| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene disruption
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 23699412
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| MR4 number |
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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 |
P. berghei ANKA 507cl1 (RMgm-7)
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| Other information parent line | P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
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| The mutant parasite was generated by |
| Name PI/Researcher | Ning, J.; Billker, O.; Snell, W.J. |
| Name Group/Department | Department of Cell Biology |
| Name Institute | University of Texas Southwestern Medical School |
| City | Dallas, Texas |
| Country | US |
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| Name of the mutant parasite |
| RMgm number | RMgm-879 |
| Principal name | gex1cl1; gex1cl2 |
| 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 | See additional information phenotype |
| Oocyst | Reduced numbers of oocysts. Mutant oocysts had only half the diameter of wild-type cysts on day 7 and, on average, had not grown by day 15. Transmission electron microscopy showed that on day 15, wild-type oocysts were forming sporozoites, while gex1 oocysts either remained small and retained a compact nucleus or became highly vacuolated but invariably failed to show signs of sporogony. |
| Sporozoite | No sporozoite formation |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of GEX1
Protein (function)
PbGEX1 is an ortholog of Chlamydomonas Cre06.g280600 (CrGEX1), a protein that functions in nuclear fusion. It is similar to an Arabidopsis gene, GEX1, previously shown to be involved in gametophyte development and embryogenesis.
CrGEX1 and PbGEX1 are members of a large, previously unrecognized family whose first-characterized member is KAR5 which is a Saccharomyces cerevisiae nuclear envelope protein, essential for nuclear fusion during yeast sexual reproduction
Phenotype
Phenotype analyses indicate a defect of oocyst development. Oocysts either remained small and retained a compact nucleus or became highly vacuolated but invariably failed to show signs of sporogony. Normal numbers of ookinetes are produced. Evidence is presented that DNA synthesis/replication during meiosis in ookinetes is normal, resulting in 4N (tetraploid) ookinetes. Evidence is presented that an uncharacterized event in the nuclear envelope was altered in the mutant zygotes (see 'Additional information').
Additional information
An ultrastructural analysis of ookinetes 18 h after fertilization suggested that meiosis in the mutant zygotes was altered at a step after DNA replication.We detected meiotic spindle poles, marked by electron-dense centriolar plaques on the nuclear envelope, more than twice as frequently in the mutant zygotes as we did in wild-type zygotes. This may indicate that an uncharacterized event in the nuclear envelope was altered in the mutant zygotes.
To localize the GEX1 protein in P. berghei, a triple HA epitope tag was fused to the 3'end of the endogenous gene (see RMgm-880). No protein was detected in asexually replicating blood stages. PbGEX1-HA was detected in a circular pattern surrounding the nucleus of the microgametocyte and macrogametocyte. Circumnuclear staining persisted to the ookinete stage. The gex1/PbGEX1-HA progeny behaved as wild type, demonstrating that the tagged protein was functional. These data indicate that GEX1 is a nuclear envelope protein.
Two independent gex1 knockout cloned lines were generated with a conventional gene targeting vector. For this, two fragments of ;500 bp flanking the coding region were amplified from genomic DNA using primer pairs olSA00021 (GCGCgggcccAAACAATTACAAAAGATAGAA)/olSA00328 (GGGgctagcATTTAACTTATGTTGGCGTG) and olSA000320 (GCGCgaattcGTTCTACATATTTGTTCTTATCG)/olSA000321 (CGCggatccGGTGGTGGTAATATGGCTTAAACG). These fragments were inserted on either side of the tgdhfr/ts expression cassette of plasmid pOB90 (Billker et al. 2004).
Other mutants |
*RMgm-879
