RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-1126
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Last modified: 15 September 2014, 18:22
*RMgm-1126| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene mutation |
| Number of attempts to introduce the genetic modification | 2 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 25204226 |
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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 | no details provided |
| Other information parent line | The parent cloned line expresses GFP under control of the hsp70 promoter. No details on the origin of this transgenic parent line are provided |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Giganti, D; Barale, JC |
| Name Group/Department | Unite´ d’Immunologie Mole´culaires des Parasites, De´partement de Parasitologie et de Mycologie |
| Name Institute | Institut Pasteur |
| City | Paris |
| Country | France |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1107100 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0507500 | ||||||||||||||||||||||||||
| Gene product | subtilisin-like protease 1 | ||||||||||||||||||||||||||
| Gene product: Alternative name | SUB1 | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | N-terminal deletion ((a-helix + belt loop, residues 21–109) | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | (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 | |||||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | See RMgm-974 for unsuccessful attempts to knockout the sub1 gene, indicating an essential role of SUB1 for development/multiplication of blood stages. In this study it was attempted to generate genetically modified P. berghei in which the whole N-terminal Plasmodium-specific insertion of PbSUB1 (a-helix + belt loop, residues 21–109) was deleted and adding a C terminal haemagglutinin (HA) tag. The unsuccessful attempts to delete this part of SUB1 indicates an essntial role of the 'belt region' for its function in blood stage development/multiplication. In this study the crystal structure of full-length SUB1 from Plasmodium vivax is reported, revealing a bacterial-like catalytic domain in complex with a Plasmodium-specific prodomain. The latter displays a novel architecture with an amino-terminal insertion that functions as a ‘belt’, embracing the catalytic domain to further stabilize the quaternary structure of the pre-protease, and undergoes calcium-dependent autoprocessing during subsequent activation. The targeting plasmid was constructed by cloning three PCR products into the plasmid described by Lacroix et al.(2011, Nat Protoc.), which carries the human dihydrofolate reductase expression cassette that confers the resistance to WR99210 drug. The pre 5-untranslated region of Pbsub1 (736 bp) DNA fragment was amplified using primers 5'-AAGCTTTAAATAAATCACCAAATATTAAATTGG-3' and 5'-GCGGCCGCATGTGTGTTATATAAGTGATTATTCTTTAAGC-3' , and cloned into the plasmid using HindIII and NotI restriction sites (underlined). The post 3'-untranslated region PbSUB1 (673 bp) DNA fragment was amplified using primers 5'-CCC GGGCATTTGGTGTATATTATTGTTTTTCTTG-3' and 5'-GGTACCTCAATGTGCATTTATTGTTATGC-3' , and inserted into the previously generated plasmid using SmaI and KpnI. The deletion of the sequence coding for the PbSUB1 B-domain and the introduction of the HA tag have been obtained using the oligonucleotides (5'-GGGCCCAAATATATGTCGGTGTAATGCACC-3', 5'-ccggCATATGAAAAATTAAATAATAATTTAAAAAAATC-3'), (5'-TACATCGTATGGGTAACCTCCTGCGTAATCTGGTACATCGTAT GGTATGCACCATTTC TTTTTTTACTTTTCCACATGCG-3' and 5'-CAGCTAGCTACCCATACGATGTACCAGATTACGCAtaaAAGGTTTGGGAAATTAAaagTTATAAAATTAATAAG-3'), respectively, and Quickchange II Xl following the manufacturer’s recommendations. The final plasmid was confirmed by sequencing and linearized using BmrI and KpnI before transfection. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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