Hello, my name is Manuel Ruiz Rubio here at the Department of Genetics of the University of Cordova in Spain. We are using the elegance as an experimental tool In the study of autism, Taking advantage of The world characterized nervous system of three elegance. We are studying mutant of the nematode, effective in genes involved in neural synapse and that has been related to autism.
Rein Regans are membrane adhesion molecules present in the synaptic. The head of C elegance contains the amfi, a sensory organ of the nematode with mediate responses to different stimuli including osmotic strength. Amfi is made of 12 sensory bipo neuron with related in height and one INAP Terminal action.
Two of these neurons Name A SH right and left are particularly important in osmotic sensory function. Detecting water soluble rebalance with high osmotic strength. Hi, A Nan River wine with sky method to measure osmotic avoidance in synapse defect mutants of The ance.
To evaluate the implications of neuro and neuro high osmotic strength avoidance, we reported the different response of C against mutants defective in neuro neural genes using a method based on a four molar fructose solution. Ring the day of the say, prepare a four molar stock solution of fructose with 1%of Congo red and dissolves completely at room Temperature. Annular Ring of one centimeter of diameter on NGM plate is subline on the center of the solid medium.
Using 15 microliters of the red four molar fructose solution. Let the fructose soak into the acre around five minutes the experiment should be carried out blindly. The plate with each strain to be S eight should be labeled by a second experimenter.
The SA is performed using these marks that they are unasked. When the experiment Has finished about 24 hours before the sale is carried out, it is necessary to pick L four larva of each genotype and transfer them to AFL NGM plate containing e coli bacteria and maintaining it at 20 degrees Celsius next day. The experiment should be started with young adults.
Put individual worm of each strain within the rest circle and follow each animal to the timing its response to the tic barrier. 10 animals of each strain and a minimum of three replica experiments should we carry out to get a conclusive result. Ones avoiding the red ring six times in a row are classified as normal.
Those exiting the ring in less than six attempt are considered defective in osmosis sensitivity control strain must be used in each SA Bristol and two animals are used as positive controls because they be avoid The ring barrier. Control strains Respond going backward when they find an osmotic barrier consisting in four molar fructose. Neuro deficient mutants bombs have a behavior similar to these respect to one type strength.
However, NEUR deficient mutants failed to detect this osmotic barrier. Double mutant strengths car in different ative neur and nout alleles recovered the wild type phenotype responding to the Osmotic strength for obtaining Of down worms by RNA interference feeding isolate colonies of e coli HD 11 five strain that has been transformed with L 44 40 vector containing a fragment corresponding to the target gene. Bacteria are isolated onto LBA or plate with carbonic the next day.
Pick a colony of bacteria and inoculate LV liquid medium. Keep growing up for about seven hours with shaking at 37 degrees. See the drop of this culture onto NGM plate with carbonic ceiling and IPTG and wai over night at room temperature, allowing bacteria to grow and beginning the induction of the expression of the fragment of the target gene.
It is necessary to use a positive and non-negative control for RNA interference. Feeding experiments. The negative control is equal.
I HT 11 five strain transformed with TL 44 40 vector. The positive controls ELI HT 11 five cell transformed with the L 44 40 vector containing a 22 gene sequence. The lockdown of this gene originate are shaking phenotype easily distinguished under the sterile microscope.
The first Day. L four stage ones from NGM PLA with bacteria are transferred on plate with bacteria and Kuwait at 20 degrees Celsius during 12 hours. This is a fasting period.
The next day move the fast young adult onto plate inoculated with bacteria expressing the specific RNA interference target gene. Leave about 48 hours at 20 degrees Celsius for obtaining the F1 progeny. Then pick a few F1 adult bombs onto another plate inoculated with the same bacteria over the next two days.
Select and isolate young adults from the F two progeny Scoring for phenotypes Bristol N two wild type strain fat with bacteria occurring empathy. L 44 40 vector was going backwards when fine. The osmotic Barrier.
Bristol And true white type strain fed with bacteria expressing a fragment of the neur gene from the elegance failed to detect the toes For molar solutions. However, neur deficient mutant unable to detect the osmotic barrier recovered this capacity when was fed with bacteria expressing a fragment of the nane gene From sea elegance. In this video, We show a simple method which allows to study the effect of genes affecting the osmotic avoidance response in sea elegance.
A ring of fructose formal tight with Congo Red is used to analyze the response of the bomb to osmotic strength. Neurology in deficient mutant are defective in this response, however, NU mutants are not impaired to detect tomo stress showing a response similar to wild type strain. This video shows that double mutant strains carrying different tive knockout alleles in ing and releasing genes recovered the wild type phenotype responding to osmotic strength.
These observations were confirmed using RNA interference, feeding lockdown approach. In this way, the wild type strain was impaired in recognizing osmotic barrier when it was fed with bacteria expressing according sequence of the ING gene. On the other hand, neur defective mutant, which are unable to detect the four more fructose solution, recovered this behavior when they were fed with bacteria expressing according sequence of the NNU gene.
The results shown in this experiment indicate that n seeing and neur might be involved in the synaptic connection of the sensor neurons of the nematode and also that they interact with each another in a way that affect the synaptic function. Okay, goodbye and good luck with your experiments.
