culture and transfection of naegleria gruberi trophozoites for molecular studies

استخدام ناقل قائم على CERE لنقل Naegleria gruberi trophozoites

The Naegleria genus contains over 45 species, although it is unlikely that all members of the species have been identified1. Naegleria can exist in different forms: as trophozoites (amoebae), as flagellates, or, when resources are severely limited, as cysts1,2,3,4. The Naegleria genus is recognized for its one particularly dangerous species, Naegleria fowleri, known as &#39;the brain-eating amoeba&#39; (reviewed in1,2,3,4,5,6,7), which is the cause of the almost universally fatal primary amoebic meningoencephalitis.
Naegleria encode their rDNA on the CERE located in the nucleolus. Complete sequencing of three Naegleria species&#39; genomes confirms the absence of rDNA in the nuclear genome8,9,10,11. Based on the limited full-length CERE sequences, the CERE ranges from approximately 10 kbp to 18 kbp in length. CERE of each species of Naegleria carry a single rDNA cistron (containing the 5.8, 18, and 28S ribosomal DNA) on each of approximately 4,000 CERE per cell12,13,14. Other than rDNA, each CERE has a large non-rDNA sequence (NRS). CERE sizes vary between species; the differences are mainly due to the varying length of the NRS, as the rDNA sequences are highly conserved across the genus1,15,16,17,18,19,20. The mapping of a single origin of DNA replication within the N. gruberi CERE NRS21 provides strong support for the hypothesis that Naegleria CERE replicates independently of the nuclear genome.
N. gruberi is a non-pathogenic amoeba often used to study Naegleria biology. We developed a methodology for transforming N. gruberi trophozoites with a CERE clone from the same species to test the hypothesis that Naegleria amoebae tolerate and independently replicate CERE within the trophozoites. This was accomplished by transforming N. gruberi with a full-length clone of N. gruberi CERE into trophozoites and following the fates of the donor CERE clone by polymerase chain reaction (PCR). A general overview of the protocol is outlined in Figure 1. The data presented herein demonstrate that the donor clone can be detected through at least seven passages in tissue culture, as well as be maintained through encystment and excystment. These studies form the basis for a means to dissect how CERE replicates, as well as explore its use as a vector to transfect Naegleria.

تسليط الضوء على المؤلف: فهم انتشار عنصر rDNA الدائري المغلق خارج الكروموسومات (CERE) في Naegleria gruberi

نقل استنساخ جزيئي من الحمض النووي الريبوزي للنيجلرية إلى N. gruberi Trophozoites

Our research represents the first step to understanding what sequence is 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 a naegleria gruberi, and what components of the CERE are necessary for effective propagation.Our protocol offers a minimalistic method of transecting 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 that 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.

ثقافة ونقل Naegleria gruberi Trophozoites للدراسات الجزيئية

culture-transfection-naegleria-gruberi-trophozoites-for-molecular

Research

JoVE Journal

Biology

Culture and Transfection of Naegleria gruberi Trophozoites for Molecular Studies

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

This protocol describes a methodology to transfect Naegleria gruberi trophozoites with a construct that is maintained throughout passaging trophozoites in vitro, as well as through encystment and excystment.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While ...

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 five 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 600 x g 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. Pipette 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 DNase in one milliliter of 10X DNase 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 1:2 ratio. Then collect the trophozoites from the remaining wells for further analysis. The Naegleria gruberi trophozoites were amoeboid 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.

Closed Circular Extrachromosomal Ribosomal DNA (rDNA) Containing Element (CERE) Isolation from Transfected Naegleria Gruberi Trophozoites and PCR Analysis

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 4 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 4 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 2 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.

عزل الحمض النووي الريبوسومي الدائري المغلق خارج الكروموسومات (rDNA) المحتوي على عنصر (CERE) من Naegleria Gruberi Trophozoites المنقولة وتحليل تفاعل البوليميراز المتسلسل