International Research Journal of Biotechnology (ISSN: 2141-5153) Vol. 2(8) pp. 173-178, November, 2011
Available online http://www.interesjournals.org/IRJOB
Copyright © 2011 International Research Journals
Full Length Research Paper
Caenorhabditis elegans as A model organism: In vivo
monitoring of the stress response with DAF-16::GFP
and blocking expression of specific genes with RNAi
*
Mahesh Kumar, R. and Catherine S. Cotillas
Nematology Faculty, Department of Biology, K. L. Ledeganckstraat 35, Ghent University, 9000 Gent, Belgium
Accepted 03 November, 2011
The nematode Caenorhabditis elegans is a model organism for research on different fields in biology. A
work carried out on stress responses of transgenic DAF-16::GFP C. elegans when exposed to ethanol
stress, osmotic stress and blocking the expression of two different genes, bli-3 and him-14, with RNA
interference (RNAi). At an exposure of 1-2% of ethanol there was an active expression of DAF-16::GFP
along the body at nuclear level and the only response by activation of DAF-16::GFP was at 0.05M of
NaCl. For C.elegans, RNAi study could be easily performed by FtFfeeding the worms with RNAi bacteria,
thereby resulting in the knock-down of a specific gene of interest. RNAi bacteria are specifically
engineered to express dsDNA corresponding to the sequence of target gene. Blistering along the cuticle
layer had more chance to develop into male adults rather than hermaphrodite because the gene bli-3
and him-14 were knocked down by RNAi.
Keywords: bli-3, him-14, mutant genes, stress response
INTRODUCTION
The nematode Caenorhabditis elegans has been used as
a model organism in biology for nearly four decades. This
simple multicellular organism has allowed researchers to
resolve and understand elementary processes in different
fields of Biology (Cutter et al., 2010).
Ageing, immunity and stress tolerance are inherent
characteristics of all organisms induce at cellular and
molecular level (Hyun-Ok et al., 2009). In animals, these
traits are regulated, at least in part, by fork head
transcription factors in response to upstream signals from
the Insulin/Insulin–like growth factor signaling (IIS)
pathway. In C. elegans this phenomenon consists of a
transmembrane protein DAF-2, several intracellular
kinases and the DAF-16 transcription factor. When
inactivated, this pathway not only extends life span but
also regulates resistance to pathogens and abiotic
stresses (Amrit et al., 2010). Mutations in this pathway,
such as inhibitory mutations in age-1 (a homologue of the
*Corresponding author E-mail: maheshkumar_r@hotmail.com;
Phone: +32492084940
mammalian phosphatidylinositol 3-OH kinase) and
several environmental stresses such as heat and
oxidative stress result in a rapid relocation of the
transcription factor DAF-16 to the nucleus (Henderson
and Johnson, 2001; Amrit et al., 2010).
An experiment was done in order to monitor in vivo the
stress responses of transgenic DAF-16::GFP C. elegans
when exposed to ethanol stress, osmotic stress and a
mixture of osmotic and heat stress.
Additionally, another set of experiments were used to
block the expression of two different genes, bli-3 and
him-14, with RNA interference (RNAi) in transgenic
nematodes DAF-16::GFP to determinate their function.
The RNAi is known as an important pathway in
experimental biology in order to study the functions of
genes both in cell cultures and in vivo in model
organisms such as C. elegans (Daneholt, 2006; Stram
and Kuzntzova, 2006). In normal conditions when the
gene bli-3 is expressed, it regulates the proper crosslinking of collagen in the cuticle of worm. Blocking of
expression leads to formation of “blisters” and
consequently making these nematodes incapable of
moving correctly. On the other hand, the gene him-14 is
174 Int. Res. J. Microbiol.
Table 1. Composition of each well used for testing the ethanol concentration
stress response in nematodes with an integrated DAF-16::GFP transgene (A –
plate A; B – plate B).
Ethanol 100% (µl)
S-buffer (µl)
0.25
0.5
0
2.5
5
1000
997.5
995
4A
5A
6A
1
2
3
10
20
30
990
980
970
7A
1B
2B
3B
5
0
5
10
50
0
50
100
950
100
950
900
4B
5B
15
50
150
500
750
500
6B
7B
75
100
750
1000
250
0
Nº of well
1A
(control)
2A
3A
Concentration
(%)
0
responsible for correct separation of chromosomes in
germ line during meiosis. The post-transcriptional knock
down of this gene leads to meiotic non-disjunction of Xchromosome which consequently makes it possible to
obtain male C.elegans. This characteristic is
transmissible to the next generation.
the fluorescence microscope to determine the stress
response by observation of the green fluorescence
protein activation when the DAF-16 transcription factor
was activated. The nematodes were then transferred to
s-buffer to recover. After 24 hours, a final observation
was made to evaluate any recovery.
MATERIALS AND METHODS
2. Transgenic C. elegans (DAF-16::GFP) response to
osmotic stress
1. Transgenic C. elegans (DAF-16::GFP) response to
ethanol stress
Two plates (A and B) with 12 wells each were used for
testing the ethanol stress response in nematodes
carrying an integrated DAF-16::GFP transgene. Each
well contained 1 ml of different concentration of ethanol
diluted according to Table 1. All wells were correctly
labeled, and the first well in each plate was considered as
control (1a and 1b).
With the help of a plastic pipette a small amount of sbuffer was placed in the agar plate which contained the
DAF-16::GFP transgenic nematodes. After covering, the
plate was gently shaken in order to let the worms float in
the s-buffer solution to make the transfer easier. With the
use of a glass pipette through capillary forces, a small
amount of this suspension was transferred to each of the
wells previously prepared. The quantity of nematode was
estimated to be similar in each well.
After 10 minutes, the first observation at the
stereoscopic microscope was done to evaluate the
mortality and movement of the nematodes. The mortality
was calculated to an approximate percentage. Twenty
(20) minutes later, the nematodes were checked under
For testing the stress response of DAF-16:GFP
nematodes when exposed to osmotic stress, 1 plate with
12 wells (C1) was prepared with different salinity
concentrations according to Table 2.
The same procedures were used for transferring and
observing the nematodes as in the previous experiment
with the ethanol stress response, but in this case the
recovery time was shorter.
3. Transgenic C. elegans (DAF-16::GFP) response to
osmotic and heat stresses combined.
After all observations were done in the previous assays,
the plate (C1) containing the nematodes exposed at
different salinities was placed in the incubator at 39.50C
for 10 minutes to test the mixed effect of salinity and heat
(rename C2). Again after 10 minutes, the first observation
was made at the stereoscopic microscope to evaluate the
mortality and movement of the nematodes and 20
minutes later at the fluorescence microscope for
determinate the stress response by observation of the
green colour when the DAF-16 transcriptions factor was
Kumar and Cotillas 175
Table 2. Composition of each well used for testing the osmotic stress response
in nematodes with an integrated DAF-16::GFP (C1 – plate C1).
Nº Well
1C (Control)
2C
3C
4C
5C
6C
Concentration (M)
0
0.05
0.1
0.25
0.5
1
4M NaCl (µl)
0
12.5
25
62.5
125
250
S-Buffer (µl)
1000
987.5
975
937.5
875
750
Table 3. General response and expression of DAF-16::GFP after exposure to different concentrations of ethanol in each plate
(A and B).
General response
DAF-16:GFP response
0.25
All alive
Almost Alive
Very active response
No response
3A
0.5
Almost Alive
No response
4A
1
Almost Alive
Active response
5A
2
Almost Alive
Active response
6A
3
Almost Alive, loss of mobility
7A
5
All dead
Very active response in some
nematodes
Very active
response in some
1B (control)
0
All alive
Very nematodes
active response
2B
5
All dead
3B
10
All dead
Very active response in some
nematodes
No
response
4B
15
All dead
No response
5B
50
All dead
No response
6B
75
All dead
No response
7B
100
All dead
No response
Nº of well
1A (control)
2A
Concentration
(%)
0
activated in the nucleus.
4. Blocking expression of bli-3 and him-14 gene with
RNAi in C. elegans mutants (DAF-16:GFP)
Three types of E.coli strains were cultured on agar plates
and used for RNAi study, coded by L4440, bli-3 and him14. Other instruments included picking needle, glass
Pasteur pipettes, 12-well plates, tips and plastic pipettes.
Two adult hermaphrodite worms were removed from an
agar media plates which are already seeded with RNAi
expressing bacteria and was transferred to a normal agar
medium free of RNAi, these plates were sealed with a
parafilm and maintained in the room temperature (16°C)
for one week. After 7 days storage, each of the plates
was checked and observed through the microscope
whether the RNAi was effective in inducing a visible
phenotype.
RESULTS
All nematodes exposed to concentrations above 5% of
ethanol died during the first 10 min. Even though the
majority of nematodes could survive at a concentration of
3% ethanol, most of them lost their normal mobility
capacity (Table 3). In both cases no recovery after 24
hours was observed. The tolerance level for normal
mobility and less than 10% mortality was observed at 2%
ethanol. At an exposure of 1-2% of ethanol there was an
active expression of DAF-16::GFP along the body and a
DAF-16::GFP expression at the nuclear level only in
some nematodes when exposed to 3-5% of ethanol.
Regarding the nematodes exposed to osmotic stress,
the lethal concentration was found when the nematodes
were exposed to concentrations above 1M of NaCl (Table
4). In this case 90% of the nematodes were estimated
dead. The effect of other concentrations ranging from 0.5
to 0.05 M NaCl can be observed on Table 4.
176 Int. Res. J. Microbiol.
Table 4. General response and expression of DAF-16::GFP after explosion to different concentrations of NaCl
(M) in plate C.
Nº Well
Concentration (M)
General response
DAF-16:GFP response
1 C1 (Control)
2 C1
0
0.05
All alive
All alive
Very active response
Small response
3 C1
0.1
Almost Alive
No response
4 C1
0.25
Most alive, loss of mobility
No response
5 C1
0.5
Half alive, loss of mobility
No response
6 C1
1
Almost dead
No response
120
100
Mortality (%)
80
60
40
20
0
0
0.25
0.5
1
2
3
5
10
Ethanol Concentration (%)
15
50
75
100
Figure 1. Mortality rate estimated after 10 min. of exposure to different concentrations of
ethanol.
Table 5. General response and expression of DAF-16::GFP after exposure to different concentrations of
NaCl (M) and heat stress (39.5ºC) in the plate C2.
Nº Well
1 C2
2 C2
3 C2
4 C2
5 C2
6 C2
Concentration(M)
+ Heat (39.5ºC)
0
0.05
0.1
0.25
0.5
1
General response
DAF-16:GFP response
Half alive
Almost dead
Almost dead
Almost dead
Almost dead
All dead
Very active response
Very active response
Active response in some nematodes
Active response in some nematodes
No response
No response
Excluding the control, the only response by activation of
DAF-16::GFP was at 0.05M of NaCl (Figure 1a and
Figure 1c). Again no recovery was observed.
When the same nematodes that were exposed to
osmotic stress and subjected to a heat stress for 10
minutes, a highest expression of DAF-16::GFP was
obtained compared to the previous assays (Table 5 and
Figure 3). Especially in the wells where the nematodes
were subjected only to heat (1 C2) and to the lowest
concentration of NaCl (2 C2) – 0.05M, the nuclear
translocalization of DAF-16::GFP was very actively
observed (Figure 3). In this case the control considered
was the result obtained from the control for testing the
response to the osmotic stress, with no NaCl (1 C1).
Since changes in localization of DAF-16 are likely to
function in the dauer decision and larval development
(Kondo et al., 2005), it is advisable to pick up L2 worms
for experiment. However, adults also showed the same
results (Henderson, 2006).
In the control plate, “L4440” no differences in
movement, behaviour and morphological characteristics
of the nematodes were observed (Figure 4a). In the plate
of “blis-3”, an abnormal formation of cuticle resulting in
“blisters” was detected (Figure 4b and Figure 4c). In the
third plate, “him-14”, no big differences were observed
compared to the control plate. A new observation after
one week revealed in opposite the presence of several
males.
Kumar and Cotillas 177
a
b
c
Figure 2. Expression of DAF-16::GFP after 30 minutes of exposure to: a- 0M of NaCl (Control); b- 0.1M of NaCl; c- 0.05M
of NaCl.
a
b
c
Figure 3. DAF-16::GFP response when the nematodes were exposed to: a- 39.5ºC; b- 0.05M of NaCl +39.5ºC;
c- 0.25M of NaCl +39.5ºC.
a
b
c
Figure 4. Transgenic C. elegans (DAF-16::GFP) in the plate: a- L4440; b- blis-3; c-blis-3
DISCUSSION
The expressions of stresses were not identified at higher
concentrations both of ethanol and salinity due to the fact
that the nematodes were dead or inactive before
responding to stress. The tolerant levels of ethanol, 3%
and salinity, 1M, where the nematodes could survive for
the first 10 min were indeed the tolerant levels found
along the time, since no recovery or differences were
observed after some time. Even when the nematodes
could survive, levels near the lethal concentrations
revealed to have effects on movement and behaviour of
these mutants nematodes, as in case of 0.25 to 0.5M of
salinity and 3% of ethanol.
Based on the experimental results, the transgenic C.
elegans exhibited the stress effects by activation of DAF16:GFP to all stresses tested, but in the case of
combined stresses the activations in the nucleus by
178 Int. Res. J. Microbiol.
translocation of the DAF-16::GFP was more evident
compared to the other results.
In the control plate all worms were like in normal
conditions as was expected, since the bacteria were not
modified. Furthermore as predicted, the severe effect
was visible on the nematode’s cuticle (blisters) and as
consequence the incapacity to move results on the
knock-down of gene bli-3. In the third plate, the
expression of him-14 gene in the nematodes was blocked
initially but in the second observation after 14 days of the
experiment, the presence of male revealed the activation
of this gene again as expected.
CONCLUSION
Therefore, it is observed during the experiment that the
salinity mixed with the heat was the stress that had more
effect in order to induce DAF-16::GFP translocation and
expression in the nucleus of certain cells of C. elegans.
It is further concluded that RNAi can indeed knock
down gene expression if it finds specific nucleotide
sequence. Using this technique it is possible to study the
functions of all genes just by blocking each gene and
from the results it showed the role of bli-3 and him-14
genes.
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