The overall goal of this procedure is to develop a simple technique for chromosome mapping of mosquito genomes. This is accomplished by first using imaginal discs of fourth instar mosquito larvae to obtain high quality chromosome spreads. The second step of the procedure is to prepare fluorescently labeled DNA probes and unlabeled repetitive DNA fractions for blocking unspecific hybridization of the probe.
The third step is to hybridize fluorescently labeled DNA probes to the chromosomes. The final step is to detect the chromosomal location of the probes using fluorescent microscopy. The advantage of using imaginal discs over cell lines or brain ganglia is that the high number of banded spreadable chromosomes can be easily obtained from force in star mosquito Using standard protocols.
Hatch mosquito eggs at 28 degrees Celsius after two to three days transfer, second or third instar larvae to 16 degrees Celsius, or 22 degrees Celsius incubators depending on the species. The instar stage of development is based on head capsule size. When fourth instar become available, immobilize them on ice, then transfer the larvae to a slide with a drop of cold hypotonic solution.
Under a stereo microscope, select larvae with oval shaped imaginal discs for further dissection. The term imaginal discs is abbreviated as IDs. Choosing the correct stage of mosquito larvae with oval shaped imaginal disc is extremely important for obtaining high quality chromosome spreads.
Using dissecting scissors, decapitate the larvae and cut the cuticle from the ventral side of the rax. Make an additional cut in the second or third abdominal segment to dissect the gut. Now open the cuticle and remove the gut and fat body from the larvae.
Using filter paper, absorb the hypotonic solution from the slide. Replace the solution with a fresh drop of hypotonic solution placed directly over the IDs. Keep the larvae in hypotonic solution for 10 minutes.
Then remove the solution using filter paper and apply car noise solution. The IDs will immediately turn white and become easily visible under the microscope. Using dissecting needles, remove the IDs from the larva.
Add a drop of 50%proponic acid, and then cover the IDs with a cover slip and wait another 10 minutes before proceeding. Now cover the slide with filter paper and squash the tissue by intensive tapping of the eraser of a pencil on the cover slip. Briefly analyze the quality of the slide using the phase contrast microscope at 100 x or 200 x magnification.
Preparations with greater than 50 chromosome spreads can be considered suitable for fish. Now, dip and hold the slide in liquid nitrogen until it stops bubbling. Remove the cover slip from the slide using a razor blade and transfer the slide immediately to a container of 70%ethanol, chilled at minus 20 degrees Celsius.
For dehydrating, collect the slides in cold, 70%ethanol before proceeding to the next step. Next, further dehydrate the slides in a series of ethanol for five minutes per bath and allow the slides to air dry at room temperature once dry. Store the slides at minus 20 degrees Celsius prior to fish extract genomic and back clone DNA and prepare the repetitive DNA fractions from the mosquito species of interest from to prepare the probe label back clone DNA using NIC translation and I-G-S-R-D-N-A using PCR.
The procedures for these two probes vary slightly for fish with back clones. Begin by incubating the ID slides in two XSSC for 30 minutes at 37 degrees Celsius. Next, transfer the slides to a jar with a Pepin solution in hydrochloric acid and incubate it for five minutes at 37 degrees Celsius.
After the Pepin bath, wash the slides in one XPBS for five minutes before placing them in a jar with formalin in PBS. Fix the chromosomes for 10 minutes at room temperature. Wash the formalin off with one XPBS for five minutes and then dehydrate the slides using the same ethanol series as before.
Finish by air drying the preparations. Now denature the slides in a jar with prewarm, 70%form amide for two minutes at 72 degrees Celsius. Then dehydrate the slides in an ethanol series followed by air drying at 37 degrees Celsius.
At this point, prepare the fish hybridization mixture. Now precipitate the DNA by adding 0.1 volume of three molar sodium acetate and two volumes of ethanol. Keep mix at minus 20 degrees Celsius for one to three hours.
Next, centrifuge the precipitated DNA aspirate the ethanol and air. Dry the pellet at room temperature for two to three minutes. Then thoroughly dissolve the DNA pellet in 10 microliters of hybridization.Buffer.
Denature the hybridization mixture for seven minutes at 97 degrees Celsius. Then immediately place it on ice for one minute. Then pre hybridize the mixture at 37 degrees Celsius for 30 minutes.
Pre hybridization of the DNA probe with unlabeled repetitive DNA fractions is vital to preventing nonspecific hybridization of the probe to the chromosomes. Place 10 microliters of the hybridization mixture on a slide and carefully cover. Remove any accidental air bubbles with gentle pressure to the cover slip, seal the cover slips around the perimeter using rubber cement.
Then place the slides in a humidified 37 degrees Celsius incubator overnight. The next day, remove the rubber cement and cover slip from the slide. Wash the slides for two minutes in Prewarm solution, one at 73 degrees Celsius, followed by five minutes in solution two at room temperature.
Counterstain the slides with yo-yo one in a dark chamber at room temperature. Finally mount the chromosomes in a small amount of prolonged gold Antifa reagent with a cover slip and analyze the preparations for fish with an I-G-S-R-D-N-A probe. Pretreat the chromosome preparation with an RNA solution.
Do not add the repetitive DNA fractions and denature the chromosome preparation and a probe simultaneously. Fish was performed on the mitotic chromosomes of eighties aegypti using back clone DNA probes. The chromosomes are counterstain with yo-yo one iodide.
For comparison. Fish using back clone DNA probes was also performed on the mitotic chromosomes of olx Quinco Fasciatus. Finally fish using an I-G-S-R-D-N.
A probe was performed on the mitotic chromosomes of anomalies, gambier After its development. This technique paved the way for researchers in the field of ology to explore chromosomes and perform genome mapping in various species of mosquitoes and other insects.
