Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation

Heidmann, Doris; Lehner, Christian F.

doi:10.1007/s004270100167

Cited by 57 publications

(54 citation statements)

References 21 publications

(20 reference statements)

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“…4B). Positive correlation between Cre-induced toxicity and proliferation was previously reported in fibroblasts (18) as well as in transgenic flies (36). In addition to hematopoietic cells, intestinal epithelial cells also proliferate rapidly, and R26CreER T2 mice occasionally demonstrated diarrhea and intestinal edema after the administration of TM, possibly due to the toxicity of CreER T2 in rapidly proliferating intestinal epithelial cells (data not shown).…”

Section: T2supporting

confidence: 69%

Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2

Higashi¹,

Ikawa

Muramatsu

et al. 2009

The Journal of Immunology

127

119

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The CreER T2 for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ER T2 ) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreER T2 in adult mice and embryos. The toxicity of Cre recombinase or CreER T2 in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreER T2 is knocked-in into the Rosa26 locus (R26CreER T2 mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreER T2 mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreER T2 toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreER T2 on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreER T2 in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreER

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Section: T2supporting

confidence: 69%

Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2

Higashi¹,

Ikawa

Muramatsu

et al. 2009

The Journal of Immunology

127

119

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“…Similar effects have been observed in mammalian systems both in vitro [18,19] and in vivo [20,21]. We explored two approaches to tighten regulation of expression of stably integrated CRE, but-considering the perpetual problem of read-through transcription [22]-our preferred solution is transient transfection of a CRE-expressing plasmid, which proved to be very effective without observable toxicity.…”

Section: Introductionmentioning

confidence: 79%

“…An earlier study showed that CRE was very effective in T. brucei but could not overcome its toxicity [17]. Indeed, toxicity from excess CRE has been reported recently in many systems [18][19][20][21]48], and, considering the potency of the GPEET promoter driving CRE expression in procyclic T. brucei, some degree of toxicity would not be unexpected. Our goal was to implement a system in which CRE would excise from the genome a combined positive and negative marker by recognizing the flanking loxP elements at a safe level of CRE expression.…”

Section: Cre/loxp Provides a Solution To The Marker Problem In T Bruceimentioning

confidence: 99%

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Scahill

Pastar

Cross

2008

Molecular and Biochemical Parasitology

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The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

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“…To overcome these complications, we developed a new (to our knowledge) lineage-tracing system ( Fig. 2A,B), which combines the main features of the TARGET, G-TRACE and inducible Cre/loxP (Heidmann and Lehner, 2001) systems. We call this new system the TARGET technique for real-time and clonal expression system, or, for simplicity, the T-TRACE system.…”

Section: Developing a New Lineage-tracing Systemmentioning

confidence: 99%

Enteroendocrine cells are generated from stem cells through a distinct progenitor in the adult Drosophila posterior midgut

2015

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Functional mature cells are continually replenished by stem cells to maintain tissue homoeostasis. In the adult Drosophila posterior midgut, both terminally differentiated enterocyte (EC) and enteroendocrine (EE) cells are generated from an intestinal stem cell (ISC). However, it is not clear how the two differentiated cells are generated from the ISC. In this study, we found that only ECs are generated through the Su(H)GBE + immature progenitor enteroblasts (EBs), whereas EEs are generated from ISCs through a distinct progenitor pre-EE by a novel lineage-tracing system. EEs can be generated from ISCs in three ways: an ISC becoming an EE, an ISC becoming a new ISC and an EE through asymmetric division, or an ISC becoming two EEs through symmetric division. We further identified that the transcriptional factor Prospero (Pros) regulates ISC commitment to EEs. Our data provide direct evidence that different differentiated cells are generated by different modes of stem cell lineage specification within the same tissues.

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Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation

Cited by 57 publications

References 21 publications

Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2

Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2

CRE recombinase-based positive–negative selection systems for genetic manipulation in Trypanosoma brucei

Enteroendocrine cells are generated from stem cells through a distinct progenitor in the adult Drosophila posterior midgut

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