RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-4583
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Last modified: 27 February 2019, 16:34
*RMgm-4583| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene disruption |
| Number of attempts to introduce the genetic modification | 3 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30700216 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | Not applicable |
| Other information parent line | |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Kumar H, Frischknecht F |
| Name Group/Department | Integrative Parasitology, Department of Infectious Diseases |
| Name Institute | University of Heidelberg Medical School |
| City | Heidelberg |
| Country | Germany |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0921300 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1127100 | ||||||||||||||||||||||||
| Gene product | deoxyuridine 5'-triphosphate nucleotidohydrolase | ||||||||||||||||||||||||
| Gene product: Alternative name | dUTPase | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||
| Restriction sites to linearize plasmid | |||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | Unsuccessful attempts to delete dUTPase indicate an essential function during growth/multiplication of asexual blood stages. In order to delete individual P. berghei house-cleaning genes (dUTPase: PBANKA_0921300, ITPase: PBANKA_0618300, Ap4AH: PBANKA_1235300, NDH: PBANKA_1361900), 5’UTR and 3’UTR regions were amplified from genomic DNA of mixed blood stages of P. berghei strain ANKA by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (New England Biolabs, USA; fragments were digested with restriction endonucleases and ligated either side of the human dihydrofolate reductase, or a yeast bifunctional enzyme cytosine deaminase fused to a uridyl phosphoribosyl transferase (hdhfr-yFCU) as selection cassettes. Linearized vectors were transfected into purified schizonts of P. berghei ANKA using standard protocols. Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism. In this paper 4 putative sanitation enzymes were identified using bioinformatic approaches ((dUTPase: PBANKA_0921300, ITPase: PBANKA_0618300, Ap4AH: PBANKA_1235300, NDH: PBANKA_1361900). Based on structural features, sanitation enzymes belong to four superfamilies: ITPases (inosine triphosphate pyrophosphatase) are defined by the Ham1 (6-n-HydroxylAMinopurine sensitive) domain consisting of a long central beta-sheet forming the floor of the active site; dUTPases (deoxy-uridine triphosphatase) which present a trimeric fold, each with an eight-stranded jelly-roll beta barrel; the NuDiX (nucleotide diphosphate linked to some other moiety, X) superfamily is defined by the NuDiX box domain G-x(5)-E-x(5)-[UA]-x-R-E-x(2)-E-E-x-G-U, where U is a hydrophobic residue and x any residue; The fourth group contains all-α-helical NTPases which are active against dNTPs as well as dNDPs with the generation of dNMPs as an end product. From the Abstract: 'While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants on the other hand are not viable suggesting an essential function for these proteins for the malaria parasite. In order to address expression timing and localization we generated transgenic berghei parasite lines expressing each protein fused to green fluorescent protein (GFP) at its C-terminus. ap4ah and ndh were tagged at the endogenous loci, while we expressed dUTPase-GFP and ITPase-GFP episomally under the control of the native promoter using 986 and 865 basepairs of genomic sequence preceding each open reading frame, respectively. Protein expression in transfected parasites was followed by live microscopy and revealed a clear cytoplasmic GFP signal for all four proteins in asexual blood stages (ring, trophozoite and schizont) as well as gametocytes. Western blot analyses showed GFP-tagged dUTPase and ITPase to run higher than their expected molecular weights, while Ap4AH::GFP and NDH::GFP were detected at their predicted positions; NDH with a clear doublet and a smaller 25 kDa band correlating with the size of the cleaved GFP tag. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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