SummaryRMgm-5143
|
Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene mutation |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 34959491 |
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 | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
top of page | |
The mutant parasite was generated by | |
Name PI/Researcher | Hirai M, Mita T |
Name Group/Department | Department of Tropical Medicine and Parasitology, Faculty of Medicine |
Name Institute | Juntendo University |
City | Tokyo |
Country | Japan |
top of page | |
Name of the mutant parasite | |
RMgm number | RMgm-5143 |
Principal name | Pb103(mutated) |
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 | Deleting either of two zinc finger (ZF) domains (ZFs) but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression. |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation To identify crucial domains for ookinete maturation, four Pb103 deletion mutants were made and designated them -ZF1, -ZF2, -C215, and -C495. The -ZF-1 or -ZF-2 lacks the first or second ZF domain, while -C215 or -C495 lacks the respective numbers of amino acids from the C-terminus. Mutants were generated using CRIPR/Cas gene editing as described for mutant RMgm-5042. Protein (function) Phenotype |
top of page | |||||||||||||||||||||||||||
Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1134900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1358600 | ||||||||||||||||||||||||||
Gene product | zinc finger protein, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | Pb103 | ||||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | Pb103 with several deletions of (zinc finger) domains | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||||
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 | Four Pb103 partial deletion mutants were generated by using CRISPR/Cas9. For the construction of -C215, two fragments covering 455 to 655 a.a. (Frag. A) and 0.5 kb of 30 UTR (Frag. B) were amplified by two pairs of primers: -C215-F/-C215-R and -C215-3UTR-F/-C215-3UTR-R. These two fragments were serially inserted as a donor into AflII/HindIII of pBS-1, which was a CRISPR/Cas9 plasmid customized to P. berghei (see mutant RMgm-5042). For the -C495 mutant, two fragments covering 189–375 a.a. (Frag. C) and 0.5 kb of 30 UTR (Frag. D) were amplified by two pairs of primers: -C495-F/-C495-R and -C495-3UTRF/-C495-3UTR-R. These two fragments were inserted into pBS-1, as described above. For the -ZF1 mutant, fragments covering the 50 UTR (0.5 kb) to the first 10 a.a. (Frag. E) and 38 to 220 a.a. motif (Frag. F) were amplified using -ZF1-F1/-ZF1-R1 and -F1-F2/-ZF1-R2. For the -ZF2 mutant construct, fragments covering the 50 UTR (0.2 kb) to the first 79 a.a. (Frag. G), and 115 a.a. to 270 a.a. (Frag. H) were amplified using -ZF2-F1/-ZF2-R1 and -ZF2-F2/-ZF2-R2, respectively. The fragment, E/F, and G/H were inserted as donors into pBS-1. The PAM sites were searched using CHOPCHOP (http://chopchop.cbu.uib.no). The double-stranded DNA coding for gRNA was inserted into the BsmBI site in pBS-1. The resultant plasmids were introduced into P. berghei, and transformants were cloned. All clones were subjected to Sanger sequencing to confirm that the expected regions in the Pb103 gene were deleted. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
| |||||||||||||||||||||||||||
top of page |