Biomedical Engineering Reference
In-Depth Information
18.2.2 Embryo Handling
Phylonix AB zebrafish were generated by natural pairwise mating in our aquaculture
facility, as described byWesterfield (1993). Four to five pairs were set up for eachmating;
on average, 50-100 embryos per mating were generated. Embryos were maintained
in embryo water (5 g of Instant Ocean Salt with 3 g of CaSO
4
in 25 L distilled water) at
28
C for approximately 24 h before sorting for viability. Because embryos receive
nourishment from an attached yolk ball, no additional maintenance was required.
18.2.3 Generation of MD Zebrafish Using
Dystroglycan-Specific siRNAs
Four smartpool siRNAs specific for zebrafish dystroglycan gene (NM_173274)
were designed and synthesized by Dharmacon RNA Technologies/Thermo
Fisher Scientific (Lafayette, CO). Target sequences were siRNA1 (SMP-1),
GTTATAGGCGATAGCAATA; siRNA2 (SMP-2), GAGGATGGAACTACCGATA;
siRNA3 (SMP-3), CGGACAAGGGCGTTCACTA; and siRNA4 (SMP-4),
GGAGATGCGCGTACGGTCA. A proprietary siRNA comprised of a nonreacting
coding sequence (AS siRNA) (Dharmacon RNA Technologies/Thermo Fisher
Scientific) was used as control. siRNA1 was directly labeled with DY547 at the 5
0
sense end. The 5
0
antisense end of each siRNAwas modified with a phosphate group.
18.2.4 Microinjection of siRNAs
To reduce variation between batches, randomized embryo samples from three or four
independent pairwisematings were used. Fertilized eggs (
1000) were microinjected
at the one-cell stage (Meng andWolf, 1997). 0.5 nL volume was delivered using an air
pressure-controlled microinjector (WPI, Sarasota, FL). Injected zebrafish were
cultured and grown as described by Westerfield (1993).
18.2.5 Zebrafish Dechorionation
Embryos hatch from a chorion at
2.5dpf stage. In order to assess zebrafish earlier
than the 2.5dpf stage, we dechorionated zebrafish. Briefly, embryos were placed on an
agar gel-coated Petri dish and treated with pronase (1mg/mL) for 3 min, followed by
extensive washing with fish water to remove residual pronase. Treated embryos
were then placed in a 28
C incubator for
30 min and removed from chorions by
gentle shaking.
18.2.6 TaqMan Quantitative PCR (qRT-PCR) Analysis
Total RNA was isolated from (a) uninjected control, (b) knockdown control (AS
siRNA injected), and (c) MD zebrafish (SMP siRNA injected) at 4, 6, and 20 h post
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