Our research represents the first step to understanding what sequences contained in the closed circular extrachromosomal ribosomal DNA containing element, or CERE, are critical in permitting replication of the CERE as well as understanding the DNA replication machinery of the CERE. This protocol addresses gaps in understanding how the CERE propagates in naegleria gruberi and what components of the CERE are necessary for effective propagation. Our protocol offers a minimalistic method of transfecting naegleria, aiming to reduce procedures that may disturb the cell.
It thereby addresses how the CERE propagates in naegleria gruberi and what components of the CERE are necessary for effective propagation. We have established that the species of naegleria contain unique CERE that varies in both sequence length and nucleotide composition. We look to provide the groundwork for analyzing the molecular dynamics of the CERE for species within the naegleria genus.
Our results have paved the way for questions regarding what components are necessary for CERE replication and whether the ORI can be utilized for the propagation of foreign constructs in naegleria species. We aim to advance our research further by branching out into other species of the naegleria genus and providing answers regarding CERE transfection at an intra-species level. To begin, place frozen naegleria gruberi trophozoites at 37 degrees Celsius for three minutes.
Culture the trophozoites in PYNFH media at 25 degrees Celsius until approximately 80%confluent. Place the confluent flask on ice for 5 to 10 minutes to release the adhered trophozoites from the plastic. Then gently swirl the flask to dislodge any remaining adherent cells.
Replace the media with sterile encystment media to encyst the naegleria gruberi cells. After 48 hours, remove the cysts from the flask. Centrifuge the cysts at 600G for 10 minutes at four degrees Celsius.
After centrifugation, resuspend the cysts in two milliliters of PYNFH media with 10%fetal calf serum to excyst the cells. Incubate the cysts at 25 degrees Celsius for 72 hours until trophozoites emerge and reach 80%confluency. To transfect the Trophozoites, first plate one milliliter of the trophozoites into a six-well flat-bottom tissue culture plate.
Next, incubate the plasmid transfection reagent mixture at room temperature for 10 minutes. Pipet out approximately 800 microliters of medium from the trophozoites monolayers just prior to transfection. Then add 200 microliters of the plasmid transfection reagent mixture to the trophozoites.
Gently swirl the plate every 15 minutes to prevent the media from aggregating at the edges of the plate and drying out. After an hour, add 10 units of DNAs in one milliliter of 10X DNAs buffer and add it to the wells. Incubate the plate at 37 degrees Celsius for 15 minutes to remove residual plasmid DNA.
Now wash the plate once with one milliliter of growth medium. Then add two milliliters of fresh media. Incubate the plate at 25 degrees Celsius for 24 to 36 hours.
When incubation is complete, split the contents of a well into a new plate at a one to two ratio. Then collect the trophozoites from the remaining wells for further analysis. The naegleria gruberi trophozoites were ameoboid and adherent to the culture flasks under standard culture conditions.
Their incubation on ice resulted in round and non-adherent trophozoites. Incubation of the trophozoites in encystment media caused them to become encysted. To begin, add 200 microliters of 100 millimolar sodium chloride to a tube containing transfected trophozoites of naegleria gruberi.
Centrifuge the tube at 600G for 10 minutes at four degrees Celsius. After removing the supernatant, resuspend the cell pellet in 100 microliters of 100 millimolar EDTA. Place the tube in a heat block for 15 minutes at 98 degrees Celsius to lyse the cells.
Centrifuge the tube at four degrees Celsius for 10 minutes at top speed to pellet the debris. Transfer the supernatant into a new tube. Next, add 0.5 volumes of ammonium acetate, three volumes of absolute ethanol, and one microliter of glycogen to the supernatant.
Invert the tube several times to mix before incubating it at minus 80 degrees Celsius for 30 minutes or overnight. After incubation, centrifuge the tubes at room temperature for 10 minutes at 16, 000G. Remove the supernatant without disturbing the pellet and add 200 microliters of 70%ethanol to wash it before centrifuging.
Resuspend the dried pellet in sterile deionized water to achieve a trophozoite density of 10, 000 trophozoites per microliter. After PCR with primer specific for the transfected DNA and detection of the transformed trophozoites, add two microliters of 6X loading dye to the CERE samples. Then load the dyed samples onto a 0.8%agarose gel.
After electrofluorescing the gel for 1.5 to two hours, incubate it in DNA dye diluted in 100 milliliters of deionized water. Transfer the stained gel into deionized water for 20 minutes to destain it. Visualize the gel on an ultraviolet light system to document the results.
PCR analysis showed that pGRUB was detected in transfected trophozoites through at least seven passages. pGEM was lost after the first passage indicating that the empty plasmid was not retained by the amoebas.
