendritic cells primary cultures and conditionally in dendritic cells of C57BL/6 mice, critically impairs antigen cross-presentation, but neither affects other antigen presentation routes nor cytokine production and secretion. Mice with Sec22b conditionally silenced in dendritic cells (Sec22b-/-) show deficient priming of CD8+ T lymphocytes, fail to control tumor growth, and are resistant to anti-checkpoint immunotherapy. In this work, we show that Sec22b-/- mice elicit a deficient specific CD8+ T cell response when challenged with sublethal doses of Trypanosoma cruzi trypomastigotes that is associated with increased blood parasitemia and diminished survival.

Whole bone subcutaneous transplantation as a strategy to study precisely the bone marrow niche.

Hematopoietic stem cells are maintained in a specialized microenvironment, known as the 'niche', within the bone marrow. Understanding the contribution of cellular and molecular components within the bone marrow niche for the maintenance of hematopoietic stem cells is crucial for the success of therapeutic applications. So far, the roles of crucial mechanisms within the bone marrow niche have been explored in transgenic animals in which genetic modifications are ubiquitously introduced in the whole body. The lack of precise tools to explore genetic alterations exclusively within the bone marrow prevents our determination of whether the observed outcomes result from confounding effects from other organs. Here, we developed a new method - 'whole bone subcutaneous transplantation'- to study the bone marrow niche in transgenic animals precisely. Using immunolabeling of CD45.1 (donor) vs. CD45.2 (recipient) hematopoeitic stem cells, we demonstrated that hematopoeitic stem cells from the host animals colonize the subcutaneously transplanted femurs after transplantation, while the hematopoietic stem cells from the donor disappear. Strikinlgy, the bone marrow niche of these subcutaneously transplanted femurs remain from the donor mice, enabling us to study specifically cells of the bone marrow niche using this model. We also showed that genetic ablation of peri-arteriolar cells specifically in donor femurs reduced the numbers of hematopoietic stem cells in these bones. This supports the use of this strategy as a model, in combination with genetic tools, to evaluate how bone marrow niche specific modifications may impact non-modified hematopoietic stem cells. Thus, this approach can be utilized for genetic manipulation in vivo of specific cell types only within the bone marrow. The combination of whole bone subcutaneous transplantation with rodent transgenic models will facilitate a more precise, complex and comprehensive understanding of existing problems in the study of the hematopoietic stem cell bone marrow niche.

Simple method to induce denaturation of fluorescent proteins in free-floating brain slices.

BACKGROUND: The generation of animals expressing reporter proteins (e.g., GFP, mCherry or tdTomato) under the control of genes of interest has become a valuable tool in neuroscience. However, the histological reuse of brain sections of these genetically modified animals in unplanned experiments is often infeasible since the constitutive expression of fluorescent reporter proteins interferes with further fluorescent staining procedures. Thus, expensive or time-demanding experiments frequently need to be repeated using additional experimental animals. NEW METHOD: To improve the reuse of tissues of reporter animals for fluorescent staining procedures, we developed fast, inexpensive and simple methods that induce denaturation of constitutively expressed fluorescent proteins in free-floating brain slices. These procedures consist of incubation of brain sections either in a 1% sodium hydroxide alkaline solution (pH 13.0) for one hour at room temperature or at 95 °C for 10-30 min. RESULTS: The strong fluorescence of tdTomato, mCherry and eGFP was completely eliminated after incubation of brain sections of different reporter mice in a pH 13.0 solution for one hour. hrGFP was resistant to denaturation in an alkaline solution, but incubation of brain sections at 95 °C for 10 min eliminated the fluorescence of hrGFP, as well as of tdTomato, mCherry and eGFP. The denaturing procedures did not prevent the reuse of brain tissues in free-floating immunofluorescence staining using multiple antibodies. Furthermore, the quality of the labeling remained unaffected. Although pretreatment in pH 13.0 solution maintained good tissue integrity, as a side effect, brain sections exhibited increased autofluorescence. However, a rinse in 0.25% Sudan Black B solution was efficient in eliminating the autofluorescence without impairing the immunofluorescence staining or DAPI counterstaining. CONCLUSIONS: The present study provides simple procedures capable of inducing denaturation of fluorescent proteins in free-floating brain slices.

Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi.

An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB.

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system.

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T(0) -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T (0) -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38') was transformed with Cre gene and the inbred/pure line ('E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T0-plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T0-plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

La proteína verde fluorescente ilumina la biociencia / The Green Fluorescent Protein that glows in Bioscience

La proteína verde fluorescente (o GFP, por sus siglas en inglés, Green Fluorescent Protein) es una proteína producida por la medusa Aequorea victoria que emite bioluminiscencia en la zona verde del espectro visible. El gen que codifica esta proteína ha sido clonado y se utiliza habitualmente en biología molecular como marcador. Los descubrimientos relacionados a la GFP merecieron el Premio Nobel de Química 2008, en conjunto a los tres investigadores, Dres Shimomura, Chalfie y Tsien que participaron escalonadamente en dilucidar la estructura y función de la proteína. El Dr. Shimomura descubrió y estudió las propiedades de GFP, el Dr. Chalfie usando técnicas de biología molecular logró introducir el gen que codificaba para la GFP en el ADN del gusano transparente C. elegans, e inició la era de GFP como marcador de procesos en células y organismos. Finalmente el Dr. Tsien modificó la estructura de la proteína para producir moléculas que emiten luz a distintas longitudes de onda, extendiendo la paleta de colores de las proteínas. Las proteínas fluorescentes, entre las cuales se encuentra la GFP, son muy versátiles y se utilizan en diversos campos como la microbiología, ingeniería genética, fisiología, e ingeniería ambiental. Permiten ver procesos previamente invisibles, como el desarrollo de neuronas, cómo se diseminan las células cancerosas, o la contaminación de agua con arsénico, por mencionar algunos usos. Con la obtención de proteínas de muchos colores complejas redes biológicas pueden ser marcadas diferencialmente, lo que permite visualizar la biología celular en acción.

Green fluorescent protein (GFP) is a protein produced by the jellyfish Aequorea victoria, that emits bioluminescence in the green zone of the visible spectrum. The GFP gene has been cloned and is used in molecular biology as a marker. The three researchers that participated independently in elucidating the structure and function of this and its related proteins, Drs. Shimomura, Chalfie and Tsien were awarded the Nobel Prize in Chemistry 2008. Dr. Shimomura discovered and studied the properties of GFP. Using molecular biological techniques, Chalfie succeeded in introducing the GFP gene into the DNA of the small, almost transparent worm C. elegans, and initiated an era in which GFP would be used as a glowing marker for cellular biology. Finally, Dr.Tsien found precisely how GFP's structure produces the observed green fluorescence, and succeeded in modifying the structure to generate molecules that emit light at slightly different wavelengths, which gave tags of different colors. Fluorescent proteins are very versatile and are being used in many areas, such as microbiology, biotechnology, physiology, environmental engineering, development, etc. They can, for example, illuminate growing cancer tumours; show the development of Alzheimer's disease, or detect arsenic traces in water. Finding the key to how a marine organism produces light unexpectedly ended up providing researchers with a powerful array of tools with which to visualize cell biology in action.