Reference - Detail
|Author||Okuyama T, Isoe Y, Hoki M, Suehiro Y, Yamagishi G, Naruse K, Kinoshita M, Kamei Y, Shimizu A, Kubo T, Takeuchi H.|
|Title||Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.|
BACKGROUND:Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.
METHODOLOGY/PRINCIPAL FINDINGS:To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0-1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.
CONCLUSIONS/SIGNIFICANCE:We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.
|MeSH||Animals Animals, Genetically Modified Brain / cytology Brain / embryology* Cell Differentiation / genetics Embryo, Nonmammalian / cytology Embryonic Development / genetics Genetic Engineering / methods* Hot Temperature Infrared Rays Integrases / genetics Integrases / metabolism* Lasers Neural Stem Cells / cytology Neural Stem Cells / metabolism Operator Regions, Genetic / genetics Oryzias / embryology* Oryzias / genetics* RNA, Messenger / genetics RNA, Messenger / metabolism Recombination, Genetic* Stochastic Processes|
|Medaka||d-rR/TOKYO (MT837) BAC clone d-rR-Tg(beta-actin-loxP-DsRed2-loxP-GFP) (TG861) pPBIS19-mgfc::BFP-8xHSE::nlsCreCherry (TG957)|