Cre recombinase is a tyrosine recombinase enzyme derived from the p1 bacteriophage. Spatial and temporal control of transgene expression in zebrafish. We found tdtomatopositive p16 high cells detectable in all organs, which were enriched with age. The ert2 sequence is an estrogen receptor targeting motif, whose. The cre recombinase protein mediates a tissue specific deletion of dna fragments that are flanked by loxp sites. The enzyme uses a topoisomerase ilike mechanism to carry out site specific recombination events. Leonard zons lab contains the insert creert2 and is published in development. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for es. Zebrafish were maintained according to university of oregon institutional guidelines.
Genetic engineering of zebrafish in cancer research. Targeted knockin of creert2 in zebrafish using crisprcas9. To generate transgenic fish expressing a cre ert2 fusion protein under the control of the dusp6 promoter, we first cloned a dusp6 promoter fragment sufficient to drive fgf dependent expression molina et al. Mutant estrogen receptor ert2 ligandbinding domain fusions with cre recombinase are a key tool for spatiotemporally controlled genetic recombination with the crelox system. We also found that these cells failed to proliferate and had halflives ranging from 2. Switch transgenics and show their use as a constitutive fluorescent lineage tracing reagent. Cre loxptechnology has been successfully applied to dissect the zebrafish genome and genomewide approaches have been conducted to create various cre effector lines 3335. Therefore, the cre mouse model allows tissue specific gene knockout based on the promoter driving cre expression. An estrogen receptor variant ert2 fused to the gal4 transcriptional activator rapidly and robustly activates transcription within 3 hours of treatment with the drug 4hydroxytamoxifen 4oht in tissue culture and transgenic zebrafish. Pdf screening pancreatic oncogenes in zebrafish using the. Functional heterogeneity within the developing zebrafish. Transdifferentiation of fast skeletal muscle into functional. Onestep efficient generation of dualfunction conditional knockout.
Targeted knockin of creer in zebrafish using crisprcas9. Next, by combining this novel driver line with a cre dependent reporter line, we show that only in the presence of tamoxifen can efficient cre loxp. Jun 17, 2015 in total, we used three different mct2ac gene trap vectors, yielding 27 novel creer t2 driver lines expressing in various tissues in the developing and adult zebrafish. Creloxcontrolled spatiotemporal perturbation of fgf. We have generated an inducible system to control the timing of transgene expression in zebrafish and chick. A research team led by professor makoto nakanishi of the institute of medical science, the university of tokyo, generated a p16 cre ert2 tdtomato mouse model to characterize in vivo p16 high.
The most commonly used version of creer is the creert2 which is. Magnuson verified mouse promoter, personal communication, a nuclear localization sequencemodified cre recombinase gene and a 2. Lgr5tm1 cre ert2 obtain from jackson labs cre ert2 insert into lgr5, wnt target gene. Oct 28, 2020 to inactivate the function of ift88, ubc cre ert2. Fulllength zebrafish fli1a and tal1, the longer alpha isoform, were used to generate the middle entry vectors without stop codons. Kryn stankunass lab contains the insert ert2 cre ert2 and is published in plos one. An inducible transgene expression system for zebrafish and chick. Aug 29, 20 we report a new open access database, the zebrafish crezoo, which contains novel creer t2 driver lines that express cre fused to the mutated human ligandbinding domain of the estrogen receptor creer t2 in several tissues.
Creert2 is efficiently activated in a concentrationdependent manner by the tamoxifen metabolite trans4ohtamoxifen trans4oht. Brand m 2015 isolation of novel creert2 driver lines in zebrafish. Protein length intermediate filament protein, conserved site intermediate filament, rod domain intermediate filament, rod domain, coil 1b keratin, type ii. Reproducible and efficient cre lox experimentation is hindered by the gradual loss of. After creloxpmediated removal of the pocassette, the two polya signals. Connie cepkos lab contains the insert ert2 cre ert2 fusion protein and is published in proc natl acad sci u s a.
Photoactivation of the creert2 recombinase for conditional. Atg5 flfl mice to determine whether autophagy is a positive or negative regulator of ol function in sci. Isolation of novel creert2driver lines in zebrafish. Genetic manipulation of brain endothelial cells in vivo. In zebrafish, genetic mutants and chemical perturbations affecting fgf. Creert2 animals if tamoxifen is not applied following heat sh.
In zebrafish, creloxp system has become increasingly effective to. Optical uncaging of the caged precursor of a nonendogeneous steroid by one or twophoton illumination was used to restore cre activity of the creer t2 fusion protein in specific target cells. Here, we created the novel transgenic line fgfr1dncargo, encoding. Tg zebrafish embryos were obtained by natural spawnings according to the zebrafish book,23 injected at onecell stage with mrna synthesized with an in vitro. Protein length homeoboxlike domain superfamily pairedbox protein 2 cterminal paired dnabinding domain paired domain pax family winged helixlike dnabinding domain superfamily. The zebrafish genetics toolkit has been missing a particularly handy piece of kit. But no longer, for in one of developments inaugural technical papers p.
In zebrafish, creloxp system has become increasingly effective to study tissue. Adult zebrafish have the remarkable ability to regenerate their heart upon. In vivo performance and properties of tamoxifen metabolites. Tgdlx5acre1mekk nervous system recombinase activity. Furthermore, the possibility of pulsed cre ert2 induction in zebrafish allows for control of the onset of cell lineage labeling and misexpression in restricted developmental time windows. Oct 29, 2016 here we describe two experimental systems based on cre loxp technology cre ert2 loxp and rttateto cre loxp, which allow the inducible, permanent labeling of mammary epithelial cells following the administration of either tamoxifen or doxycycline. Tgdlx5acre1mekk respiratory system recombinase activity. In addition, use of ert2 gal4 in conjunction with tissuespecific enhancers enables the control of transgene expression in both space and time.
For this, we first generated a double transgenic zebrafish line by crossing tgmyl7. Ubiquitous transgene expression and crebased recombination. Spatial and temporal control of transgene expression. Guidance on the housing and care of zebrafish, danio rerio. Genetic lineage labeling in zebrafish uncovers novel. Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid x receptor alpha mediated by a tamoxifeninducible chimeric cre recombinase cre ert2 in adipocytes. Cre driver transgenic lines now exist for an increasing number of tissues and cell types table s1 in the supplemental information online. Endoxifen potently induces creert2 activity in zebrafish and ert2. This method labels single cells irreversibly by inducing recombination in. In these transgenic fish, gfp is expressed only in cms when a recombination event is induced by tamoxifen administration. With novel technologies being regularly developed, the zebrafish is poised to. A cre gene was placed under the control of the i56i and i56ii enhancer elements of zebrafish dlx5a and a 3.
Zebrafish danio rerio is an excellent model to study a wide. Download scientific diagram endoxifen potently induces creert2 activity in zebrafish and ert2 reporters in cell culture. We have generated a broadly expressed inducible ert2 gal4 zebrafish line using the ubiquitin ubi enhancer. Apr 14, 2016 mutant estrogen receptor ert2 ligandbinding domain fusions with cre recombinase are a key tool for spatiotemporally controlled genetic recombination with the cre lox system. May 01, 2018 subsequently, cre encoding transgenes, such as heatinducible egfptagged cre hsp70. To activate the kras gene in zebrafish pancreatic progenitor cells, we used the inducible tg ptf1a. A ciliumindependent role for intraflagellar transport 88 in. Efficient generation of a dualfunction ki allele at the zebrafish tbx5a locus. One report in zebrafish described using crispr and 1 kb of homology cloned in front of a targeting cassette to integrate cre ert2 upstream of the otx2 translation initiation codon kesavan et al. The enzyme uses a topoisomerase i like mechanism to carry out site specific recombination events. Dlx56 cre, dlx56i cre, line 8561, tgdlx6a cre 1mars, tgdlx6a cre 1mekk molecular description. Brand m 2015 isolation of novel creert2driver lines in zebrafish. Fish, affected genomic regions, phenotype, gene expression. A ciliumindependent role for intraflagellar transport 88.
Alb cre, alb cre, albcre, alb cre, alb cre2, albumin cre, cr alb, cre alb, tgalb cre 21mg. Tgdlx5acre1mekk sensory organs recombinase activity. Abstract we report a new open access database, the zebrafish crezoo. An additional technology adapted for use in zebrafish is the crelox system. Zhao y, yang z, phelan jk, wheeler da, lin s, mccabe er 2006 zebrafish dax1. Blocking autophagy in oligodendrocytes limits functional.
Cell senescence plays a key role in ageassociated organ dysfunction, but the in vivo pathogenesis is largely unclear. Cre mediated recombination removes an intervening sequence and places the oncogene. Endodermderived myeloidlike metaphocytes in zebrafish gill. Gene manipulation using the cre loxprecombinase system has been successfully employed in zebrafish to study gene functions and lineage. Tol2 vectors engineered with a splice acceptormcherry2a cre ert2 gene trap are driven by endogenous promoter and regulatory elements after inframe integration within a gene jungke et al. Subsequently, cre encoding transgenes, such as heatinducible egfptagged cre hsp70.
The ert2 sequence is an estrogen receptor targeting motif, whose nuclear. We further demonstrate the powerful application of olactbdriven tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of. Similar experiments in mouse embryos are technically challenging and ambiguous in their readouts, as tam needs to be injected or administered in the drinking. Pdf isolation of novel creerdriver lines in zebrafish. This method allows one to control gene activity in space and time in almost any tissue of the mouse, thus opening new avenues for studying gene function and for establishing sophisticated animal models of human diseases. Switch doublepositive zebrafish embryos at approximately 3 dpf for baseline gfp expression b and mcherry indicating successful loxp excision b. Broad expression of cre creer t2 can be achieved using the inducible heat shock cognate 70kd protein, like hsp70l 21, 37 or the ubiquitin b. Austin smiths lab contains the insert cre ert2 and is published in cell. Zebrafish as a developmental model organism for pediatric. Isolation of novel creert2driver lines in zebrafish using. Ift88 flfl mice were administrated with 20 mgml tamoxifen.
Isolation of novel creert2driver lines in zebrafish using an. We report a new open access database, the zebrafish crezoo, which contains novel creer t2 driver lines that express cre fused to the mutated human ligandbinding domain of the estrogen receptor creer t2 in several tissues. Fate predetermination of cardiac myocytes during zebrafish heart. M 4oht, ethanol etoh carrier, or a short 15 minute pulse of 5. Here, we generated a p16 cre ert2 tdtomato mouse model to analyze the in vivo characteristics of p16 high cells at a singlecell level. Generation and characterization of a zebrafish muscle specific.
Myelopoiesis and myeloid leukaemogenesis in the zebrafish. Middle entry vector containing tamoxifeninducible estrogen receptor ert2 fused to both ends of cre recombinase for. An inducible transgene expression system for zebrafish and. Sigma, st louis, mo, usa was dissolved in ethanol at a final stock concentration of 10 mm and kept in singleuse aliquots in the dark at 20c. Single cell dynamics of embryonic muscle progenitor cells. Temporallycontrolled sitespecific recombination in zebrafish. Pdf screening pancreatic oncogenes in zebrafish using. Hydroxytamoxifen 5 m was added immediately after heat shock to the fish water to induce recombination in. Creert2 is efficiently activated in a concentrationdependent manner by the tamoxifen metabolite trans4. Fate predetermination of cardiac myocytes during zebrafish. Mar 01, 2016 the tie1 cre line generated by gustafsson and colleagues used a 1. Cre ert2 line, which induces the expression of the transgene in ptf1aexpressing cells of the. Lineage tracing of mammary stem and progenitor cells.
We implemented a noninvasive optical method for the fast control of cre recombinase in single cells of a live zebrafish embryo. Single cell dynamics of embryonic muscle progenitor cells in. Limited dedifferentiation provides replacement tissue. However, currently the number of available cell and tissuespecific cre driver lines to transactivate the before mentioned cre effector lines is limited 36. Mar 09, 2020 to establish a lineage relationship between epi2 and the epicardium beyond tbx18 expression, we performed a tracing experiment using a tcf21driven cre tgbaccryaa.
The authors also created inducible ubidriven cre ert2 transgenes and loxp lineagetracer transgenes that give strong reporter activity upon cre exposure, which further enhances and expands the zebrafish transgenesis toolkit. The cre lox sitespecific recombination system has emerged as an important tool for the generation of conditional somatic mouse mutants. All applicable institutional andor national guidelines for the care and use of animals were followed. The use of mice was approved by the animal care and use committee of nankai university. But they can also react strongly to sudden loud noises or novel vibrations so steps. Novel insights into the conservation of haematopoietic lineages and.
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