Protocols

RNAi In Honeybee Brain

Posted on Saturday, July 01, 2006

Description
RNA Interference (RNAi) is a simple, rapid and specified method for silencing gene function. It is also called genetic interference by double-stranded RNA (dsRNA). dsRNA directs the specific degradation of mRNA by a process known as RNAi. dsRNA is first taken up by cells and then cleaved by dsRNA-specific nuclease into 21-23 nucleotide segments. These short segments are further dissociated by ATP-dependent helicase. dsRNAs are bound by RNAi specific proteins. The small dsRNA fragments guide for the degradation of the mRNA that matches the sequence. This results in degradation of specific endogenous mRNA and consequent suppression of individual gene functions. In vivo, one can use RNAi by simply injecting dsRNA in adult germ line, large intestinal cells, body cavity and in brain. In vitro, one can induce RNAi phenomenon by pre-incubating dsRNA with cell lysate at 37 degrees Celsius. RNAi reduces expression by causing degradation of the target mRNA. The resulting decrease in specific protein will depend on its stability and turnover.
One point to be remembered is that the inhibitory effects on gene function are present throughout the life span of the animal. Secondly, after injecting dsRNA into a localized region of the body, it may spread to other regions resulting in overtime amplified effect. When using RNAi to reduce gene expression, it is important that the targeted receptor mRNA level is specifically reduced without affecting levels of closely related family members. For that reason, the dsRNA used for RNAi should map to the least conserved regions of the related genes. RNAi has been used to knock out several closely related genes so that the functions of different members of the family can be evaluated alone or in combination. I have used this technique to silence AmOA1 gene expression in the antennal lobes of honeybee brain to evaluate the role of AmOA1 receptors in olfactory learning and memory.


Procedure
1. Protocol For PCR Template Method To Synthesize dsRNA
2. Microsurgery
3. Proboscis Extension Response (PER) Conditioning
4. Evaluation Of RNAi
I. Effect Of AmOA1 dsRNA On Acquisition And Recall
II. Effect Of Pre-conditioning on dsRNA-mediated Recall
Response
III.Effect Of AmOA1 dsRNA ON AmOA1 Translation In
Honeybee Brains

Recipes
1. Protocol For PCR Template Method To Synthesize dsRNA
We have modified PCR-template method of Kennerdell and Carthew (Cell 95:1017-1026, 1998) to synthsize octopamine receptor (AmOA1) dsRNA as following:

1. Subclone a fragment of AmOA1.
2. Choose optimal primer sequences for forward and reverse primers that allow amplification of the region of interest for AmOA1 dsRNA (Farooqui et al., J Neurosci. 23: 5370-5380, 2003; J Insect Physiol 50:701-713, 2004).
3. To facilitate the direct transcription of RNA from the PCR product, attach T7-promoter sequence (TAATACGACTCACTATAGGGAGAC- CAC) to the 5' end of each primer sequence.
4. Perform PCR in 50 ml volume using 100 ng recombinant plasmid DNA template, 25 pmol of each T7-linked primer, 8 mM MgCl2, 0.5 mM each dNTP and 2.5 U Taq DNA polymerase. Amplify DNA was by 35 cycles of 1 min at 94 degrees Celsius, 1 min at 58 degrees Celsius, and 1 min at 72 degrees Celsius followed by an additional extension for 10 min at 72 degrees Celsius.
5. Load PCR product on 1.5% agarose gel. Excise DNA fragment from the gel and purified by QIAquick Gel Extraction Kit protocol (Qiagen, Valencia, CA).
6. Carry out transcription reaction in 20 ul transcription reaction including 1 ug DNA template, 0.5 mM NTPs and 10 U T7 RNA polymerase at 37 degrees Celsius for 1 hr. RNA becomes dsRNA during its synthesis.
7. Perform phenol-chloroform extraction and precipitate dsRNA with NH4OAc/EtOH.
8. Resuspend the pellet in 25 ul of injection buffer containing 5 mM KCl, 10 mM NaH2PO4, pH 7.8.
9. Check the quality of AmOA1 dsRNA by agarose gel electrophoresis.
10. Check the yield of AmOA1 dsRNA by spectrophotometer. Store AmOA1 dsRNA at - 80 degrees Celsius.

2. Microsurgery
1. After harnessing, cut a small window in the head capsule just above the antennae to expose the antennal lobes.
2. After surgery, feed each subject 0.4 ul droplet of 1.25 M sucrose solution and leave it undisturbed for at least 1 hour.
3. Proboscis Extension Response (PER) Conditioning.
PER conditioning assay was used to assay the responses of honeybees to odors. This procedure has been previously described in detail and used to monitor olfactory learning and memory (Hosler et al., Behav Neurosci 114: 514-525, 2000).
1. Place 3 ul of odorant onto a small strip of filter paper and insert strip into 1 ml glass
tuberculin syringe. Place the odorant cartridge in the front of stage and connect it to a pump for air supply.
2. Test subjects for motivation by touching one antenna with sucrose. Discard the subjects if they do not extend their proboscis.
3. Move each subject individually to a conditioning arena through which air is continuously
drawn into an exhaust system.
4. Approximately 30 sec after placement, inject odor-laden air for 4 sec into the exhaust stream that is drawn over the subject’s antennae.
5. Three seconds after odor onset, stimulate subject's antennae with a 0.4 ul droplet of 1.25 M sucrose solution. Allow all subjects to receive six acquisition trials with the conditioned odor, 1-octanol.
6. Separate each acquisition trial by a 30 sec intertrial interval.
7. Give a positive score if subject extends its proboscis after the onset of odor but prior to presentation of the sucrose.
8. Perform recall testing identical to acquisition trials except no sucrose reinforcement will be allowed. Perform test trials with odorants 1-octanol, 1-hexanol, and geraniol at 3 minute interval in randomized order.

4. Evaluation Of RNAi
RNAi technique provides the opportunity to investigate the role of target receptors by specifically reducing its mRNA levels in subjects. Following protocols were used to evaluate RNAi in honeybees ((Farooqui et al., J Neurosci. 23: 5370-5380, 2003; J Insect Physiol 50:701-713, 2004).

4.I. Effect Of AmOA1 dsRNA On Acquisition And Recall
1. Inject 4 nl of injection buffer (Spradling, 1986) or 4 nl of injection buffer containing AmOA1 dsRNA (500 pg) in each antennal lobe of honeybee brain.
2. After surgery, Leave honeybees undisturbed at room temperature in a humidified
box.
3. Twenty-four hours later, condition subjects with 1-octanol.
4. Ninety minutes later, test subjects with 1-octanol, 1-hexanol and geraniol respectively.

4.2. Effect Of Pre-conditioning on dsRNA-mediated Recall Response
1. Two hours after surgery, condition honeybees with 1-octanol. After conditioning, inject injection buffer or injection buffer containing AmOA1 dsRNA in each antennal lobe of honeybee brain.
2. Leave honeybees undisturbed at room temperature in a humidified box.
3. Twenty-four hours later, test honeybees with 1-octanol, 1-hexanol and geraniol
respectively.

4.3. Effect Of AmOA1 dsRNA ON AmOA1 Translation In Honeybee Brains
dsRNA directs RNA-mediated interference (RNAi) by degrading sequence specific mRNA, which results in specific loss-of-function due to depletion of protein.
1. From control, 24 hours and 48 hours post dsRNA-injected honeybee brains (antennal lobes and/or rest of the brains) were dissected and homogenized in Laemeli buffer. Homogenates were centrifuged at 4 degrees Celsius at 6000xg for 15 min.
2. Pellets were discarded and supernatants were used for protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
3. Nitrocellulose membranes were then blocked with 5% bovine serum albumin (BSA; Sigma) in Tris-buffered saline (TBS; 10 mM Tris, 150 mM NaCl, pH 8.0) for 2 hours at room temperature.
4. Membranes were then probed for 1 hr at room temperature with polyclonal anti-peptide antiserum (anti-AmOAR; Alpha Diagnostic) at 1:500 dilution in 0.2% Tween/TBS (TTBS) plus 5% BSA.
5. Membranes were washed three times with TTBS followed by incubation with secondary antibody horseradish peroxides (HRP)-conjugated goat anti-rabbit Gig (Habersham Pharmacia Biotech, Piscataway, NJ) for 1 hour at room temperature at 1:15,000 dilution in TTBS plus 5% BSA.
6. Protein bands were visualized by an enhanced chemiluminescence's detection system (ECL; Habersham Pharmacia Biotech, Piscataway, NJ).


Supplies
Subjects:
Honeybees (Apis mellifera) used for this study were from a genetically closed breeding population maintained at The Rothenbuhler Honeybee Research Laboratory at The Ohio State University.
Preparation Of dsRNA:
In vitro transcription reaction MAXIscriptTM kit (Ambion, Austin, TX), forward and reverse primers (synthesized by invitrogen), dNTPs, Taq DNA polymerase (invitrogen), QIAquick Gel Extraction Kit protocol (Qiagen, Valencia, CA).
Proboscis Extension Response (PER) Conditioning
Odorants 1-octanol, 1-hexanol and geraniol were purchased from Sigma.
Western Blot Analysis:
Nitrocellulose membranes (Bio-Rad, Hercules, CA); enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Piscataway, NJ), secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ), primary polyclonal anti-peptide antiserum (anti-AmOAR; Alpha Diagnostic). Antibody against an octopamine receptor (OAMB) from fruit fly mushroom bodies was a gift from Dr. K.A. Han, Pennsylvania State University, PA.


Tips
1. In the transcription reaction, SP6 or T3 RNA polymerase was not used. Since T7 promoter sequence was attached on 5' end of forward and reverse primers therefore T7 RNA polymerase should read T7 on both sides. It was unnecessary to perform an annealing reaction because these single-stranded RNA strands would automatically be self-annealed during synthesis.
2. It is necessary to check out the specificity of dsRNA. I have used different controls such as injection buffer, dsRNA from genes which have no role in olfactory learning and memory, tRNA from Ecoli and/or yeast. In tRNA, since hairpin turns and brings complementary stretches of the chain in contact so double helical regions are formed; therefore it should be a good control. All controls failed to reproduce the inhibitory effect observed with AmOA1 dsRNA in honeybee brain.
3. dsRNA diffuses with time. Therefore it necessary to perform the time- and dose- dependent study to monitor its optimum effect. For these studies, 4 nl = 500 pg dsRNA was injected in each antennal lobe and duration was maintained between 24-48 hours depending on the experiment.
4. AmOA1 dsRNA aliquots can be stored as NH4OAc and ethanol precipitate at 80 degrees Celsius as well as a resuspension in buffer containing 5 mM KCl, 10 mM NaH2PO4, pH 7.8.
5. A potent inhibitory effect of AmOA1 dsRNA was observed on learning and test responses in honeybees suggesting that octopamine is responsible for behavioral plasticity in learning and memory. Furthermore, four bands were recognized by specific anti-octopamine receptor antibody by western blotting. The intensity of these bands was reduced by AmOA1 dsRNA suggesting that these bands may represent AmOA1 splice variants.
6. At present RNAi therapeutics is long way from entering into clinic. For these studies information is needed about the stability, specificity, potency, toxicity, half life, safety, convenience and target-site-specificity before even considering RNAi as a therapeutic agent. Therefore, it is too early to comment on "how soon, how safe and how valuable" RNAi will be to show its therapeutic importance.



Submitted by: buckeyetf