Cre-mediated, loxP independent sequential recombination of a tripartite transcriptional stop cassette allows for partial read-through transcription

Bapst, Andreas M.; Dahl, Sophie L.; Knöpfel, Thomas; Wenger, Roland H.

doi:10.1016/j.bbagrm.2020.194568

Cited by 15 publications

(13 citation statements)

References 27 publications

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“…We believe that in our mouse model, the low level of Tek in the dim tdT cells translates into a fewer recombination events (fewer Cre molecules excising a small percentage of transgene integrants), resulting in two different fluorescence intensity signals from identical locus. Consistent with our findings, Bapst AM et al demonstrated the presence of low and high intensity tdTomato expressing cells in the kidney from the identical promoter 34 .…”

Section: Discussionsupporting

confidence: 93%

Glomerular endothelial cell heterogeneity in Alport syndrome

Soloyan

Thornton

Villani

et al. 2020

Sci Rep

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Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. How GEC affect the pathology of Alport syndrome (AS) however, is unclear. We characterized GEC from wild type (WT) and col4 α 5 knockout AS mice, a hereditary disorder characterized by progressive renal failure. We used endothelial-specific Tek-tdTomato reporter mice to isolate GEC by FACS and performed transcriptome analysis on them from WT and AS mice, followed by in vitro functional assays and confocal and intravital imaging studies. Biopsies from patients with chronic kidney disease, including AS were compared with our findings in mice. We identified two subpopulations of GEC (dim tdT and bright tdT ) based on the fluorescence intensity of the Tek tdT signal. In AS mice, the bright tdT cell number increased and presented differential expression of endothelial markers compared to WT. RNA-seq analysis revealed differences in the immune and metabolic signaling pathways. In AS mice, dim tdT and bright tdT cells had different expression profiles of matrix-associated genes ( Svep1, Itgβ6 ), metabolic activity ( Apom, Pgc1α) and immune modulation ( Apelin, Icam1 ) compared to WT mice. We confirmed a new pro-inflammatory role of Apelin in AS mice and in cultured human GEC. Gene modulations were identified comparable to the biopsies from patients with AS and focal segmental glomerulosclerosis, possibly indicating that the same mechanisms apply to humans. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans. This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets.

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Section: Discussionsupporting

confidence: 93%

Glomerular endothelial cell heterogeneity in Alport syndrome

Soloyan

Thornton

Villani

et al. 2020

Sci Rep

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“…1 B ). Since the flexon exon is a single, short, nonrepetitive sequence, it is relatively easy to clone and adjust ( SI Appendix ) and presumably would not be subject to incomplete recombination of repetitive stop sequences ( 21 ). If the initial premature stop is read through, the subsequent frameshift mutations would be expected to create a nonfunctional protein.…”

Section: Resultsmentioning

confidence: 99%

“…Unwanted expression of the gene can occur from incomplete abrogation of transcription and translational initiation downstream of the stop cassette. Leaky expression has also been observed from read-through transcription that results from incomplete recombination between sequence repeats within the stop cassette ( 21 ). While leaky expression is the chief concern for stop cassette usage in transgenes, further limitations apply when using a stop cassette in an endogenous context.…”

mentioning

confidence: 99%

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

Shaffer

Greenwald

2022

Proc. Natl. Acad. Sci. U.S.A.

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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.

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“…Research into the frequency and level of this leaky expression (in invertebrates) would be of great interest. Inversely, incomplete removal of the stop cassette leading to variable levels of expression has also been demonstrated in mice (Bapst et al, 2020), and the incidence of this phenomenon in invertebrates would also be useful to quantify. Although the irreversibility of the ultimate result of recombinase systems can be advantageous in some cases, it can also be a major disadvantage in and of itself as it is a one-way street: once Cre or Flp has been induced and executed the recombination reaction, target expression is permanently turned on or off.…”

Section: Site-directed Recombination Unidirectionally Switches Expression On or Off Using Heat Compounds Or Lightmentioning

confidence: 99%