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are obtained. The animals are then collected from the P0 plates; one
half is passed through the worm sorter to isolate mutants, and the other
half is kept for recovery of possible lethal/sterile mutants.
(iii) Sorting: The sorter is gated to exclude eggs, small L1, and debris using
gating parameters time of flight versus extinction. Worms are then
sorted based on fluorescence parameters (green fluorescence intensity
versus Red fluorescence intensity [GI/RI]). Any mutants that affect GI/
RI ratio are kept for further analysis.
(iv) Confirmation: For the sorted positive candidates, visual inspection
under compound microscopes is used to confirm the phenotypes.
(3) Analysis of mutants: the phenotypes of mutants should be confirmed for two
generations to make sure they are true-breeding. Further analysis of the mutants
follows standard guideline, including backcrossing and genetic mapping.
C. Gene Expression Profiling of Neurons
GFP labeling at transgenic C. elegans lines is now widely used to isolate sub-
groups of neurons for transcriptome analysis. This is largely due to the maturation of
the procedure for in vitro culturing of dissociated neurons (
Strange et al., 2007
). Four
steps for isolating GFP positive neurons are illustrated in
Fig. 5
and described below.
(1) C. elegans culture: Multiple methods can be used to achieve large-scale culture.
One common method is a combination of dauer liquid culture and growth on
large NGM plates (15 cm). In brief, worms are cultured in 500 mL s-Basal
medium (100 mM NaCl, 50 mM KH
2
PO
4
.KOH [pH 6.0], 10 mg/mL choles-
terol) with 35 g/mL streptomycin and fed with 10 mL streptomycin-resistant
HB101 until they become dauer. These dauer animals are plated on 2-4 mL
HB101-seeded 15-cm NGM plates and grown until they reach young gravid
adults.
(2) Dissection and cell culture: Synchronized adult hermaphrodites are lysed for
5 min using bleach solution (0.5 M NaOH and 1% NaOCl). Eggs are then
collected and washed three times in sterile egg buffer (118 mM NaCl, 48 mM
KCl, 2 mMCaCl
2
, 2 mMMgCl
2
, and 25 mMHEPES [pH 7.3]). Eggs are floated
on 30% sucrose by centrifugation to remove adult carcasses. The egg layer is
collected and washed twice with the egg buffer and eggs are treated with 0.1 U/
mL chitinase (in egg buffer) for 30 min to destroy eggshells. Embryos are
pelleted to remove chitinase, and the pellet is resuspended in 2% fetal bovine
serum (FBS) in the egg buffer. Embryos are dissociated by trituration. Intact
embryos, larvae, and clumps are removed by passing the cell suspension through
a5
m
m filter. Filtered cells are plated onto poly-D-lysine-coated cell culture
plates in Leibovitz
'
s L-15 medium containing 10% FBS and antibiotics. 16-18
hours later, cells are collected by gentle wash and trituration, centrifuged for 5
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