The overall goal of this procedure is to demonstrate the technique of RNA interference in ticks by injection. This is accomplished by first synthesizing double stranded RNA of the gene or genes of interest. The RNA is then injected into ticks, after which they're held in a humidity chamber for 24 hours.
After this holding period, the ticks are allowed to feed on an animal such as a sheep finally tick. Feeding parameters such as weight, molting and ova position, and the expression of the targeted gene or genes of interest by real-time R-T-P-C-R are determined. Ultimately, results can be obtained that demonstrate the effect of silencing targeted genes on tick biology by evaluation of tick biological parameters and gene expression by real-time R-T-P-C-R.
Our group works with ticks trying to characterize molecular events at the tick pathogen interface to use this basic information to develop methods for control of tick infestation and the transmission of pathogens to humans and animals. RNA interference has emerged as an essential tool in this research because it is the only method available for genetic manipulation of ticks. The function of many tick genes is unknown.
RNA interference allows us to define the role of tick genes and gene expression in many aspects of tick biology, including mating, reproduction, feeding, digestion, salivary gland function, and the tick vector competency for the transmission of pathogens. Demonstrating the procedure will be the tick RNA interference team from our laboratory, Dr.Ed Lewin, graduate student and Busby and myself. To generate double stranded RNA first synthesize oligonucleotide primers containing T seven promoter sequences for in vitro transcription of RNA.
For example, derma center vari lesin. Use the oligonucleotide primers D eight A a T 75, and D eight DVT 73 shown here. Amplify the target gene by R-T-P-C-R using 10 picomoles of each oligonucleotide primer and one to 10 nanograms of tick total RNA.
Next, purify the PCR product. Then use eight microliters or approximately 100 nanograms to synthesize double stranded RNA. To prepare the ticks for injection first, wash the ticks in the following series of solutions, tap water 3%hydrogen peroxide distilled water twice, 70%ethanol and two final washes with distilled water.
Perform each wash in a 50 milliliter disposable centrifuge tube by shaking. Then decanting the solution through a fine mesh wire screen. Block the ticks dry on paper towels, then aliquot the ticks into groups of 20 to 50 depending upon the experiment.
Place each group into a 1.25 ounce plastic cup with a tight fitting lid and label each cup with the experimental group. Number next, assemble an RNAi team consisting of three people that will carry out the injection process. The first person positions each tick on double sticky tape affixed to a three by six inch sheet of red dental wax.
The second person injects the ticks and the third monitors the ticks after injection breathes carbon dioxide on the ticks to activate them and counts and transfers the living ticks into cups labeled with the experimental group number. All team members must wear disposable gloves To begin the tick injection process, capture a tick using dumont fine forceps and place it ventral side up on double sticky tape affixed to the sheet of red dental Wax closely Position the ticks into groups of five. Place a small strip of masking tape over the mouth parts of all five ticks.
In order to further restrain them, leave most of the body exposed so that the injection process can be observed by the tick injector. To inject the tick pierce a hole in the lower right quadrant of the ventral surface of the exoskeleton using a mono eject insulin syringe fitted with a 29 gauge half inch needle. Next, immediately inject the ticks with 0.2 to 0.5 microliters of double stranded RNA solution using a custom made Hamilton syringe and a 33 gauge one inch needle with a 45 degree beveled point, place the needle well into the tick cavity to ensure the placement and retention of the double stranded RNA.
Even though fluid is released after injection, deep placement of the needle for injection will ensure that sufficient double stranded RNA is deposited into the tick cavity to cause gene silencing care should be taken not to overin. Inject the ticks, which will cause loss of hemolymph and death of the tick. Immediately after injecting the ticks, pick them up from the double sticky tape with the fine forceps and place them in a plastic recovery container.
The ticks will briefly remain inactive after injection, but should soon begin to crawl around the dish. Activate the ticks by breathing carbon dioxide onto them. Once the ticks are crawling and active, the injection wound will heal rapidly and they will most likely survive.
Count the ticks in each experimental group and place each group in a labeled plastic cup with a tightly fitted lid. Replace any ticks that die in the current group before injecting the next experimental group. Clean the Hamilton syringe before injecting the next experimental group by filling and then expelling the syringe with fresh 3%hydrogen peroxide.
15 times followed by 15 washes with sterile water. Take care not to bend the plunger of the Hamilton syringe, otherwise the plunger will not move smoothly and respond to the gentle touch required for tick injection. After the injecting, place the ticks in a chamber with a container filled with water and potassium sulfate, which maintains high humidity and them for one day.
Next place individual ticks into tick feeding cells glued to a sheep and allow them to feed with an equal number of unin injected male or female ticks, whichever sex was not injected. Collect and wave female ticks from the feeding cell after they complete feeding for approximately 10 days or when the control females have dropped off, the host incubate each group of ticks in the humidity chamber until the completion of ova position. Evaluate ova position by group by weighing the eggs mass produced by all the ticks in a group to evaluate the tick phenotype after feeding.
Determine the number of ticks that survived and calculate the tick weight as well as the ova position and the fertility of the eggs. Depending on the targeted gene and objectives of the study, other analyses may be performed to confirm gene silencing by R-T-P-C-R. After feeding, dissect the salivary glands and guts from individual ticks from the control injected and double stranded RNA injected groups, and then extract total RNA from the individual tissue samples.
Analyze the target gene transcripts in individual tissues by real-time R-T-P-C-R and normalize the RNA levels against tick 16 s ribosomal RNA. Using the gene norm method, run dissociation curves at the end of the reaction to ensure that only one amplicon is formed and that the amplicons consistently denature in the same temperature range for every sample, compare the normalized CT values for mRNA levels between control injected and double stranded. RNA injected ticks using the student's T-test.
The protocol described here has been used in our laboratory for RNAi in many different exot tick species. The amount of double stranded RNA injected into the ticks varies with the size of the tick. Larger tick species can accommodate a larger volume.
The references can be found in the accompanying written portion of the protocol. If the protocol is done correctly, less than 5%mortality should be obtained from the injection procedure after 24 hours. A typical phenotype after gene knockdown in ticks is shown here where a panel of ticks were injected with pools of double stranded RNA that were used to screen for tick protective antigens.
In ticks that are examined by light microscopy, cellular degeneration in tick tissues may be observed. Phenotypic changes may also be accompanied by loss of function. For example, RNAI of SUBIN results in sterile males that cannot mate successfully with the females Following RNA interference and ticks.
Other methods can be used to determine the impact of gene silencing on gene and protein expression and on tick biology, including real-time R-T-P-C-R, light electron or confocal microscopy. Genomics or proteomics. RNA interference can also be done in tick cell cultures and provides an in vitro method for the study of tick cell pathogen interactions.
