Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites
Summary
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.
Abstract
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).
Introduction
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 'the brain-eating amoeba' (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' 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.
Protocol
Representative Results
Discussion
The protocol outlined herein is very straightforward, although every construct will likely require some degree of optimization, particularly of the DNA-transfection reagent ratio, depending on the nature of the construct and the species of Naegleria used. We have only tested one commercially available transfection reagent using this protocol, but it is likely that several others may be effective. Given that a full-length clone of the CERE is used, containing a functional origin of replication and thus replicatin…
Disclosures
The authors have nothing to disclose.
Acknowledgements
These studies were partially funded by a grant from the George F. Haddix Fund of Creighton University (KMD). Figure 1 is generated in biorender.com, and Figure 3 is generated in benchling.com.
Materials
| Agarose | Bio Rad | 161-3102 | |
| Ammonium Acetate | Sigma Aldrich | A-7330 | |
| Calcium Chloride | Sigma Aldrich | C-4901 | |
| Crushed ice | |||
| Culture Flasks, T-75 | Thermo Scientific | 130190 | |
| Culture Plate, 6-well | Corning | 3506 | |
| DNAse | Sigma Aldrich | D-4527 | |
| EDTA, 0.5 M | Affymetrix | 15694 | |
| Electropheresis Gel Apparatus | Amersham Biosciences | 80-6052-45 | |
| Eppendorf Tubes, 1.5 mL | Fisher Scientific | 05-408-129 | |
| Eppendorf Tubes, 2 mL | Fisher Scientific | 05-408-138 | |
| Ethanol, 100% | Decon Laboratories | 2716 | |
| Ethidium Bromide | Sigma Aldrich | E-8751 | |
| Fetal Bovine Serum | Gibco | 26140 | |
| Folic Acid | Sigma Aldrich | F7876-25G | |
| GeneRuler 1 kb Plus Ladder | Thermo Scientific | SM1331 | |
| Glacial Acetic Acid | Fisher Scientific | UN2789 | |
| GoTaq Green PCR Master Mix | Promega | M7122 | |
| Heating Block | Thermo Scientific | 88871001 | |
| Hemacytometer | Hausser Scientific | 1483 | |
| Hemin | Sigma Aldrich | 51280 | |
| Iron Chloride | Sigma Aldrich | 372870-256 | |
| Ligase | NEB | M2200S | |
| Magneisum Chloride | Fisher Scientific | M33-500 | |
| Microfuge | Thermo Scientific | MySpin 12 | |
| Microscope | Nikon | TMS | |
| N. gruberi | ATCC | 30224 | |
| Nucleic Acid | Chem Impex Int’l | #01625 | |
| Peptone | Gibco | 211677 | |
| pGEM | Promega | P2251 | |
| Potassium Phosphate | Sigma Aldrich | P0662-500G | |
| PowerPac HC Electropharesis Power Supply Unit | Bio Rad | 1645052 | |
| Sodium Chloride | MCB Reagents | SX0420 | |
| Sodium Phosphate, dibasic | Sigma Aldrich | S2554 | |
| Tabletop Centrifuge | eppendorf | 5415R | |
| Tris, base | Sigma Aldrich | T1503-1KG | |
| Trypan Blue, 0.4% | Gibco | 15250-061 | |
| ViaFect Reagent | Promega | E4981 | |
| Weigh Scale | Denver Instruments | APX-60 | |
| Yeast Extract | Gibco | 212750 |
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