Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene disruption,
Introduction of a transgene
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 29440367 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone |
RMgm-689
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Other information parent line | RMgm-689 (1971cl1) is a P.y.yoelii 17XNL mutant expressing the fusion protein GFP-Luciferase under the control of the constitutive P. berghei eef1a promoter. The mutant does not contain a drug-selectable marker. |
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The mutant parasite was generated by |
Name PI/Researcher | Vaughan AM; Kappe SHI |
Name Group/Department | Center for Infectious Disease Research |
Name Institute | Center for Infectious Disease Research |
City | Seattle |
Country | Washington |
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Name of the mutant parasite |
RMgm number | RMgm-4448 |
Principal name | LISP2(-) |
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 | Reduced liver stage development resulting in prolonged prepatent period.
We chose to study LISP2 because it is expressed on the middle-to-late liver stage parasitophorous vacuole membrane and deletion of P. berghei LISP2 leads to incomplete late liver stage growth arrest
To determine if P. yoelii lisp2− arrests during liver stage development, groups of BALB/cJ mice were i.v. challenged with either 1,000 marker-free GFP-luciferase-expressing 1971cl1 parent parasites (here referred to as wild type) or 1,000 lisp2− sporozoites, and time to blood stage patency was determined. All wild-type-infected mice became blood stage patent on day 3 after challenge, whereas two of seven P. yoelii lisp2−-infected mice did not become patent and the remaining mice showed severe delays to patency, becoming patent between 5 and 7 days after infection. When mice were challenged with 10,000 lisp2−sporozoites, all mice became blood stage patent from days 4 through 6, demonstrating the incomplete attenuation of P. yoelii lisp2−. |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of LISP2
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).
Protein (function)
The protein contains a predicted Plasmodium 6-cysteine motif. Several studies analysing expression of this protein in P. berghei provide evidence for specific expression in liver stages. The protein has been named LISP2 and sequestrin.
Phenotype
Reduced liver stage development resulting in prolonged prepatent period.
We chose to study LISP2 because it is expressed on the middle-to-late liver stage parasitophorous vacuole membrane and deletion of P. berghei LISP2 leads to incomplete late liver stage growth arrest
To determine if P. yoelii lisp2− arrests during liver stage development, groups of BALB/cJ mice were i.v. challenged with either 1,000 marker-free GFP-luciferase-expressing 1971cl1 parent parasites (here referred to as wild type) or 1,000 lisp2− sporozoites, and time to blood stage patency was determined. All wild-type-infected mice became blood stage patent on day 3 after challenge, whereas two of seven P. yoelii lisp2−-infected mice did not become patent and the remaining mice showed severe delays to patency, becoming patent between 5 and 7 days after infection. When mice were challenged with 10,000 lisp2−sporozoites, all mice became blood stage patent from days 4 through 6, demonstrating the incomplete attenuation of P. yoelii lisp2−.
Additional information
The lisp2 gene was deleted using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, which allows efficient editing of the parasite genome. The advantage to this system is that transgenic parasites do not carry a drug susceptibility marker and thus can easily undergo further genetic manipulation.
Deletion of P. yoelii LISP2 was achieved based on the previously reported CRISPR/Cas9 strategy using plasmid pYC. In brief, LISP2 was deleted using double-crossover homologous recombination following a double-stranded DNA break mediated by Cas9 containing a guide RNA targeting the gene of interest. Complementary regions upstream and downstream of the open reading frame were ligated into plasmid pYC, as was the 20-nucleotide guide RNA sequence, resulting in the creation of plasmid pYC_LISP2. The pYC plasmid was transfected into the blood stage schizonts of P. yoelii line 1971cl1, a marker-free parasite that behaves as the wild type and expresses a green fluorescent protein (GFP)-luciferase fusion throughout the life cycle under the control of the elongation factor 1 alpha promoter. This led to the creation of the P. yoelii lisp2−. Two separate knockout clones from two independent transfections were initially phenotypically analyzed throughout the life cycle.
Other mutants |