Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The overall goal of this procedure is to provide a step-by-step demonstration of the biotic transformation of a linear construct that contains a fluorescent tag. This is accomplished by first coating gold microcar beads with the DNA of interest. In the second step, the DNA is transferred to the C neo forin cells by gene gun.

The transformed cells are then plated, and the integration of the DNA construct is assessed by PCR and R-T-P-C-R. Ultimately, fluorescent microscopy is used to confirm the proper integration of the DNA product via homologous recombination. The main advantage of this technique over existing methods, such as electroporation, is that BIC transformation results in a stable integration into the genome of crypto office.

Near performance. Demonstrating this technique is Tanya Taylor, a PhD student in my laboratory who's become proficient at this technique. Begin by using forceps to submerge orange macro carrier BIC discs in 100%ethanol, then transfer the discs into a large Petri dish containing dryer, right taking care that the discs do not touch the desiccant.

Once dry press the discs into ethanol wiped silver disc holders, then vortex a tube of gold beads and aliquot 12 microliters of the bead solution into one 1.5 milliliter micro centrifuge tube per transformation. Next sequentially, add two micrograms of DNA 10 microliters of calcium chloride, and two microliters of sperm aine freebase into each tube of beads. Vortex each sample.

Then incubate the tubes at ambient temperature for five minutes with occasional gentle flicking to resus. Suspend the settled beads at the end of the incubation pellet the DNA coated gold beads and carefully aspirate the snat. Slowly resuspend the beads completely in 600 microliters of 100%ethanol, then pellet the cells again.

This time resus suspending the beads in eight microliters of ethanol. Now dispense each tube of beads onto the center of an individual BIC disc in one centimeter diameter circles. If a sufficient concentration of beads is present, a gold circle will be visible in the center of each disc.

Once the beads are dry, to operate the gene gun, first turn on the vacuum pump, then turn the helium tank knob counterclockwise until a pressure of approximately 2200 PSI is reached on the pressure gauge. Next, flip the left red switch to turn on the gene gun and adjust the flow rates for the vacuum and the vent so that the vacuum will reach 28 inches of mercury within 15 seconds. Confirm that the distance between the rupture disc and macro carrier is approximately three eighths of an inch at this time as well.

When everything is in position, wipe down the entire chamber with 70%ethanol and submerge the rupture discs in 100%ethanol. After drying the discs on a sterile surface, use a torque wrench to loosen the rupture disc holder and insert a clean disc. Screw the rupture disc holder back into place and turn it once to the right with the torque wrench to tighten it.

Next submerge the mesh screens in 100%ethanol. Once dry, place a screen onto the white plastic mounting plate and place the macro carrier disc holder DNA side down into the disc chamber screw on the silver cap and place the mounting plate in the highest slot. Then place a YPD auger plate containing one molar sorbitol onto the bottom plate.

Now shut the chamber door and lock it into place. Then push and hold the middle red switch in the up position to engage the vacuum and to allow it to reach 28 inches of mercury. Once the proper vacuum level is reached and the middle switch has been moved to the down position, hold down the right red switch to fire.

When the rupture disc pops, immediately release the fire button and push the middle red switch to the middle position. In order to vent the chamber to zero PSI if the shot was successful, a gold ring will be visible on the plate. Clean out the rupture disc and macro carrier disc debris and turn off the gene gun.

Then turn the helium tank knob clockwise to turn off the gas and turn off the vacuum pump. After a four hour rest period, add 700 microliters of YPD to each plate. Then use a cell scraper to gently lift the cells off of the auger and transfer the cell suspension into a sterile 1.5 milliliter micro centrifuge tube.

After a second YPD wash to recover all of the cells, spin them down and resuspend the pellet. In 500 microliters of YPD, then dispense 100 microliters of the cell suspension onto the center of YPD plus antibiotic plates and spread the cells with glass beads. Finally incubate the plates upside down at room temperature for three to four days.

As colonies appear, patch them onto new YPD plus antibiotic plates. Both DNA and RNA can be isolated from the individual c neoformans colonies and analyzed to confirm a proper integration and expression of the constructs. For example, if this protocol is used for tag, gene fusion PCR would be conducted utilizing a primer set in which one primer and neos to the outside of the construct and within the surrounding genome to confirm the correct recombination into the desired locus.

Lanes one and two are PCR products obtained. Using this primer set with genomic DNA from wild type C neoformans H 99, and the A CKM cherry transformed strain, R-T-P-C-R would then be used to confirm that both the gene of interest and the tag are expressed.Indeed. This R-T-P-C-R fusion product indicates that the tag was properly fused to the gene at the RNA level.

Fluorescent microscopy can then be used to confirm that the recombination into the desired locus was successful, and that the RNA was translated into the protein of interest. After watching this video, you should have a good understanding of how to set up a Balistic Jing gun and use it for successful homologous integration of a linear construct into the genome of cryptococcus. Neo formm.