Embryonic expression of an <i>Nkx2‐5/Cre</i> gene using <i>ROSA26</i> reporter mice

Moses, Kelvin A.; DeMayo, Francesco J.; Braun, Renee M.; Reecy, James M.; Schwartz, Robert J.

doi:10.1002/gene.10022

Cited by 266 publications

(316 citation statements)

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“…1C,D). By crossing ROSA‐Neo‐EGFP‐Cetn1 mice to cardiac‐specific Nkx2.5‐Cre mice (Moses et al ., 2001), EGFP‐Centrin1 was expressed specifically in heart, as expected (Fig. 1E).…”

Section: Resultssupporting

confidence: 83%

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

Hirai

Chen

Evans

2016

Genesis

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SummaryCentrosomes are major microtubule organizing centers (MTOCs) that play an important role in chromosome segregation during cell division. Centrosomes provide a stable anchor for microtubules, constituting the centers of the spindle poles in mitotic cells, and determining the orientation of cell division. However, visualization of centrosomes is challenging because of their small size. Especially in mouse tissues, it has been extremely challenging to observe centrosomes belonging to a specific cell type of interest among multiple comingled cell types. To overcome this obstacle, we generated a tissue‐specific centrosome indicator. In this mouse line, a construct containing a floxed neomyocin resistance gene with a triplicate polyA sequence followed by an EGFP‐Centrin1 fusion cassette was knocked into the Rosa locus. Upon Cre‐mediated excision, EGFP‐Centrin1 was expressed under the control of the Rosa locus. Experiments utilizing mouse embryo fibroblasts (MEFs) demonstrated the feasibility of real‐time imaging, and showed that EGFP‐Centrin1 expression mirrored the endogenous centrosome cycle, undergoing precisely one round of duplication through the cell cycle. Moreover, experiments using embryo and adult mouse tissues demonstrated that EGFP‐Centrin1 specifically mirrors the localization of endogenous centrosomes. genesis 54:286–296, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

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Section: Resultssupporting

confidence: 83%

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

Hirai

Chen

Evans

2016

Genesis

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“…Nkx2.5-Cre is a knockin of Cre into the Nkx2.5 locus (Moses et al, 2001). When crossed with the general ROSA26lacZ reporter mouse, lacZ expression is weak in the heart crescent at headfold stages, but is robust in the developing heart tube at early somite stages (Moses et al, 2001). In accordance with this expression pattern, we found that ROSA26 eGFP-DTA/+ ;Nkx2.5 Cre/+ compound heterozygous embryos showed a fully penetrant phenotype characterised by the absence of the heart at the 12-14 somite stage (n = 11) ( Fig.…”

Section: Ukpmc Funders Group Author Manuscriptmentioning

confidence: 99%

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confidence: 99%

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In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A

Ivanova

Signore

Caro

et al. 2005

genesis

222

236

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SummaryWe generated a ROSA26-eGFP-DTA mouse line by introducing an eGFP-DTA (enhanced green fluorescent protein -diphtheria toxin fragment A) cassette into the ROSA26 locus by homologous recombination in ES cells. This mouse expresses eGFP ubiquitously, but DTA expression is prevented by the presence of eGFP, a Neo cassette, and a strong transcriptional stop sequence. Mice carrying this construct are normal and fertile, indicating the absence of DTA expression. However, upon Cre-mediated excision of the floxed region DTA expression is activated, resulting in the specific ablation of Cre-expressing cells. As an example of this approach, we ablated Nkx2.5 and Wnt1-expressing cells by using the Nkx2.5-Cre and Wnt1-Cre mouse lines, respectively. We observed loss of the precise tissues in which Nkx2.5 and Wnt1 are expressed. Apart from being a general GFP reporter, the ROSA26-GFP-DTA mouse line should provide a useful resource for genetic ablation of specific groups of cells. KeywordsCre; loxP; diphtheria toxin; eGFP; Nkx2.5; Wnt1; genetic ablation; heart; midbrain The role of individual cells in complex tissues or within the whole organism can be examined by specific deletion of appropriate lineages. For example, during embryogenesis specific groups of cells (signalling centres) control the fate of neighbouring cells by emanating diffusible molecules (Meinhardt, 1983;Martinez et al., 1991;Shimamura and Rubenstein, 1997;Placzek and Briscoe, 2005). Mechanical ablation of restricted embryonic regions has been useful in identifying these signalling centres and their role during development (Placzek et al., 1995;Shimamura and Rubenstein, 1997;Thomas and Beddington, 1996). However, these experiments require very precise surgery on the developing embryo, which can often be a difficult task. In adulthood the loss of small numbers of strategically placed cells may result in conditions such as Parkinson's disease (Moore, 2005), Type I diabetes (Butler et al., 2003), and Hirschsprung's disease (Amiel and Lyonnet, 2001). Thus, a versatile system to specifically ablate cells of any lineage during embryogenesis or in adulthood would be of substantial benefit for studies of development, as well as to model human diseases of various aetiologies. UKPMC Funders Group Author Manuscript UKPMC Funders Group Author ManuscriptWith the aim of developing such a system, we generated a ROSA26-eGFP-DTA mouse line, which combines the use of enhanced green fluorescent protein (GFP), diphtheria toxin A subunit (DTA), and Cre recombinase. eGFP is a mutated version of GFP that displays enhanced fluorescence in mammalian cells in vivo and in vitro (Srinivas et al., 2001). Diphtheria toxin is secreted by pathogenic strains of Corynebacterium diphtheriae and is composed of two subunits, A and B. Subunit B is responsible for the internalisation of the toxin upon binding to its receptor. Once inside the cell, subunit A catalyses the inactivation of elongation factor 2, resulting in termination of protein synthesis and apoptosis of the target cell (Maxw...

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“…The reporter gene is favored by many researchers for marking a specific cell population and its offspring. Many studies have elaborated on reporter gene expression, including research into transplantation and stem cells (Hu et al, 2002;Wong et al, 2002), differentiation of the urogenital system (Nielsen and Byskov, 2001;Bex et al, 2002), cardiovascular development (Gutstein et al, 2001;Moses et al, 2001), tumors (Kohlhepp et al, 2001), neural crest cell tracking a weakening or disappearance of staining. This may be due to a loss of the marker, but it may also reflect developmental processes, including changes in compartmentalization of the protein.…”

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Changing intracellular compartmentalization of β‐galactosidase in the ROSA26 reporter mouse during embryonic development: A light‐ and electron‐microscopic study

Aoyama¹,

Molin²,

Mentink³

et al. 2004

Anat. Rec.

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The beta-geo (LacZ) reporter gene encodes for ␤-galactosidase (␤-gal) in all cells of the ROSA26 mouse during embryonic development. As such, ␤-gal activity constitutes an excellent marker for in situ labeling of expressing cells. However, the intracellular distribution of ␤-gal differs between cells, and changes during embryonic development. Therefore, we studied LacZ-encoded ␤-gal using light and electron microscopy in the heart, lung, liver, and small intestine on days 13 and 16 of gestation, and the kidney on day 16 of gestation in ROSA26 mice. The Bluo-gal method was carried out under standardized conditions, including fixation, washing, and incubation procedures. Intracellular ␤-gal staining is encountered in a combination of membranous compartments, including the nuclear envelope, the endoplasmic reticulum, and the plasma membrane. Its exact localization depends on the cell type and is regulated during development. Therefore, one must take the compartmental transition of intracellular ␤-gal staining into consideration when interpreting results obtained from experiments using ROSA26 mice.

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Embryonic expression of an Nkx2‐5/Cre gene using ROSA26 reporter mice

Cited by 266 publications

References 32 publications

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

Generation and Characterization of a Tissue‐Specific Centrosome Indicator Mouse Line

In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A

Changing intracellular compartmentalization of β‐galactosidase in the ROSA26 reporter mouse during embryonic development: A light‐ and electron‐microscopic study

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