RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-1113
|
||||||||||
*RMgm-1113| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene mutation, Introduction of a transgene |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 25166051 |
| MR4 number | |
| top of page | |
| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei NK65 |
| Name parent line/clone | RMgm-278 |
| Other information parent line | |
| top of page | |
| The mutant parasite was generated by | |
| Name PI/Researcher | Lehmann C; Heussler V |
| Name Group/Department | Institute of Cell Biology |
| Name Institute | University of Bern |
| City | Bern |
| Country | Switzerland |
| top of page | |
| Name of the mutant parasite | |
| RMgm number | RMgm-1113 |
| Principal name | PbICP(cond) |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
| top of page | |
| Phenotype | |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Not different from wild type |
| Fertilization and ookinete | Not different from wild type |
| Oocyst | Not different from wild type |
| Sporozoite | Mutant sporozoites show reduced invasion of heptocytes in vitro (sporozoites of this mutant still produce ICP from mRNA produced before excision of the icp locus by Flp; mutants lacking expression of ICP in sporozoites (see RMgm-970; RMgm-1114) show strongly reduced invasion of salivary gland invasion and impaired gliding motility) |
| Liver stage | Sporozoites show reduced invasion of heptocytes in vitro. Liver stages show impaired growth resulting in smaller sized parasites. Liver stages exhibited an abnormal nuclear distribution and did not develop into the characteristic cytomere stage. Liver stages show strongly impaired detachment and merozoite egress in vitro. Mutant sporozoites have a strongly reduced infectivity in vivo. |
| Additional remarks phenotype | Mutant/mutation This mutant has been generated by replacement of the endogenous icp gene by a FRTed icp locus in the mutant RMgm-278 that expresses FlpL. |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0813000 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0911900 | ||||||||||||||||||||||||||
| Gene product | falstatin | inhibitor of cysteine proteases | ||||||||||||||||||||||||||
| Gene product: Alternative name | falstatin; PbICP; ICP | ||||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||||
| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | The mutant contains a FRTed ORF of the icp locus | ||||||||||||||||||||||||||
| Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | The ORF of icp is removed in the sporozoite stage by the Flp/FRT system. | ||||||||||||||||||||||||||
| 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 | pbdhfr | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | We first introduced FRT sites into the 3'regulatory sequence (UTR) of the pbicp locus by double crossover homologous recombination. However, even though excision of the FRTed 3'UTR occurred with great efficiency in salivary gland sporozoites, it was not sufficient to completely suppress expression of the pbicp gene. Therefore, using an alternative strategy, one FRT site was introduced between the sequence coding for the C-terminal inhibitor domain and the N-terminal domain of the protein. Following integration by double crossover homologous recombination, Flp-mediated excision of the sequence between both FRT sites (pbicp 3'-coding region and 3'UTR) was expected to block expression of the functional inhibitor. The plasmid pPbICP/FRT contains the following elements, listed with the numbers of the relevant primer pairs in parentheses. In pPbICP/FRT, the first 0.85 kb (1) (PbICP-N) of the PbICP coding sequence (PlasmoDB: PBANKA_081300) is immediately followed by 16 nucleotides (tcgttttcgtttaact), a first FRT site, the last 0.7 kb (2) (PbICP-C) of the PbICP coding sequence, the hdhfr 3'regulatory sequence (3) (0.45 kb), the hdhfr cassette, the second FRT site, the plasmid backbone and 0.76 kb (4) of the PbICP 3'regulatory sequence. The coding and regulatory sequences of pbicp and hdhfr were cloned from gDNA of P. berghei NK65 wildtype blood stages or Phdhfr/FRT-Flp plasmid using Phusion Taq High-Fidelity DNA polymerase (Finnzyme) and the following primer pairs: (1) fw, 5'-ATGCATGCCGTGTTTAATATATGfCTCCATCCTAGCC-3', and (1) rv, 5'-ATATGCGGCCGCAGTTAAACGAAAACGATATATCTTCGCTATTATCAGAAAAATTACTTGCTG-3'; (2) fw, 5'-ATGATATCGAAGATAATCAAAAATACCCAACTAC-3', and (2) rv, 5'-ATGATATCTTATTGGACAGTCACGTATATAATTTTAGTG-3'; (3) fw, 5'-ATATGGGCCCCGTTTTTCTTACTTATATATTTATACCAAT-3', and (3) rv, 5'-ATATGGGCCCATTGAAGGAAAAAACATCATTTG-3'; (4) fw, 5'-ATATAAGCTTGTATATATGCGTATATATAATATATGCAATAATAATTTTTTTTTATGCC-3', and (4) rv, 5'-ATGCATGCGAAATTGTGGAAAGAATGAAAAAGGGGTG-3'. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
| |||||||||||||||||||||||||||
| 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 | FlpL recombinase of yeast (FlpL) | ||||||||||||||||||
| top of page | |||||||||||||||||||
| Details of the genetic modification | |||||||||||||||||||
| Inducable system used | Flp/FRT | ||||||||||||||||||
| Additional remarks inducable system | The mutant expresses the yeast FlpL recombinase under the control of the promoter of uis4. The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. | ||||||||||||||||||
| 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 | eef1a | ||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||
| Additional remarks genetic modification | The flpl gene was cloned between 1.5 kb of 5′ regulatory sequence of uis4 and 0.6 kb of 3′ regulatory sequence of trap. The plasmid was linearized in the 5′ uis4 regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous uis4 gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. | ||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||
| top of page | |||||||||||||||||||
| Other details transgene | |||||||||||||||||||
| top of page | |||||||||||||||||||
| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0501200 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | Not available | ||||||||||||||||||
| Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||
| Gene product: Alternative name | |||||||||||||||||||
| |||||||||||||||||||
| top of page | |||||||||||||||||||
| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
| Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
| Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
| |||||||||||||||||||
| Insertion/Replacement locus | |||||||||||||||||||
| Replacement / Insertion | Insertion locus | ||||||||||||||||||
| Gene Model of Parasite | PBANKA_0501200 | ||||||||||||||||||
| Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||
| Gene product: Alternative name | |||||||||||||||||||
| |||||||||||||||||||
| top of page | |||||||||||||||||||