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Ligand-Controlled Site-Specific Recombination in ZebrafishBrand, Michael, Chekuru, Avinash, Kuscha, Veronika, Hans, Stefan 17 September 2019 (has links)
Cre-mediated site-specific recombination has emerged as an indispensable tool for the precise manipulation of genomes allowing lineage-tracing studies, temporal and spatial misexpressions, and in particular the generation of conditional knockout alleles. Previously, we and others showed that Cre and its ligand-inducible variant CreERT2 are also highly efficient in the developing and adult zebrafish. The number of Cre driver and effector lines is currently still limited in zebrafish. However, the recent advent of novel genome editing tools such as TALEN and CRISPR/Cas will significantly increase interest in the conditional Cre/lox-technology in this organism. The considerations of basic transgene design and subsequent transgenesis have been addressed elsewhere. Here we outline practical experimental steps for transient functionality tests of CreERT2 driver and effector constructs. In addition, we introduce detailed protocols to elicit CreERT2-mediated recombination in vivo at embryonic as well as adult stages.
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Targeted knock-in of CreERT2 in zebrafish using CRISPR/Cas9Kesavan, Gokul, Hammer, Juliane, Hans, Stefan, Brand, Michael 26 April 2019 (has links)
New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreERT2 transgene at the otx2 gene locus to generate a conditional Cre-driver line.We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreERT2 upstream of the endogenous ATG of otx2, we utilized this gene’s native promoter and enhancer elements to perfectly match CreERT2 and endogenous otx2 expression patterns. 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-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreERT2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult
zebrafish.
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Transcriptional and Distributional Profiling of Microglia in Retinal Angiomatous ProliferationSchlecht, Anja, Wolf, Julian, Boneva, Stefaniya, Prinz, Gabriele, Braunger, Barbara M., Wieghofer, Peter, Agostini, Hansjürgen, Schlunck, Günther, Lange, Clemens 07 February 2024 (has links)
Macular neovascularization type 3, formerly known as retinal angiomatous proliferation
(RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of
myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However,
the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions
remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse
model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and
determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most
abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late
stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes
mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP
and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly,
MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In
summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG
significantly altered their transcriptional profile during RAP formation, activating immune-associated
processes and exhibiting enhanced proliferation, however, without showing substantial upregulation
of angiomodulatory factors.
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