Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus

Yang, George; Banks, Kathleen G.; Bonaguro, Russell J.; Wilson, Gary; Dreolini, Lisa; Leeuw, Charles N. de; Liu, L.; Swanson, Douglas J.; Goldowitz, Dan; Holt, Robert A.; Simpson, Elizabeth M.

doi:10.1016/j.ygeno.2008.09.014

Cited by 40 publications

(55 citation statements)

References 41 publications

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“…We selected the CAG ubiquitous promoter as well as five previously characterized multicopy random-insertion promoters that drive expression in a subset of neurons or glial cells, generated vector constructs, and targeted insertion to the Hprt locus (Dataset S1 "Control"). The CAG promoter drives ubiquitous reporter gene activity, demonstrating that widespread, adult expression can be achieved from the locus, consistent with previous studies (10).…”

Section: Validation Of the Approach Using A Set Of Previously Charactsupporting

confidence: 88%

“…The MiniP sequences are cloned 5′ of a reporter gene, such as EGFP, lacZ, or an EGFP/cre fusion protein. The constructs are then introduced by homologous recombination immediately 5′ of the Hprt locus on the X chromosome, as previously described (9,10), providing a single-copy knockin insertion for reproducible, predictable expression (11).…”

Section: Resultsmentioning

confidence: 99%

“…S5 and at http://www.pleiades.org. (8,10,11) and all others (5-7, 9) identify EGR1 (e; red) and FOS (f; green) binding sites putatively responsible for Ple151-specific expression. The conservation plots were captured from the University of California Santa Cruz Genome Browser.…”

Section: Discussionmentioning

confidence: 97%

“…The pipeline, involving five specialized laboratories, is summarized below. The general methods used have been described previously (10). Modifications or additions specific to this work are available in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

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A regulatory toolbox of MiniPromoters to drive selective expression in the brain

Portales-Casamar

Swanson

Liu

et al. 2010

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

112

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The Pleiades Promoter Project integrates genomewide bioinformatics with large-scale knockin mouse production and histological examination of expression patterns to develop MiniPromoters and related tools designed to study and treat the brain by directed gene expression. Genes with brain expression patterns of interest are subjected to bioinformatic analysis to delineate candidate regulatory regions, which are then incorporated into a panel of compact human MiniPromoters to drive expression to brain regions and cell types of interest. Using single-copy, homologous-recombination “knockins” in embryonic stem cells, each MiniPromoter reporter is integrated immediately 5′ of the Hprt locus in the mouse genome. MiniPromoter expression profiles are characterized in differentiation assays of the transgenic cells or in mouse brains following transgenic mouse production. Histological examination of adult brains, eyes, and spinal cords for reporter gene activity is coupled to costaining with cell-type–specific markers to define expression. The publicly available Pleiades MiniPromoter Project is a key resource to facilitate research on brain development and therapies.

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Section: Validation Of the Approach Using A Set Of Previously Charactsupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A regulatory toolbox of MiniPromoters to drive selective expression in the brain

Portales-Casamar

Swanson

Liu

et al. 2010

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

112

View full text Add to dashboard Cite

show abstract

“…in these loci [1,18,20,51,63,72,77]. Since expression of the targeted transgene from these loci is highly reproducible, they are frequently used for rigorous analysis of transgene expression [13,38,55,65,77].…”

Section: Es Cell Targeting By Homologous Recombinationmentioning

confidence: 99%

PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

et al. 2012

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Transgenic (Tg) mice have been extensively used as valuable tools for analyses of gene function and have also served as models for many human diseases. Typically, a transgenic mouse is created by microinjection of DNA into pronuclei in which the DNA gets integrated at random locations in the genome. Frequently however, the random integration of multiple copies of a transgene results in transgene silencing, probably because of a positional effect and/or repeat-induced gene silencing. The transgene silencing issue has been overcome by single-copy transgene integration into a predetermined locus through ES cell-mediated transgenesis, despite it being expensive and more time-consuming compared with pronuclear injection (PI)-mediated transgenesis. Recently, several groups have reported novel approaches that employ PI for targeted transgenesis. They are based on site-specific recombination catalyzed by a recombinase or an integrase or homologous recombination enhanced by a zinc-finger nuclease via PI. These next-generation transgenesis methods, which we termed as PI-based Targeted Transgenesis (PITT), are more convenient and faster than ES cell-based transgenesis. Furthermore, the Tg mice generated by these newer methods contain a single-copy transgene and exhibit reliable expression of the transgene. The objective of this review is to present the recent progress in mouse targeted transgenesis.

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An oocyte‐specific Cas9‐expressing mouse for germline CRISPR/Cas9‐mediated genome editing

Lanza,

Mao,

Lorenzo

et al. 2024

Genesis

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SummaryCas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene‐edited animals creates unique issues including ill‐defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte‐specific, Gdf9 promoter driven, Cas9 transgene (Gdf9‐Cas9) targeted as a single copy into the Hprt1 locus. The X‐linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9‐Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9‐Cas9 does not mediate genome editing, confirming that Gdf9‐Cas9 is not expressed in embryos. Finally, we demonstrate that off‐target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9‐Cas9 transgene is a viable alternative to exogenous Cas9.

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Next generation tools for high-throughput promoter and expression analysis employing single-copy knock-ins at the Hprt1 locus

Cited by 40 publications

References 41 publications

A regulatory toolbox of MiniPromoters to drive selective expression in the brain

A regulatory toolbox of MiniPromoters to drive selective expression in the brain

PITT: Pronuclear Injection-Based Targeted Transgenesis, a Reliable Transgene Expression Method in Mice

An oocyte‐specific Cas9‐expressing mouse for germline CRISPR/Cas9‐mediated genome editing

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