Time course study of the response to LPS targeting the pig immune gene networks

Background: Stress is a generic term used to describe non-specific responses of the body to all kinds of challenges. A very large variability in the response can be observed across individuals, depending on numerous conditioning factors like genetics, early influences and life history. As a result, there is a wide range of individual vulnerability and resilience to stress, also called robustness. The importance of robustness-related traits in breeding strategies is increasing progressively towards the production of animals with a high level of production under a wide range of climatic conditions and management systems, together with a lower environmental impact and a high level of animal welfare. The present study aims at describing blood transcriptomic, hormonal, and metabolic responses of pigs to a systemic challenge using lipopolysaccharide (LPS). The objective is to analyze the individual variation of the biological responses in relation to the activity of the HPA axis measured by the levels of plasma cortisol after LPS and ACTH in 120 juvenile Large White (LW) pigs. The kinetics of the response was measured with biological variables and whole blood gene expression at 4 time points. A multilevel statistical analysis was used to take into account the longitudinal aspect of the data. Results: Cortisol level reaches its peak 4 h after LPS injection. The characteristic changes of white blood cell count to LPS were observed, with a decrease of total count, maximal at t = +4 h, and the mirror changes in the respective proportions of lymphocytes and granulocytes. The lymphocytes / granulocytes ratio was maximal at t = +1 h. An integrative statistical approach was used and provided a set of candidate genes for kinetic studies and ongoing complementary studies focused on the LPS-stimulated inflammatory response. Conclusions: The present study demonstrates the specific biomarkers indicative of an inflammation in swine. Furthermore, these stress responses persist for prolonged periods of time and at significant expression levels, making them good candidate markers for evaluating the efficacy of anti-inflammatory drugs

Development of a GAL4-VP16/UAS trans-activation system for tissue specific expression in <i>Medicago truncatula</i>

<div><p>Promoters with tissue-specific activity are very useful to address cell-autonomous and non cell autonomous functions of candidate genes. Although this strategy is widely used in <i>Arabidopsis thaliana</i>, its use to study tissue-specific regulation of root symbiotic interactions in legumes has only started recently. Moreover, using tissue specific promoter activity to drive a GAL4-VP16 chimeric transcription factor that can bind short upstream activation sequences (UAS) is an efficient way to target and enhance the expression of any gene of interest. Here, we developed a collection of promoters with different root cell layers specific activities in <i>Medicago truncatula</i> and tested their abilities to drive the expression of a chimeric GAL4-VP16 transcription factor in a trans-activation UAS: β-Glucuronidase (GUS) reporter gene system. By developing a binary vector devoted to modular Golden Gate cloning together with a collection of adapted tissue specific promoters and coding sequences we could test the activity of four of these promoters in trans-activation GAL4/UAS systems and compare them to “classical” promoter GUS fusions. Roots showing high levels of tissue specific expression of the GUS activity could be obtained with this trans-activation system. We therefore provide the legume community with new tools for efficient modular Golden Gate cloning, tissue specific expression and a trans-activation system. This study provides the ground work for future development of stable transgenic lines in <i>Medicago truncatula</i>.</p></div

Comparison of GUS staining efficiency and tissue specificity of promoter:GUS and transactivatable (GAL4-VP16/ UAS) GUS constructs.

<p>Comparison of GUS staining efficiency and tissue specificity of promoter:GUS and transactivatable (GAL4-VP16/ UAS) GUS constructs.</p

References and expected tissular expression of all tested promoters.

<p>References and expected tissular expression of all tested promoters.</p

New pCambia Golden Gate vector.

<p>Schematic backbone of the pCAMBIA_CR1 vector showing the BsaI cloning sites (in red) that allow insertion of the oriented blocks using the Golden Gate strategy. Cloning sites (single cutter restriction enzymes or BsaI cloning sites) disrupt the LacZ gene (blue arrow) upon cloning, allowing blue/ white screening with the X-Gal substrate. For <i>E</i>.<i>coli</i> selection, a chloramphenicol (Cm) resistance gene can be used (yellow arrow outside the T-DNA fragment). A kanamycin resistance (kanR) gene, driven by a NOS promoter (yellow box and arrow), enables both selection for the presence of the plasmid in <i>A</i>. <i>rhizogenes</i> and transformed roots on selective medium. The T-DNA contains a <i>pAtUbi</i>:<i>DsRED</i> selection gene (red box and arrow) that allows detection of transformed roots using DsRED fluorescence. RB/LB: T-DNA right border and left border.</p

GoldenGate cloning strategy and consensus “sticky end” adapters used.

<p>Schematic representation of the consensus sequences used as “sticky ends” for oriented cloning of each specific block. The pCAMBIA_CR1 binary vector backbone is shown in purple. Here, “AB” blocks are promoter regions, the “BC” block was separated as “BN” and “NC” fragments (where N is a chosen sequence, here TTCA) for the GAL4-VP16 chimeric transcription factor and for the “transcriptional terminator 5xUAS_minimal promoter” blocks, respectively. The β-glucuronidase (GUS) coding region was introduced as a CD block for UAS constructs or BD block for the direct promoter fusions. Note that the “B” adapter was designed to provide an optimized dicotyledon start codon context and the “C” adapter to provide a linker for in frame tag fusions, respectively. X is a chosen spacer nucleotide that will be removed after BsaI digeston. GOI: gene of interest.</p