RMgmDB - Rodent Malaria genetically modified Parasites
Back to search resultsSummaryRMgm-4920
|
||||||||||
Last modified: 20 November 2020, 16:05
*RMgm-4920| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene disruption, Introduction of a transgene |
| Reference (PubMed-PMID number) | Not published (yet) |
| MR4 number | |
| top of page | |
| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | RMgm-1116 |
| Other information parent line | OVA::HEP17(hep17)(RMgm-1116; 2030cl1) expresses a fusion protein of OVA (ovalbumin) and HEP17/EXP1 (PBANKA_092670) under the hep17 promoter. The mutant does not contain a drug-selectable marker. |
| top of page | |
| The mutant parasite was generated by | |
| Name PI/Researcher | Franke-Fayard B, Janse C.J |
| Name Group/Department | Leiden Malaria Research Group, Parasitology |
| Name Institute | Leiden University Medical Centre |
| City | Leiden |
| Country | The Netherlands |
| top of page | |
| Name of the mutant parasite | |
| RMgm number | RMgm-4920 |
| Principal name | OVA::HEP17(hep17)-s1GIMO |
| Alternative name | 3256cl2 |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
| top of page | |
| Phenotype | |
| Asexual blood stage | Not tested |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | Not tested |
| Sporozoite | Not tested |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation The mutant has been generated in the OVA::HEP17(hep17) line (RMgm-1116; 2030cl1) that expresses a fusion protein of OVA (ovalbumin) and HEP17/EXP1 (PBANKA_092670) under the hep17 promoter. The mutant does not contain a drug-selectable marker. The mutant expresses a fusion of a drug resistance gene and a drug sensitivity gene, the so called positive-negative selectable marker (SM), constitutively expressed by the P. berghei eef1α promoter. Specifically, the mutant contains a fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the neutral S1 locus through double cross-over recombination. The GIMO mother line is used for introduction of transgenes into the modified S1 locus through transfection with constructs that target the s1 locus. These constructs insert into the s1 locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice. Additional information |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1206800 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1008600 | ||||||||||||||||||||||||
| Gene product | zinc finger (CCCH type) protein, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | S1 | ||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||
| 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 | We deleted the s1 coding sequence (CDS) and replaced it with the positive-negative selectable marker, to create a P. berghei s1 deletion GIMO line (OVA::hep17(hep17) s1 GIMO). In order to do this, we generated the pL1928 construct that is based on the standard GIMO DNA construct pL0034 (MRA-849, www.beiresources.org). This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette, and was used to insert both the s1 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences respectively. The s1 5’ gene targeting region was PCR amplified using the primer pairs p1/p2 and ligated into pL0034 using ApaI and KpnI restriction sites. The s1 3’ gene targeting region which was PCR amplified using the primer pairs p3/p4 and ligated using KasI and NotI restriction sites, resulting in plasmid pL1928. The construct was linearized using KasI and ApaI restriction sites outside of the 5’ and 3’ TRs before transfection. The construct pL1928 was used to transfect OVA::hep17(hep17) parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Positive selection results in selection of transgenic parasites where the s1 gene of OVA::hep17(hep17) parasites is replaced by the hdhfr::yfcu, resulting in line 3256. Transfected parasites of line 3256 were cloned by limiting dilution, resulting in the OVA::hep17(hep17) s1-GIMO line (line 3256cl2). | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
|
Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
| |||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||
Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
| Transgene name | Ovalbumin (OVA ) fused to HEP17/EXP1 | ||||||||||||||||||
| top of page | |||||||||||||||||||
| Details of the genetic modification | |||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||
| 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/yfcu | ||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||
| Selection (positive) procedure | No | ||||||||||||||||||
| Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
| Additional remarks genetic modification | |||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||
| top of page | |||||||||||||||||||
| Other details transgene | |||||||||||||||||||
| top of page | |||||||||||||||||||
| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0926700 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1121600 | ||||||||||||||||||
| Gene product | exported protein 1 | circumsporozoite-related antigen | parasitophorous vacuole membrane antigen QF 116 | ||||||||||||||||||
| Gene product: Alternative name | HEP17; EXP1 | ||||||||||||||||||
| |||||||||||||||||||
| top of page | |||||||||||||||||||
| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0926700 | ||||||||||||||||||
| Gene product | exported protein 1, putative circumsporozoite-related antigen | ||||||||||||||||||
| Gene product: Alternative name | HEP17; EXP1 | ||||||||||||||||||
| |||||||||||||||||||
| Insertion/Replacement locus | |||||||||||||||||||
| Replacement / Insertion | Replacement locus | ||||||||||||||||||
| Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
| Gene product | 6-cysteine protein | ||||||||||||||||||
| Gene product: Alternative name | 230p | ||||||||||||||||||
| |||||||||||||||||||
| top of page | |||||||||||||||||||