scholarly journals Conditional Gene Expression/Deletion Systems forMarchantia polymorphaUsing its Own Heat-Shock Promoter and Cre/loxP-Mediated Site-Specific Recombination

2015 ◽  
Vol 57 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Ryuichi Nishihama ◽  
Sakiko Ishida ◽  
Hiroko Urawa ◽  
Yasuhiro Kamei ◽  
Takayuki Kohchi
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Heat Shock Promoter ◽  
Conditional Gene Expression

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 Site-specific recombination between inverted target sites generates dicentric/acentric chromosomes

2020 ◽  
Author(s):  
Simon W. A. Titen ◽  
Makenna T. B. Johnson ◽  
Mario Capecchi ◽  
Kent G. Golic
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Cell Biology ◽  
Negative Effects ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Dicentric Chromosomes ◽  
Sister Chromatids ◽  
Target Sites ◽  
And Behavior

ABSTRACTSite-specific recombinases are widely used tools for analysis of genetics, development and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the consequence of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

Use of Synchronous Site-Specific Recombination in Vivo to Regulate Gene Expression

1984 ◽  
Vol 2 (12) ◽  
pp. 1045-1049 ◽  
Author(s):  
K. Backman ◽  
M. J. O'Connor ◽  
A. Maruya ◽  
M. Erfle
Keyword(s):  
Gene Expression ◽  
Site Specific ◽  
Regulate Gene Expression ◽  
Site Specific Recombination ◽  
Regulate Gene

scholarly journals Site-Specific Recombination with Inverted Target Sites: A Cautionary Tale of Dicentric and Acentric Chromosomes

Genetics ◽  
2020 ◽  
Vol 215 (4) ◽  
pp. 923-930
Author(s):  
Simon W. A. Titen ◽  
Makenna T. B. Johnson ◽  
Mario Capecchi ◽  
Kent G. Golic
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Cautionary Tale ◽  
Negative Effects ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Dicentric Chromosomes ◽  
Sister Chromatids ◽  
Target Sites ◽  
And Behavior

Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

Engrailed-mediated repression of Ultrabithorax is necessary for the parasegment 6 identity in Drosophila

Development ◽  
1994 ◽  
Vol 120 (11) ◽  
pp. 3205-3212 ◽  
Author(s):  
R.S. Mann
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Homeotic Gene ◽  
Homeotic Genes ◽  
Posterior Compartment ◽  
Anterior Compartment ◽  
Heat Shock Promoter ◽  
Sensory Structures ◽  
Different Levels ◽  
Homeotic Gene Expression

The homeotic genes of Drosophila are expressed in overlapping domains along the anterior-to-posterior axis and specify the distinct morphological patterns of each parasegment. Within single parasegments, the levels of homeotic gene expression are often modulated, in part because of cross-regulation by other homeotic gene products. However, the functional significance of different levels of homeotic gene expression is unclear. Here modulations in Ultrabithorax (Ubx) expression within parasegment 6 are examined. Specifically, Ubx is shown to be down-regulated in the posterior compartment of this parasegment by engrailed (en). The significance of Ubx repression by en was demonstrated by characterizing the expression of the Ubx target gene, Distal-less (Dll). In the posterior compartment of parasegment 6, Dll is normally expressed in a small cluster of cells. If Ubx is expressed uniformly via a heat-shock promoter, Dll is inappropriately repressed in these posterior compartment cells. In the anterior compartment of parasegment 6, Dll is normally repressed by high levels of Ubx. However, if en is expressed uniformly via a heat-shock promoter, Ubx is repressed and Dll is derepressed. Because Dll is required for the development of larval sensory structures, these results demonstrate that en-mediated repression of Ubx in the posterior compartment is necessary for the morphology of parasegment 6. Thus, different levels of homeotic gene expression can be important for their segmental patterning functions.

Identification of a Functional Medaka Heat Shock Promoter and Characterization of Its Ability to Induce Exogenous Gene Expression in Medaka in Vitro and In Vivo

2010 ◽  
Vol 27 (5) ◽  
pp. 410-415 ◽  
Author(s):  
Shoji Oda ◽  
Sachi Mikami ◽  
Yusuke Urushihara ◽  
Yasuhiko Murata ◽  
Yasuhiro Kamei ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Promoter ◽  
Exogenous Gene
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