scholarly journals A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

2021 ◽  
Author(s):  
Justin M Shaffer ◽  
Iva Greenwald
Keyword(s):  
Gene Expression ◽  
Specific Protein ◽  
Specific Gene ◽  
Coding Region ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Nonsense Mediated Decay ◽  
Conditional Gene Expression ◽  
Stop Cassette

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.

scholarly journals Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

2022 ◽  
Vol 119 (3) ◽  
pp. e2117451119
Author(s):  
Justin M. Shaffer ◽  
Iva Greenwald
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Specific Protein ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Site Specific ◽  
Tissue Specific ◽  
Endogenous Gene ◽  
Conditional Gene Expression ◽  
Stop Cassette

Conditional gene expression is a powerful tool for genetic analysis of biological phenomena. In the widely used “lox-stop-lox” approach, insertion of a stop cassette consisting of a series of stop codons and polyadenylation signals flanked by lox sites into the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been successfully used in many experimental systems, this design has certain limitations. Here, we describe the Floxed exon (Flexon) approach, which uses a stop cassette composed of an artificial exon flanked by artificial introns, designed to cause premature termination of translation and nonsense-mediated decay of the mRNA and allowing for flexible placement into a gene. We demonstrate its efficacy in Caenorhabditis elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression of green fluorescent protein (GFP) is obtained in specific lineages. We also demonstrate its efficacy in an endogenous gene context: we inserted a flexon into the Argonaute gene rde-1 to abrogate RNA interference (RNAi), and restored RNAi tissue specifically by expression of Cre. Finally, we describe several potential additional applications of the Flexon approach, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation, and generation of genetic mosaics. The Flexon approach should be feasible in any system where a site-specific recombination-based method may be applied.

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.

scholarly journals Starvation resistance and tissue-specific gene expression of stress-related genes in a naturally inbred ant population

2016 ◽  
Vol 3 (4) ◽  
pp. 160062 ◽  
Author(s):  
Nick Bos ◽  
Unni Pulliainen ◽  
Liselotte Sundström ◽  
Dalial Freitak
Keyword(s):  
Gene Expression ◽  
Stress Responses ◽  
Specific Gene ◽  
Starvation Resistance ◽  
Specific Expression ◽  
Tissue Specific ◽  
Trade Offs ◽  
Stress Related Genes ◽  
The Individual ◽  
Different Tissues

Starvation is one of the most common and severe stressors in nature. Not only does it lead to death if not alleviated, it also forces the starved individual to allocate resources only to the most essential processes. This creates energetic trade-offs which can lead to many secondary challenges for the individual. These energetic trade-offs could be exacerbated in inbred individuals, which have been suggested to have a less efficient metabolism. Here, we studied the effect of inbreeding on starvation resistance in a natural population of Formica exsecta ants, with a focus on survival and tissue-specific expression of stress, metabolism and immunity-related genes. Starvation led to large tissue-specific changes in gene expression, but inbreeding had little effect on most of the genes studied. Our results illustrate the importance of studying stress responses in different tissues instead of entire organisms.

scholarly journals A non-coding genetic variant maximally associated with serum urate levels is functionally linked to HNF4A-dependent PDZK1 expression

2018 ◽  
Author(s):  
Sarada Ketharnathan ◽  
Megan Leask ◽  
James Boocock ◽  
Amanda J. Phipps-Green ◽  
Jisha Antony ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Genetic Variant ◽  
Fluorescent Protein ◽  
Serum Urate ◽  
Specific Gene ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Tissue Specific

ABSTRACTSeveral dozen genetic variants associate with serum urate levels, but the precise molecular mechanisms by which they affect serum urate are unknown. Here we tested for functional linkage of the maximally-associated genetic variant rs1967017 at the PDZK1 locus to elevated PDZK1 expression.We performed expression quantitative trait locus (eQTL) and likelihood analyses followed by gene expression assays. Zebrafish were used to determine the ability of rs1967017 to direct tissue-specific gene expression. Luciferase assays in HEK293 and HepG2 cells measured the effect of rs1967017 on transcription amplitude.PAINTOR analysis revealed rs1967017 as most likely to be causal and rs1967017 was an eQTL for PDZK1 in the intestine. The region harboring rs1967017 was capable of directly driving green fluorescent protein expression in the kidney, liver and intestine of zebrafish embryos, consistent with a conserved ability to confer tissue-specific expression. The urate-increasing T-allele of rs1967017 strengthens a binding site for the transcription factor HNF4A. siRNA depletion of HNF4A reduced endogenous PDZK1 expression in HepG2 cells. Luciferase assays showed that the T-allele of rs1967017 gains enhancer activity relative to the urate-decreasing C-allele, with T-allele enhancer activity abrogated by HNF4A depletion. HNF4A physically binds the rs1967017 region, suggesting direct transcriptional regulation of PDZK1 by HNF4A.With other reports our data predict that the urate-raising T-allele of rs1967017 enhances HNF4A binding to the PDZK1 promoter, thereby increasing PDZK1 expression. As PDZK1 is a scaffold protein for many ion channel transporters, increased expression can be predicted to increase activity of urate transporters and alter excretion of urate.

scholarly journals Predicting tissue-specific gene expression from whole blood transcriptome

2020 ◽  
Author(s):  
Mahashweta Basu ◽  
Kun Wang ◽  
Eytan Ruppin ◽  
Sridhar Hannenhalli
Keyword(s):  
Gene Expression ◽  
Whole Blood ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Complex Disorders ◽  
Tissue Specific Gene ◽  
Tissue Specific Expression ◽  
Specific Gene Expression ◽  
Blood Transcriptome

AbstractComplex diseases are systemic, largely mediated via transcriptional dysregulation in multiple tissues. Thus, knowledge of tissue-specific transcriptome in an individual can provide important information about an individual’s health. Unfortunately, with a few exceptions such as blood, skin, and muscle, an individual’s tissue-specific transcriptome is not accessible through non-invasive means. However, due to shared genetics and regulatory programs between tissues, the transcriptome in blood may be predictive of those in other tissues, at least to some extent. Here, based on GTEx data, we address this question in a rigorous, systematic manner, for the first time. We find that an individual’s whole blood gene expression and splicing profile can predict tissue-specific expression levels in a significant manner (beyond demographic variables) for many genes. On average, across 32 tissues, the expression of about 60% of the genes is predictable from blood expression in a significant manner, with a maximum of 81% of the genes for the musculoskeletal tissue. Remarkably, the tissue-specific expression inferred from the blood transcriptome is almost as good as the actual measured tissue expression in predicting disease state for six different complex disorders, including Hypertension and Type 2 diabetes, substantially surpassing predictors built directly from the blood transcriptome. The code for our pipeline for tissue-specific gene expression prediction – TEEBoT, is provided, enabling others to study its potential translational value in other indications.

scholarly journals Intronic enhancers regulate the expression of genes involved in tissue-specific functions and homeostasis

2020 ◽  
Author(s):  
Beatrice Borsari ◽  
Pablo Villegas-Mirón ◽  
Hafid Laayouni ◽  
Alba Segarra-Casas ◽  
Jaume Bertranpetit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Specific Gene ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Specific Gene Expression ◽  
Expression Of Genes ◽  
Genomic Location

AbstractTissue function and homeostasis reflect the gene expression signature by which the combination of ubiquitous and tissue-specific genes contribute to the tissue maintenance and stimuli-responsive function. Enhancers are central to control this tissue-specific gene expression pattern. Here, we explore the correlation between the genomic location of enhancers and their role in tissue-specific gene expression. We found that enhancers showing tissue-specific activity are highly enriched in intronic regions and regulate the expression of genes involved in tissue-specific functions, while housekeeping genes are more often controlled by intergenic enhancers. Notably, an intergenic-to-intronic active enhancers continuum is observed in the transition from developmental to adult stages: the most differentiated tissues present higher rates of intronic enhancers, while the lowest rates are observed in embryonic stem cells. Altogether, our results suggest that the genomic location of active enhancers is key for the tissue-specific control of gene expression.

A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression

1992 ◽  
Vol 12 (1) ◽  
pp. 120-127
Author(s):  
M Schickler ◽  
S A Lira ◽  
R A Kinloch ◽  
P M Wassarman
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
Small Region ◽  
Specific Protein ◽  
Luciferase Gene ◽  
Flanking Sequence ◽  
Coding Region ◽  
Mobility Shift ◽  
Specific Expression ◽  
Minimal Sequence

The gene encoding mZP3, the mouse sperm receptor, is expressed exclusively in growing oocytes during oogenesis. To investigate the molecular basis of oocyte-specific mZP3 gene expression, we generated several lines of mice harboring a transgene that contains 470 bp of mZP3 gene 5'-flanking sequence (nucleotides -470 to +10) fused to the firefly luciferase gene coding region. Three of four expressing transgenic lines exhibited luciferase activity only in growing oocytes, suggesting that the 470-bp fragment is sufficient to direct Iocyte-specific expression of the luciferase gene. Results of DNase I footprinting and gel mobility shift assays suggested the presence of an ovary-specific protein that binds to a small region (nucleotides-99 to -86) within the 470-bp fragment of the mZP3 promoter, with 5'-G(G/A)T(G/A)A-3' representing the minimal sequence required for binding. Southwestern (DNA-protein) gel blots revealed the presence of an oocyte-specific, approximately 60,000-Mr protein, called OSP-1, that binds to the minimal sequence. Changes in levels of OSP-1 during oogenesis and early cleavage are consistent with the pattern of mZP3 gene expression during these developmental stages in mice. Therefore, OSP-1 may be a mammalian oocyte-specific transcription factor involved in regulating oocyte-specific mZP3 gene expression.

Melanocyte-specific gene expression: role of repression and identification of a melanocyte-specific factor, MSF

1994 ◽  
Vol 14 (5) ◽  
pp. 3494-3503
Author(s):  
U Yavuzer ◽  
C R Goding
Keyword(s):  
Gene Expression ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Specific Factor ◽  
Specific Gene ◽  
Binding Motif ◽  
Specific Expression ◽  
Tissue Specific ◽  
Specific Gene Expression

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocyte-specific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene.

A modified pod specific promoter for high level heterologous expression of genes in legumes

2019 ◽  
Author(s):  
Aravind Kumar Konda ◽  
Pallavi Singh ◽  
Khela Ram Soren ◽  
Narendra Pratap Singh
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Expression ◽  
Tissue Specific ◽  
Cis Acting ◽  
Inducible Promoters ◽  
Expression Of Genes ◽  
Enhanced Expression ◽  
High Level

Promoters are cis-acting regulatory elements that are usually present upstream to the coding sequences and determine the gene expression. Deployment of tissue specific and inducible promoters are constantly increasing for development of successful and stable multiple transgenic plants. To this end, as a strategy for enhanced expression of cis or transgenes, promoter engineering of the native msg promoter from soya bean has been carried out for executing pod specific expression of genes. Cis regulatory elements such as 5’UTR and poly (A) tract have been incorporated for imparting mRNA stability and translational enhancement to generate the modified 1.285 Kb pod specific promoter. Further to attain transcriptional enhancement the modified promoter has been cloned to generate Bi-directional Duplex Promoters (BDDP). The engineered msg promoter gene constructs can be deployed for high level tissue specific gene expression of cis/trans genes along with chosen terminator in chickpea. soybean and other legumes as well.

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