Genome-wide host responses against infectious laryngotracheitis virus vaccine infection in chicken embryo lung cells

<p>Abstract</p> <p>Background</p> <p>Infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) infection causes high mortality and huge economic losses in the poultry industry. To protect chickens against ILTV infection, chicken-embryo origin (CEO) and tissue-culture origin (TCO) vaccines have been used. However, the transmission of vaccine ILTV from vaccinated- to unvaccinated chickens can cause severe respiratory disease. Previously, host cell responses against virulent ILTV infections were determined by microarray analysis. In this study, a microarray analysis was performed to understand host-vaccine ILTV interactions at the host gene transcription level.</p> <p>Results</p> <p>The 44 K chicken oligo microarrays were used, and the results were compared to those found in virulent ILTV infection. Total RNAs extracted from vaccine ILTV infected chicken embryo lung cells at 1, 2, 3 and 4 days post infection (dpi), compared to 0 dpi, were subjected to microarray assay using the two color hybridization method. Data analysis using JMP Genomics 5.0 and the Ingenuity Pathway Analysis (IPA) program showed that 213 differentially expressed genes could be grouped into a number of functional categories including tissue development, cellular growth and proliferation, cellular movement, and inflammatory responses. Moreover, 10 possible gene networks were created by the IPA program to show intermolecular connections. Interestingly, of 213 differentially expressed genes, BMP2, C8orf79, F10, and NPY were expressed distinctly in vaccine ILTV infection when compared to virulent ILTV infection.</p> <p>Conclusions</p> <p>Comprehensive knowledge of gene expression and biological functionalities of host factors during vaccine ILTV infection can provide insight into host cellular defense mechanisms compared to those of virulent ILTV.</p

Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line

BACKGROUND: The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years. RESULTS: The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21(WAF1 )gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15(INK4b )expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells. CONCLUSION: The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21(WAF1), may have contributed to the immortalization of the SC-2 CEF cell line

Progesterone signalling in broiler skeletal muscle is associated with divergent feed efficiency

Background: We contrast the pectoralis muscle transcriptomes of broilers selected from within a single genetic line expressing divergent feed efficiency (FE) in an effort to improve our understanding of the mechanistic basis of FE. Results: Application of a virtual muscle model to gene expression data pointed to a coordinated reduction in slow twitch muscle isoforms of the contractile apparatus (MYH15, TPM3, MYOZ2, TNNI1, MYL2, MYOM3, CSRP3, TNNT2), consistent with diminishment in associated slow machinery (myoglobin and phospholamban) in the high FE animals. These data are in line with the repeated transition from red slow to white fast muscle fibres observed in agricultural species selected on mass and FE. Surprisingly, we found that the expression of 699 genes encoding the broiler mitoproteome is modestly–but significantly–biased towards the high FE group, suggesting a slightly elevated mitochondrial content. This is contrary to expectation based on the slow muscle isoform data and theoretical physiological capacity arguments. Reassuringly, the extreme 40 most DE genes can successfully cluster the 12 individuals into the appropriate FE treatment group. Functional groups contained in this DE gene list include metabolic proteins (including opposing patterns of CA3 and CA4), mitochondrial proteins (CKMT1A), oxidative status (SEPP1, HIG2A) and cholesterol homeostasis (APOA1, INSIG1). We applied a differential network method (Regulatory Impact Factors) whose aim is to use patterns of differential co-expression to detect regulatory molecules transcriptionally rewired between the groups. This analysis clearly points to alterations in progesterone signalling (via the receptor PGR) as the major driver. We show the progesterone receptor localises to the mitochondria in a quail muscle cell line. Conclusions: Progesterone is sometimes used in the cattle industry in exogenous hormone mixes that lead to a ~20% increase in FE. Because the progesterone receptor can localise to avian mitochondria, our data continue to point to muscle mitochondrial metabolism as an important component of the phenotypic expression of variation in broiler FE

Enzymatic engineering of the porcine genome with transposons and recombinases

<p>Abstract</p> <p>Background</p> <p>Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs.</p> <p>Results</p> <p>Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- <it>Sleeping Beauty</it>, <it>Tol2</it>, <it>piggyBac</it>, and <it>Passport </it>in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons.</p> <p>Conclusion</p> <p>We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.</p

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

<p>Abstract</p> <p>Background</p> <p>Infection by infectious laryngotracheitis virus (ILTV; <it>gallid herpesvirus 1</it>) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays.</p> <p>Results</p> <p>Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death.</p> <p>Conclusion</p> <p>The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections.</p

B cells Using Calcium Signaling for Specific and Rapid Detection of Escherichia coli O-157:H-7

A rapid and sensitive detection technology is highly desirable for specific detection of E. coli O-157:H-7, one of the leading bacterial pathogens causing foodborne illness. In this study, we reported the rapid detection of E. coli O-157: H-7 by using calcium signaling of the B cell upon cellular membrane anchors anti-E. coli O-157: H-7 IgM. The binding of E. coli O-157:H-7 to the IgM on B cell surface activates the B cell receptor (BCR)-induced Ca2+ signaling pathway and results in the release of Ca2+ within seconds. The elevated intracellular Ca2+ triggers Fura-2, a fluorescent Ca2+ indicator, for reporting the presence of pathogens. The Fura-2 is transferred to B cells before detection. The study demonstrated that the developed B cell based biosensor was able to specifically detect E. coli O-157:H-7 at the low concentration within 10 min in pure culture samples. Finally, the B cell based biosensor was used for the detection of E. coli O-157:H-7 in ground beef samples. With its short detection time and high sensitivity at the low concentration of the target bacteria, this B cell biosensor shows promise in future application of the high throughput and rapid food detection, biosafety and environmental monitoring

Regulacija sile hidrauličke preše pomoću programa LabVIEW i uređaja NI-myRIO

U ovom završnom radu načinjen je eksperiment regulacije sile hidrauličke preše pomoću programa LabVIEW i uređaja NI myRIO. LabVIEW je grafički programski jezik namijenjen lakšem sakupljanju podataka iz ljudske okoline sa raznih instrumenata. Najčešće se koristi za prikupljanje, analizu i obradu podataka te kontrolu instrumenata i opreme. Programi rađeni pomoću LabVIEW alata nazivaju se Virtualni Instrumenti, skraćeno VI (engl. Virtual Instruments), jer njihov izgled i rad imitira stvarne instrumente. Svaki VI se sastoji od prednjeg panela i blok dijagrama. Prednji panel (engl. Front panel) namijenjen je za izradu grafičkog sučelja koji korisnik vidi kada je sustav pokrenut, a blok dijagram (engl. Block diagram) sadrži programske elemente preko kojih se odvija obrada podataka. S elektro-hidrauličkim servo sustavom povezan je uređaj NI myRIO na kojem se izvršava upravljački program za regulaciju sile hidrauličke preše primjenom servo ventila. NI myRIO je laboratorijski uređaj koji omogućuje izvršavanje algoritma u stvarnom realnom vremenu. Za vizualizaciju i nadzor rada elektro-hidrauličkog servo sustava koristi se prijenosno računalo na kojem se nalazi grafičko sučelje