RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4408

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PBANKA_1432300; Gene model (P.falciparum): PF3D7_1216600; Gene product: cell traversal protein for ookinetes and sporozoites (CelTOS, S4) Details mutation: 'promoter swap mutant': the promoter of CelTOS replaced with the ctrp promoter
Phenotype	Sporozoite;

Last modified: 2 January 2018, 15:18

*RMgm-4408

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) : 29253313
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Steel RWJ, Kappe SHI
Name Group/Department	Center for Infectious Disease Research
Name Institute	formerly Seattle Biomedical Research Institute
City	Seattle
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-4408
Principal name	Pb s4/celtos(ctrp)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not tested
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Gliding trails for Pb S4/CelTOS(CTRP) sporozoites were shorter than Pb WT.
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation In the 'promoter swap mutant': the promoter of CelTOS replaced with the ookinete-specific ctrp promoter (PBANKA_0412900; circumsporozoite- and TRAP-related protein). Protein (function) CelTOS is localized to the micronemes of ookinetes and (salivary gland) sporozoites and is secreted. It plays a role in migration of ookinetes through mosquito midgut cells and traversal of sporozoites through hepatocytes Phenotype CelTOS has essential/important role in traversal of ookinetes through the midgut epithelium. In order to analyse the role of CelTOS in sporozoites in more detail in this study two promoter-swap mutants, Py s4/celtos^ctrp and Pb s4/celtos^ctrp, were generated, where celtos is under control of the ookinete-specific ctrp promoter, resulting in rescue of the ookinete phenotype in mutants lacking celtos. Most phenotype analyses has been performed with the Py s4/celtos^ctrp mutant (RMgm-4407). In this paper only the 'gliding' phenotype of Pb s4/celtos^ctrp sporozoites is reported. ' We sought to confirm the role for S4/CelTOS during gliding for other Plasmodium parasite species, and to this end we generated a Pb S4/CelTOS(CTRP) line. As for Py S4/CelTOS(CTRP), we observed that gliding trails for Pb S4/CelTOS(CTRP) sporozoites were shorter than Pb WT'. Additional information We were unable to detect S4/CelTOS protein in Py S4/CelTOS(CTRP) salivary gland sporozoites by either indirect immunofluorescence assay (IFA) or western blot, while expression of other adhesion and motility motor proteins such as CSP, TRAP and MTIP were unaffected. Collectively these data demonstrate that the CTRP promoter swap strategy substantially rescued the deficit in midgut infection associated with the complete S4/CelTOS knockout, yielding robust salivary gland sporozoite numbers that were deficient in S4/CelTOS. Other mutants

Mutated: Mutant parasite with a mutated gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1432300

Gene Model P. falciparum ortholog

PF3D7_1216600

Gene product

cell traversal protein for ookinetes and sporozoites

Gene product: Alternative name

CelTOS, S4

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Details of the genetic modification

Short description of the mutation

'promoter swap mutant': the promoter of CelTOS replaced with the ctrp promoter

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

DNA fragments containing approximately 800 bp of the 5′ UTR of the Pb S4/CelTOS gene, approximately 700 bp of the 5’ UTR immediately upstream of the start codon of Pb CTRP gene (CTRP promoter), the entire Pb S4/CelTOS open reading frame with stop codon, and the 3’ UTR of Pb S4/CelTOS were amplified from WT Pb NK65 genomic DNA before cloning into the b3D.DT^H.^D vector (BEI Resources).

Additional remarks selection procedure

Primer information: Primers used for amplification of the target sequences Click to view information

Primer information: Primers used for amplification of the target sequences Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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