Nancy Rodríguez

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals.

Transgenic farm animals are attractive alternative mammalian models to rodents for the study of developmental, genetic, reproductive and disease-related biological questions, as well for the production of recombinant proteins, or the assessment of xenotransplants for human patients. Until recently, the ability to generate transgenic farm animals relied on methods of passive transgenesis. In recent years, significant improvements have been made to introduce and apply active techniques of transgenesis and genetic engineering in these species. These new approaches dramatically enhance the ease and speed with which livestock species can be genetically modified, and allow to performing precise genetic modifications. This paper provides a synopsis of enzyme-mediated genetic engineering in livestock species covering the early attempts employing naturally occurring DNA-modifying proteins to recent approaches working with tailored enzymatic systems.

Adrenal response of male rats exposed to prenatal stress and early postnatal stimulation

Abstract Stress in pregnant rats caused by chronic immobilization alters the pattern of secretion of corticosterone and modifies the hypothalamic-pituitary-adrenal axis (HPA) of the fetus. Early postnatal handling, however, may reverse the effects of increased secretion of corticosterone. We investigated the effects of prenatal stress and postnatal handling on the activity of the HPA axis of male offspring of stressed female rats. Male 90-day-old rats from four groups were investigated: prenatally stressed animals without postnatal handling, prenatally stressed animals with postnatal handling, unstressed control animals with postnatal handling, and unstressed control animals without postnatal handling. After sacrifice, the adrenal glands were weighed to determine the adrenal-somatic index. Apoptosis was evaluated by TUNEL assay and active caspase-3 expression. We found that the adrenal gland cortex:medulla ratio increased in animals with prenatal stress and that eventually the stress caused apoptosis. Handling newborns to simulate maternal activity ameliorated some of the negative effects of prenatal stress.

Physicochemical properties of ionic and non-ionic biocompatible hydrogels in water and cell culture conditions: Relation with type of morphologies of bovine fetal fibroblasts in contact with the surfaces

Cationic, anionic and non-ionic hydrogels having acrylamide polymer backbones were synthesized via free radical polymerization with N,N-methylenebisacrylamide (BIS) as crosslinker. The chemical structures of the hydrogels were characterized by Fourier Transform Infrared Spectroscopy (FTIR). Physicochemical properties such as swelling kinetic, maximum swelling capacity, volume phase transition temperature (VPTT) and wettability (static water contact angle) of hydrogels swollen in aqueous and cell culture medium, at room and cell culture temperatures were studied. In order to correlate the surface properties of the hydrogels and cellular adhesivity of bovine fetal fibroblasts (BFFs), cellular behaviour was analyzed by inverted fluorescence optical microscopy and atomic force microscopy (AFM). MTT assay demonstrated that the number of viable cells in contact with hydrogels does not significantly change in comparison to a control surface. Flattened and spindle-shaped cells and cell spheroids were the adopted morphologies during first days of culture on different hydrogels. Cell spheroids were easily obtained during the first 5days of culture in contact with PNIPAM-co-20%HMA (poly (N-isopropylacrylamide-co-20%N-acryloyl-tris-(hydroxymethyl)aminomethane)) hydrogel surface. After 15days of culture all hydrogels showed high adhesion and visual proliferation. According to obtained results, non-ionic and hydrophilic surfaces with moderated wettability induce the formation of BFFs cell spheroids. These hydrogel surfaces could be used in clinical and biochemical treatments at laboratory level to cell growth and will allow generating the base for future biotechnologic platform.

203 Genome Modifications by Sleeping Beauty Transposition and CRISPR/Cas9 to Improve Cow Milk Composition for Human Consumption

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim of this study was to introduce the sequence for a desaturase construct (mFAT-2, from C. elegans), which is able to catalyse the synthesis of ω-3 and ω-6 fatty acids, into the bovine genome by Sleeping Beauty (SB) transposition, and simultaneously knocking out the bovine β-lactoglubulin gene using CRISPR/Cas9 system. The sgRNA (AAGTGCCTCCTGCTTGCCC) targeted to BLG exon 1 was synthesised as an oligo linker and cloned into the px459-Cas9. The mutation activity of the designed sgRNA at the target locus was determined by T7 endonuclease assay I (T7EI) mismatch detection assay. Briefly, bovine fetal fibroblasts (BFF) were seeded at 0.5 × 105 cells per well of a 24-well plate in triplicate, when the cells reached 80% confluence (12–24 h), cultures were transfected with 1 μg of px459-Cas9::BLG plasmid co-expressing Cas9 and sgRNA using polyethylenimine reagent (PEI; 3 ng μL−1). After 3 days of puromycin selection, genomic DNA from transfected cells were extracted and the sequence of interest was PCR-amplified and digested by T7EI restriction enzyme. Digestion products showed a mutation efficiency at the target locus of 29%. Subsequently, we chemically cotransfected 0.5 × 105 BFF with 0.5 μg of knockout vector (px459-Cas9::BLG) and 0.5 μg of SB plasmids (carrying mFAT-2 cDNA for mammary gland-specific expression) using 3 ng μL−1 PEI in triplicate. At 48 h post-transfection, cell cultures were subjected to 3 days of puromycin and 21 days of neomycin selection. PCR analysis of antibiotic resistant colonies revealed the presence of mFAT-2 transgene in almost 70% of the analysed cells lines. Genotyping of BLG exon 1 was performed by direct sequencing of PCR amplicons using primers flanking the target site. Despite the appreciable gene mutation activity of the sgRNA sequence previously determined by T7EI assay (29%), none of the cell lines analysed showed modification in the BLG target locus. We speculate that the SB vector might have disrupted the activity of targeting vector. We are currently performing additional experiments to accomplish gene addition (mFAT) and gene knockout (BGL) in one step using these highly efficient and precise transgenic tools. Genetically modified cells will be used as nuclear donor to produce transgenic cows by somatic cells nuclear transfer. The financial support of CONICET, UNRC and FONCYT is gratefully acknowledged.

Attenuation effects of prenatal stress by early postnatal stimulation in different research models

The prenatal and early postnatal life stages are both dynamic and vulnerable phases during mammalian development. Exposure to adverse factors that interfere with this critical sequence of events places the exposed individual to a higher risk of developing various disorders in adult life.1–3 For instance, high blood glucocorticoids concentration in pregnant stressed females crossing the placenta and the fetal blood-brain barriers,4 can affect brain development, birth weight and HPA axis function in offspring.5

Stress versus immunity

Stress can be defined as a real or supposed threat to physical or psychological integrity of an individual, resulting in a physiological and /or behavioral response.1 The degree of damage caused by the stress depends on the nature, intensity and duration of the stimuli as well as the stage of gestation at which the stressor is applied.2 Dhabhar & McEwen3 showed that the activation of the physiologic stress response systems can also enhance immune function as evidenced by increase in allergic contact sensitivity or delayed-type hypersensitivity. In contrast, acute stress has no effect on the course of irritant contact sensitivity, an immune reaction that does not involve an Ag-specific memory response. Herbert & Cohen4 suggested that objective stressful events leas to larger immune changes than subjective self-reports of stress and that interpersonal events are related to different immune outcomes than non-social events.

IL-2 is involved in immune response of prenatally stressed rats exposed to postnatally stimulation

espanolRESUMEN: Las condiciones ambientales influyen en el crecimiento y desarrollo de los mamiferos durante la vida prenatal, posnatal temprana y pueden ejercer efectos a largo plazo en la vida adulta. Altas concentraciones circulantes de glucocorticoides durante el embarazo (estres prenatal) afecta a la actividad del eje hipotalamo-pituitario-adrenal (HPA) de la descendencia y se encuentra en relacion con alteraciones de las respuestas del sistema inmunologico. La estimulacion postnatal temprana de los animales estresados prenatalmente genera efectos beneficiosos a largo plazo en la reactividad del eje HPA y en la funcion del sistema inmunologico. El objetivo del estudio fue investigar el efecto de las manipulaciones posnatales tempranas sobre la respuesta inmune de ratas machos y dilucidar la posible relacion con la actividad del eje HPA. Crias controles y estresadas prenatalmente (PS) por inmovilizacion (IMO) se manipularon durante la primera semana de vida. Los animales controles y PS fueron sometidos a estres agudo por la IMO. Los niveles de corticosterona (COR) plasmatica se midieron por el ensayo de RIA, la proliferacion de linfocitos T por [3H] timidina y los niveles de IL-2 por la tecnica de ELISA directa. El estres prenatal cronico por IMO aumento los niveles plasmaticos basales de COR en la madre, mientras que las crias PS sometidas a una sesion de estres agudo posnatal mostraron disminuida la proliferacion de celulas T y niveles menores de IL-2. En conclusion, las estimulaciones postnatales tempranas revirtieron los efectos negativos provocados por el estres prenatal sobre la proliferacion de los linfocitos T y la liberacion de IL-2. EnglishABSTRACT: Environmental cues influence growth and development of mammals during prenatal and particularly early postnatal life and can exert long-lasting effects in adult life. High circulating concentration of glucocorticoids during pregnancy (prenatal stress) affects the activity of the hypothalamic-pituitary-adrenal axis (HPA) of offspring and has been linked to alter immune system responses. Early postnatal stimulation (handling) of prenatally stressed animals generates long-term beneficial effects on the reactiveness of the HPA axis and immune system function. The aim of this study was to further investigate the effect of handling on immune response in prenatally stressed male rats and to elucidate a possible relationship with the HPA axis activity. Control and prenatally stressed (PS) offspring by immobilization (IMO) were handled during the first week of life. Animals from both treatments were subjected to acute stress by IMO. Corticosterone (COR) plasma concentration was measured by RIA assay, T lymphocyte proliferation by [3H] thymidine assay and IL-2 levels by direct ELISA technique. Chronic IMO prenatal stress caused an increase in mother plasma COR basal levels. Furthermore, prenatally stressed rats subjected to an acute stress session had lower T cell proliferation and decreased IL-2 release. In addition, early postnatal stimulation reversed the negative effects of prenatal stress on proliferation of T lymphocytes and IL-2 release.