SummaryRMgm-4455
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 29774536 |
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 | RMgm-166 |
Other information parent line | This mutant expresses GFP under the 5'- and 3'-regulatory sequences of HSP70 |
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The mutant parasite was generated by | |
Name PI/Researcher | Soga A, Fukumoto S |
Name Group/Department | National Research Center for Protozoan Diseases |
Name Institute | Obihiro University of Agriculture and Veterinary Medicine |
City | Inada-cho, Obihiro, Hokkaido |
Country | Japan |
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Name of the mutant parasite | |
RMgm number | RMgm-4455 |
Principal name | HSP70-pac |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Mutant blood stages have decreased sensitivity to blasticidin compared to wild type blood stages (see additional information) |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Phenotype We constructed a targeting vector that contained a bsd expression cassette which replaced the gfp expression cassette of the GFP-expressing mutant (PbDHFR-GFP) through double crossover homologous recombination. The targeting vector was transfected to PbDHFR-GFP, and the gfp negative mutant ratio was monitored after each in vitro selection. The ratio was 4.74 ± 0.99, 6.88 ± 0.69, and 98.57 ± 2.35 (Mean ± S.D.) % after each selection, respectively. It took 2-3 days from transfection to 1st selection (parasitemia 1.09 ± 0.92%), and 5-6 days from 1st to 2nd selection (parasitemia 1.80 ± 0.86%). Two clones were isolated and Southern blot analysis confirmed that bsd was integrated into the genome. A typical transfection line was also analyzed using flow cytometry. More than 99% of the parasites did not express GFP after the second selection. Therefore, bsd can be used for gene targeting. Other mutants |
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | blasticidin S deaminase (bsd) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
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 | blasticidin S deaminase (bsd) | ||||||||||||||||||
Promoter of the selectable marker | hsp70 | ||||||||||||||||||
Selection (positive) procedure | blasticydin in vitro | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | Elements of the pXL/hdhfr-bsd-egfp plasmid were sequentially ligated into a pXL-BacII-DHFR plasmid backbone. Firstly, the hdhfr expression cassette was excised from pXL-BacII-DHFR, namely pXL-BacII-DHFR (-). Next, hdhfr was cloned into pXL-BacII-DHFR (-) under control of the P. berghei elongation factor 1 alpha (ef-1α) promoter and the pbdhfr-ts terminator. Then, egfp was excised from the pCX-EGFP Vector under control of the P. berghei hsp70 promoter and terminator. This was cloned into the plasmid pXL-BacII-DHFR (-) that also contained the hdhfr expression cassette. The bsd gene was then excised using pCMV/Bsd (Invitrogen, CA, USA) under control of the hsp70 promoter and terminator and cloned into the pXL-BacII-DHFR (-) that contained the hdhfr and egfp expression cassettes. The promoters and terminators were excised from a Yuda 2 plasmid (obtained from Dr. M. Yuda, Mie University). pXL/hdhfr-bsd-egfp was transected with the transposase expression vector EGF-pgT. Elements of pBS/bsd were generated into a pBS backbone. The bsd expression cassette was flanked by the hsp70 promoter and terminator. In addition: We constructed a targeting vector that contained a bsd expression cassette which replaced the gfp expression cassette of the GFP-expressing mutant (PbDHFR-GFP) through double crossover homologous recombination. | ||||||||||||||||||
Additional remarks selection procedure | We analyzed the IC50 values for blasticidin of wild type parasites (WT), pyrimethamine resistant pbdhfr-ts marker integrated parasites (PBDHFR), and pXL/hdhfr-bsd-egfp transfected parasites that were selected by pyrimethamine (BSD). The IC50 values were 29.87 ± 12.55 μg ml-1 for WT, 27.13 ± 8.47 μg ml-1 for PBDHFR, and 159.15 ± 103.54 μg ml-1 for BSD. The IC50 value of BSD tended to be increased, as compared with that of WT (p=0.0684) and PBDHFR (p=0.0637). BSD showed significantly decreased susceptibility to blasticidin at the concentrations above the IC70 value compared with WT and PBDHFR (p < 0.05). Drug sensitivity test described above confirmed that the optimum concentration of blasticidin was 500 μg ml-1 (approximately IC70 value of BSD and 5-fold IC90 value of PBDHFR). The in vitro selection procedure for the pXL/hdhfr-bsd-egfp transfected parasite was performed three times with the 500 μg ml-1 concentration of blasticidin, and the egfp-expressing mutant ratio was monitored after each selection. The ratio was 3.29 ± 1.57, 92.33 ± 1.97, and 94.64 ± 1.98 (Mean ± S.D.) % after each selection, respectively. It took 1-2 days from transfection to 1st selection (parasitemia 1.89 ± 1.53%), 6-7 days from 1st to 2nd selection (parasitemia 1.46 ± 0.23%), and 5 days (parasitemia 0.98 ± 0.51%) from 2nd to 3rd selection. Two clones were isolated from the mutant parasites population selected in Fig.1C by in vivo limiting dilution method to confirm that bsd was integrated into the genome. Southern blot analysis confirmed that bsd was integrated into the genome. We constructed a targeting vector that contained a bsd expression cassette which replaced the gfp expression cassette of the GFP-expressing mutant (PbDHFR-GFP) through double crossover homologous recombination. The targeting vector was transfected to PbDHFR-GFP, and the gfp negative mutant ratio was monitored after each in vitro selection. The ratio was 4.74 ± 0.99, 6.88 ± 0.69, and 98.57 ± 2.35 (Mean ± S.D.) % after each selection, respectively. It took 2-3 days from transfection to 1st selection (parasitemia 1.09 ± 0.92%), and 5-6 days from 1st to 2nd selection (parasitemia 1.80 ± 0.86%). Two clones were isolated and Southern blot analysis confirmed that bsd was integrated into the genome. A typical transfection line was also analyzed using flow cytometry. More than 99% of the parasites did not express GFP after the second selection. Therefore, bsd can be used for gene targeting. | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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