Embryo

Example 2

About 5 μM fluorescein (F1300, Invitrogen, Carlsbad, CA) solution in ethanol was prepared. For imaging, the solution was transferred into a sealed 10 mm glass bottom dish (P35G-1.5-10-c, MatTek Corporation, Ashland, MA, USA) and mounted in an inverted confocal microscope. Imaging was performed on a Zeiss LSM780 inverted confocal microscope with QUASAR detector (Carl Zeiss, Jena, Germany). A typical dataset consists of 32 images, each of dimensions 512×512 pixels, corresponding to different wavelengths from about 410.5 nm to about 694.9 nm with about 8.9 nm bandwidth. The measurement is repeated 10 times using C-Apochromat 40×/1.20 W Korr Zeiss objective at any given imaging parameter. Fluorescein was imaged with about 488 nm laser at different acquisition parameters (Table 1).

For in vivo imaging 5-6 zebrafish embryos at appropriate stage were placed into about 1% agarose (Catalog No. 16500-100, Invitrogen™) moulds created in an imaging dish with #1.5 coverglass bottom, (Catalog No. D5040P, WillCo Wells) using a custom designed negative plastic mould [29]. Embryos were immobilized by adding about 2 ml of about 1% UltraPure™ Low Melting Point Agarose (Catalog No. 16520-050, Invitrogen™) solution prepared in about 30% Danieau (about 17.4 mM NaCl, about 210 μM KCl, about 120 μM MgSO₄.7H₂O, about 180 μM Ca(NO₃)₂, about 1.5 mM HEPES buffer in water, pH about 7.6) with about 0.003% PTU and about 0.01% tricaine. This solution was then added on top of the embryos already placed in the mold. Following solidification of agarose at room temperature (1-2 minutes), the imaging dish was filled with about 30% Danieau solution and about 0.01% Tricaine, at about 28.5° C. Subsequent imaging was performed on an inverted confocal microscope by positioning the petridish appropriately on the microscope stage. Samples were obtained by crossing Gt(desm-citrine)^ct122a/+ with Tg(kdrl:eGFP) fish for two color imaging. Samples with four fluorescent proteins result from same crossing followed by injection of about 100 pg per embryo of mRNA encoding H2B-cerulean and membrane-mCherry. Samples of Gt(desm-citrine)^ct122a/+;Tg(kdrl:eGFP) were imaged with about 488 nm laser to excite both Citrine and eGFP and a narrow about 488 nm dichroic to separate excitation and fluorescence emission. Samples of Gt(desm-citrine)^ct122a/+;Tg(kdrl:eGFP) with H2B-cerulean and membrane-mCherry labels were imaged with about 458 nm laser to excite Cerulean, eGFP and Citrine with a narrow about 488 nm dichroic, following an about 561 nm laser to excite mCherry with an about 458-561 nm dichroic.

For in vivo time-lapse imaging 5-6 zebrafish at appropriate stage were immobilized in an imaging dish with #1.5 coverglass bottom using about 0.5% Low Melting Point Agarose agarose (same as above) to allow for development and with about 0.003% PTU and about 0.01% tricaine. Subsequent imaging was performed on the same confocal-two photon inverted microscope at about 28.5° C. A solution of Egg Water was added every hour to the imaging dish to ensure proper hydration of the sample. Samples with five fluorescent proteins were obtained by crossing Tg(kdrl: eGFP) with Tg(ubiq:membrane-Cerulean-2a-H2B-tdTomato) zebrafish followed by injection of about 120 pg and about 30 pg per embryo of mRNA encoding Rab9-YFP and Rab11-mCherry, respectively. Volumetric data was acquired using about 950 nm to excite Cerulean, eGFP, YFP and (weakly) tdTomato with a 760+ bandpass filter, following an about 561 nm laser to excite mCherry and tdTomato with an about 458-561 nm dichroic.

Table 3 provides the detailed description of the imaging parameters used for all images presented in this work.

Example 20

2.3 nL of a solution containing 20 μg/nL plasmid DNA and 20 μg/nL tol2 mRNA was injected into the one-cell stage embryo obtained through crossing AB with Casper zebrafish. The injected F0 embryos were raised and crossed to casper zebrafish for screening. The F1 embryos for prospective Tg(hsp70I:Cerulean-P2A-CreER^T2) line and Tg(fli1:mKO2) were screened for ubiquitous Cerulean expression after heat shock for 30 min at 37° C., and mKO2 expression restricted in vasculatures, respectively. Positive individual F1 adults were subsequently outcrossed to casper zebrafish, and their offspring with casper phenotype were then used for experiments when 50% transgene transmission was observed in the subsequent generation, indicating single transgene insertions.

Example 18

Lines were raised and maintained following standard literature practice and in accordance with the Guide for the Care and Use of Laboratory Animals provided by the University of Southern California. Fish samples were part of a protocol approved by the IACUC (permit number: 12007 USC).

Transgenic FlipTrap Gt(desm-Citrine) ct122a/+ line is the result of previously reported screen, Tg(kdrl:eGFP)^s843 line was provided by the Stainier lab (Max Planck Institute for Heart and Lung Research). The Tg(ubi:Zebrabow) line was a kind gift from Alex Schier. Controllable recombination of fluorophores was obtained by crossing homozygous Tg(ubi:Zebrabow) adults with a Tg(hsp70I:Cerulean-P2A-CreER^T2) line. Embryos were raised in Egg Water (60 μg/ml of Instant Ocean and 75 μg/ml of CaSO4 in Milli-Q water) at 28.5° C. with addition of 0.003% (w/v) 1-phenyl-2-thiourea (PTU) around 18 hpf to reduce pigment formation.

Zebrafish samples with triple fluorescence were obtained by crossing Gt(desm-Citrine)ct122a/+ with Tg(kdrl:eGFP) fish followed by injection of 100 μg per embryo of mRNA encoding H2B-Cerulean at one cell stage as described in previous work²⁹. Samples of Gt(desm-Citrine)ct122a/+;Tg(kdrl:eGFP); H2B-Cerulean were imaged with 458 nm laser to excite Cerulean, Citrine and eGFP and narrow 458-561 nm dichroic for separating excitation and fluorescence emission.