Analysis of an approach to oviduct-specific expression of modified chicken lysozyme genes

2005 ◽  
Vol 83 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Mamiko Shimizu ◽  
Jan K Losos ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Gene Targeting ◽  
Stop Codon ◽  
Specific Gene ◽  
Specific Expression ◽  
Endogenous Gene ◽  
Lysozyme Gene ◽  
Tubular Gland ◽  
Chicken Lysozyme

The –2.7 kb enhancer (E) element of the chicken lysozyme gene domain appears to govern expression of the gene in macrophages but not in oviduct tubular gland cells, the only other site of lysozyme expression. The ultimate goal of our research was to determine whether lysozyme domain variants could be developed that would mainly be expressed in the oviduct so that transgenic birds could be produced that would deposit exogenous protein in the egg white. Accordingly, precise mutations were made by poxvirus-mediated gene targeting in FEF/PU.1 and CCAAT/enhancer-binding protein (C/EBP) transcription factor binding sites in the –2.7 kb E of cloned copies of a specific lysozyme gene variant that includes a hydrophobic pentapeptide tail encoding sequence inserted immediately prior to the stop codon. This variant contains the entire lysozyme domain and is cloned in a λ bacteriophage vector (λDIILys-HT); the novel tail sequence enables distinction in cell-based expression systems between transcripts of the variant and those of the endogenous gene. These various lysozyme domain mutants, in bacteriophage vector form, were tested for expression in cultured chicken blastodermal cells cotransfected with plasmids encoding the transcription factors C/EBP and v-Myb. In the absence of these plasmids, barely detectable levels of endogenous lysozyme gene transcription resulted in the blasto dermal cells. In the presence of the plasmids, however, transcripts of the endogenous gene could be detected as well as varying levels (as evaluated by quantitative real-time PCR) of transcripts of all of the lysozyme domain mutants. These results are discussed in the context of the known role and occurrence of various transcription factors involved in gene expression in differentiating macrophage cells. The ultimate test of expression of the variants in macrophages vs. oviduct cells will be to use them to produce transgenic birds.Key words: lysozyme, macrophage-specific gene expression, poxvirus-mediated gene targeting.

scholarly journals The Th1 cell regulatory circuitry is largely conserved between human and mouse

2021 ◽  
Author(s):  
Stephen Henderson ◽  
Venu Pullabhatla ◽  
Arnulf Hertweck ◽  
Emanuele de Rinaldis ◽  
Javier Herrero ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Binding Site ◽  
Inflammatory Diseases ◽  
Specific Gene ◽  
Type I ◽  
Th1 Cell ◽  
Specific Expression ◽  
A Genome ◽  
Species Specific

ABSTRACTGene expression programmes controlled by lineage-determining transcription factors are often conserved between species. However, infectious diseases have exerted profound evolutionary pressure, and therefore the genes regulated by immune-specific transcription factors might be expected to exhibit greater divergence due to exposure to species-specific pathogens. T-bet (Tbx21) is the immune-specific lineage-defining transcription factor for T helper type I (Th1) immunity, which is fundamental for the immune response to intracellular pathogens but also underlies inflammatory diseases. We therefore compared T-bet genomic targets between mouse and human CD4+ T cells and correlated T-bet binding patterns with species-specific gene expression. Remarkably, we show that the vast majority of T-bet regulated genes are conserved between mouse and human, either via preservation of a binding site or via an alternative binding site associated with transposon-linked insertion. We also identified genes that are specifically targeted by T-bet in humans or mice and which exhibited species-specific expression. These results provide a genome-wide cross-species comparison of T-bet target gene regulation that will enable more accurate translation of genetic targets and therapeutics from pre-clinical models of inflammatory disease into human clinical trials.

scholarly journals The combined detection of Amphiregulin, Cyclin A1 and DDX20/Gemin3 expression predicts aggressive forms of oral squamous cell carcinoma

2021 ◽  
Author(s):  
Ekaterina Bourova-Flin ◽  
Samira Derakhshan ◽  
Afsaneh Goudarzi ◽  
Tao Wang ◽  
Anne-Laure Vitte ◽  
...  
Keyword(s):  
Gene Expression ◽  
Squamous Cell ◽  
Large Scale ◽  
Biomarker Discovery ◽  
Gene Expression Signature ◽  
Predictive Biomarkers ◽  
Specific Gene ◽  
Specific Expression ◽  
Intrinsic Nature ◽  
Independent Cohort

Abstract Background Large-scale genetic and epigenetic deregulations enable cancer cells to ectopically activate tissue-specific expression programmes. A specifically designed strategy was applied to oral squamous cell carcinomas (OSCC) in order to detect ectopic gene activations and develop a prognostic stratification test. Methods A dedicated original prognosis biomarker discovery approach was implemented using genome-wide transcriptomic data of OSCC, including training and validation cohorts. Abnormal expressions of silent genes were systematically detected, correlated with survival probabilities and evaluated as predictive biomarkers. The resulting stratification test was confirmed in an independent cohort using immunohistochemistry. Results A specific gene expression signature, including a combination of three genes, AREG, CCNA1 and DDX20, was found associated with high-risk OSCC in univariate and multivariate analyses. It was translated into an immunohistochemistry-based test, which successfully stratified patients of our own independent cohort. Discussion The exploration of the whole gene expression profile characterising aggressive OSCC tumours highlights their enhanced proliferative and poorly differentiated intrinsic nature. Experimental targeting of CCNA1 in OSCC cells is associated with a shift of transcriptomic signature towards the less aggressive form of OSCC, suggesting that CCNA1 could be a good target for therapeutic approaches.

scholarly journals Methylation of the Cyclin A1 Promoter Correlates with Gene Silencing in Somatic Cell Lines, while Tissue-Specific Expression of Cyclin A1 Is Methylation Independent

2000 ◽  
Vol 20 (9) ◽  
pp. 3316-3329 ◽  
Author(s):  
Carsten Müller ◽  
Carol Readhead ◽  
Sven Diederichs ◽  
Gregory Idos ◽  
Rong Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Promoter Methylation ◽  
Cpg Island ◽  
Trichostatin A ◽  
Specific Gene ◽  
Cyclin A1 ◽  
Specific Expression ◽  
Tissue Specific ◽  
Tissue Specific Expression

ABSTRACT Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

Stage-specific expression of a developmentally regulated gene in Dirofilaria immitis

1992 ◽  
Vol 66 (1) ◽  
pp. 62-67 ◽  
Author(s):  
S. Sun ◽  
T. Matsuura ◽  
K. Sugane
Keyword(s):  
Gene Expression ◽  
Cdna Clone ◽  
Dirofilaria Immitis ◽  
Mrna Level ◽  
Specific Antigen ◽  
Specific Gene ◽  
Developmentally Regulated ◽  
Specific Expression ◽  
Specific Gene Expression

ABSTRACTA previously reported cDNA clone encoding 34 kDa antigenic polypeptide of Dirofilaria immitis (λ cD34) was studied to elucidate the mechanism of stage-specific gene expression. The 34 kDa polypeptide was a larva-specific antigen and the mRNA was detectable in microfilariae but not in adult worms and eggs. The λ cD34 gene was not sex linked and was contained in the genome of D. immitis at each stage. The stage-specific expression of the developmentally regulated gene in D. immitis may be controlled primarily at the mRNA level.

Transcription Factors Controlling Muscle-Specific Gene Expression

1993 ◽  
pp. 93-115 ◽  
Author(s):  
John J. Schwarz ◽  
James F. Martin ◽  
Eric N. Olson
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Specific Gene ◽  
Specific Gene Expression

scholarly journals Promoter-specific regulation of PPARGC1A gene expression in human skeletal muscle

2015 ◽  
Vol 55 (2) ◽  
pp. 159-168 ◽  
Author(s):  
Daniil V Popov ◽  
Evgeny A Lysenko ◽  
Tatiana F Vepkhvadze ◽  
Nadia S Kurochkina ◽  
Pavel A Maknovskii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Stop Codon ◽  
Constitutive Expression ◽  
Alternative Promoter ◽  
Specific Expression ◽  
Post Exercise ◽  
Specific Regulation ◽  
Intensity Of Exercise

The goal of this study was to identify unknown transcription start sites of thePPARGC1A(PGC-1α) gene in human skeletal muscle and investigate the promoter-specific regulation ofPGC-1αgene expression in human skeletal muscle. Ten amateur endurance-trained athletes performed high- and low-intensity exercise sessions (70 min, 70% or 50%o2max). High-throughput RNA sequencing and exon–exon junction mapping were applied to analyse muscle samples obtained at rest and after exercise.PGC-1αpromoter-specific expression and activation of regulators of PGC-1α gene expression (AMPK, p38 MAPK, CaMKII, PKA and CREB1) after exercise were evaluated using qPCR and western blot. Our study has demonstrated that during post-exercise recovery, human skeletal muscle expresses thePGC-1αgene via two promoters only. As previously described, the additional exon 7a that contains a stop codon was found in all samples. Importantly, only minor levels of other splice site variants were found (and not in all samples). Constitutive expressionPGC-1αgene occurs via the canonical promoter, independent of exercise intensity and exercise-induced increase of AMPKThr172phosphorylation level. Expression ofPGC-1αgene via the alternative promoter is increased of two orders after exercise. This post-exercise expression is highly dependent on the intensity of exercise. There is an apparent association between expression via the alternative promoter and activation of CREB1.

scholarly journals Genetic influences on cell type specific gene expression and splicing during neurogenesis elucidate regulatory mechanisms of brain traits

2020 ◽  
Author(s):  
Nil Aygün ◽  
Angela L. Elwell ◽  
Dan Liang ◽  
Michael J. Lafferty ◽  
Kerry E. Cheek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
Regulatory Mechanisms ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Risk Variants ◽  
Allele Specific ◽  
Cell Type Specific ◽  
Regulatory Effects

SummaryInterpretation of the function of non-coding risk loci for neuropsychiatric disorders and brain-relevant traits via gene expression and alternative splicing is mainly performed in bulk post-mortem adult tissue. However, genetic risk loci are enriched in regulatory elements of cells present during neocortical differentiation, and regulatory effects of risk variants may be masked by heterogeneity in bulk tissue. Here, we map e/sQTLs and allele specific expression in primary human neural progenitors (n=85) and their sorted neuronal progeny (n=74). Using colocalization and TWAS, we uncover cell-type specific regulatory mechanisms underlying risk for these traits.

60 CRISPR-on, a new tool for activation of endogenous gene expression in bovine embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 155
Author(s):  
V. Savy ◽  
V. Alberio ◽  
N. Canel ◽  
L. Ratner ◽  
M. Gismondi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Regulatory Region ◽  
Bovine Embryos ◽  
Endogenous Gene ◽  
Cell Fate Decisions ◽  
Guide Rnas ◽  
Endogenous Expression ◽  
Mammalian Embryos

The CRISPR-Cas9 system enables precise genome editing in mammalian somatic cells and embryos at a very high efficiency. A modified version of Cas9 (dCas9) was engineered, resulting in a DNA binding protein capable of site-specific target recognition but unable to cut the DNA. By means of dCas9 fusion to heterologous domains, including transcriptional activators or repressors, specific modulation of gene expression has successfully been achieved invitro, making possible the modulation of the cell-differentiation state. However, CRISPR-mediated transcriptional activation (CRISPR-on) has been mainly used invitro, and to our knowledge, there are no reports regarding its use for the activation of endogenous gene expression in mammalian embryos. As a proof of principle, we evaluated the CRISPR-on system in bovine embryos for modulation of endogenous expression of SMARCA4 and TFAP2C, transcription factors implicated in trophoblast lineage commitment. We hypothesised that CRISPR-on may induce SMARCA4 or TFAP2C endogenous expression, enabling the design of strategies to induce trophectoderm proliferation of invitro-derived embryos. To this aim, we designed and synthesised 4 non-overlapping single guide RNAs to target the regulatory region of each of these target genes. Presumptive zygotes were cytoplasmically microinjected with a mix containing dCas9-VP160 mRNA and a pool of 4 single guide RNAs targeting SMARCA4 (dCas9_SM group) or TFAP2C (dCas9_TF group). As control, a non-injected group was also included. Analysis was carried out in pools of 10 early embryos or 5 blastocysts and at least 3 biological replicates were included. Gene expression was assessed by RTqPCR at Days 2, 4, and 7 after microinjection and data were normalized to that obtained for the non-injected group. The CRISPR-on system was efficient to induce expression of SMARCA4 two days after microinjection (dCas9_SM group, Mann-Whitney t-test; P<0.05), but failed to significantly increase TFAP2C expression (dCas9_TF group). Surprisingly, CDX2, which is a downstream effector for trophectoderm maintenance, was induced both in dCas9_SM and dCas9_TF groups, supporting the CRISPR-mediated induction of targeted transcription factors. However, no changes were observed in the endogenous level of NANOG. Additional analysis is currently ongoing to determine whether CRISPR-on mediated induction of SMARCA4 and/or TFAP2C expression affects lineage specification and regulation. To our knowledge, this is the first report on the use of CRISPR-on for modulation of endogenous gene expression in mammalian embryos. Our study lays the foundations for CRISPR-on application in embryos as a useful tool to understand key cell fate decisions and will enable unprecedented studies of significance to embryo development, cell differentiation, and segregation.

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