SummaryRMgm-972
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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) : 24348254 |
MR4 number | |
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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 | |
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The mutant parasite was generated by | |
Name PI/Researcher | C. Suarez; O. Billker; M.J. Blackman |
Name Group/Department | Division of Parasitology |
Name Institute | Medical Research Council National Institute for Medical Research, Mill Hill |
City | London |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-972 |
Principal name | condSUB1(short); condSUB1 |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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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 | Not different from wild type |
Liver stage | GFP expression in mutant oocysts/sporozoites showed high levels (>90%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed ORF of SUB1. The mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites and produced liver stages at 28 hour after incubation of mutant sporozoites with HepG2 cells that were comparable in number and size to that of wild type sporozoites. However, 48 hour mutant liver stages were significantly smaller than wild type liver stages. The majority of the PbSUB1-deficient parasites completely lacked a detectable MSP1 signal. Multiple nuclei were observed in the early PbSUB1-deficient schizonts, indicating normal nuclear replication, but in later stages many of the schizont nuclei appeared condensed and abnormal. Mutant liver stages were deficient in merosome formation. SUB1-deficient liver stages are unable to establish a blood stage infection. |
Additional remarks phenotype | Mutant/mutation See RMgm-973 for a mutant mutant expressing a HA-tagged version of SUB1. Staining of PbSUB1-HA schizonts with the anti-HA mAb again produced a clear punctate signal. The foci were associated with but distinct from individual merozoite nuclei, and similar to the exoneme-specific signal previously observed in P. falciparum blood stage schizonts. No PbSUB1-HA IFA signal was detected in salivary gland sporozoites or early liver stage schizonts. |
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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 | The mutant contains a FRTed ORF of the sub1 locus | ||||||||||||||||||||||||||
Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | The ORF of sub1 is removed in the sporozoite stage by the Flp/FRT system. | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | PCR construct 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 | Working from a P. berghei genomic DNA library clone from the PlasmoGEM resource (http://plasmogem.sanger.ac.uk/) containing 9.6 kb of the pbsub1 locus and neighbouring genes, we constructed allelic exchange vectors designed to flank the entire pbsub1 coding sequence with FRT sites, while at the same time inserting a C-terminal HA epitope tag into pbsub1. The 59 FRT site was introduced into one of two alternative positions in the large upstream intergenic region together with a constitutive promoter sequence of the P. berghei hsp70 gene. A promoterless GFP coding sequence was positioned immediately downstream of the second FRT site. Precise placement of the FRT sites and other exogenous sequence both upstream and downstream of pbsub1 was achieved in 4 steps by recombinase mediated genetic engineering in E. coli, using both the improved Red/ET recombinase system of lambda phage and transient expression of Flp. The constructs were designed such that, following integration by ends-out homologous recombination into the parasite genome, correct recombinase-mediated excision of the sequence lying between the FRT sites (which included the epitope-tagged pbsub1 gene) would reposition the GFP reporter adjacent to the hsp70 promoter, driving constitutive GFP expression only in those parasites in which deletion of the pbsub1 gene had occurred. To prevent the FRT site from interfering with initiation of translation, the start codon for GFP expression was placed just upstream of the 59 FRT site, such that after excision the FRT sequence would be translated into a 12 residue N-terminal extension of GFP. The final constructs, called pJazz-FRTed-pbsub1 and pJazz-FRTed-pbsub1short (which differed only in the placement of the 59 FRT site at either ,2.3 kb or ,1.8 kb respectively upstream of the start ATG of the pbsub1 gene) contained,7 kb and 700 bp regions of homology respectively at their 59 and 39 ends for homologous integration into the P. berghei genome. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | FlpL recombinase of yeast (FlpL) | ||||||||||||||||||
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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 | Plasmid single 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 | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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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 | |||||||||||||||||||
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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 | ||||||||||||||||||
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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 | |||||||||||||||||||
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