RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-623
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0412900; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein (CTRP; circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP))
Details mutation: Mutated ctrp lacking the six A-domains
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 28 July 2011, 14:10
  *RMgm-623
Successful modificationThe 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) : 21729699
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherC. Ramakrishnan; R.E. Sinden
Name Group/DepartmentDivision of Cell and Molecular Biology, Sir Alexander Fleming Building
Name InstituteImperial College London, South Kensington Campus
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-623
Principal nameΔA6
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteOokinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts.
OocystOokinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of CTRP protein (circumsporozoite protein [CSP] and thrombospondin-related adhesive protein [TRAP]-related protein; CSP and TRAP-related protein (CTRP). The endogenous ctrp gene is replaced with a mutated form which lacks the six A domains.

Protein (function)
CTRP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). CTRP has six tandemly arrayed A domains followed by seven TSP type 1-like domains. CTRP is located in the micronemes of ookinetes. This structure is similar to that of TRAP (PF13_0201; PB000374.03.0), a malaria sporozoite protein critical for sporozoite motility and invasion into host cells.
CTRP plays a role in the motility of ookinetes and invasion of midgut epithelial cells. Ookinetes lacking expression of CTRP (RMgm-140, RMgm-141) show impaired motility and invasion of midgut epithelium and do not develop into oocysts.

Phenotype
Ookinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts. This phenotype is comparable to mutants lacking expression of CTRP (RMgm-140, RMgm-141) and indicates that the A domains are essential for ookinete infectivity. See also RMgm-622 for a mutant expressing a mutated form  of CTRP which lacks all 7 TS domains. This mutant  showed normal production of oocysts in both A stephensi and A. gambiae. These results indicate that the seven TS domains are dispensable for infectivity of ookinetes.

Additional information
Less than 50% of the mutant ookinetes displayed any gliding motility and the velocity of those that moved was significantly reduced compared with wild type ookinetes (median of 0.0 (mean 0.6) body lengths/10 min).
Mutant ookinetes were never observed inside the midgut epithelium by confocal microscopy

The same  ΔA6 and ΔTS7 mutants were also generated in the P. berghei ANKA clone 507cl1. 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).

Other mutants
RMgm-140: A mutant lacking expression of CTRP   
RMgm-141: A mutant lacking expression of CTRP
RMgm-622: A mutant expressing a mutated form of CTRP protein which lacks all seven TS domains.


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0412900
Gene Model P. falciparum ortholog PF3D7_0315200
Gene productcircumsporozoite- and TRAP-related protein
Gene product: Alternative nameCTRP; circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP)
Details of the genetic modification
Short description of the mutationMutated ctrp lacking the six A-domains
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid NheI, SbfI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA 682 bp fragment encompassing the 3’ untranslated region (UTR) and the 3’ region of ctrp was amplified from P. berghei genomic DNA using the primers 9-CTMCT3’/U (5’-GGTACCGGTAGTAGTCAGGAAATTCAAATGG-3’) and 10-CTMCT3’/D (5’-ACGCGTCGAATAATGGTGAAAATAGTGAAG-3’) and excised from an intermediate vector using MluI and KpnI digestion. The signal sequence of ctrp together with its 5’ UTR was amplified as a 2,158 bp fragment using 3-CPROSIG/U (5’-
ACGCGTGCCTTCTTGATATATTTTTTTGAAG-3’) and 4-CPROSIG/D (5’-GAATTCGTGAAAATATGTGTATTTTTTCTTAAATTTG-3’) and was excised from an intermediate vector by EcoRI and MluI digestion. Both fragments were ligated into EcoRI-KpnI digested pDb.Dh.^ Db (de Koning-Ward et al., 1998), resulting in pCN-CTRP.

All seven TS domains were then amplified with 7-CTSP/U (5'-ACGCGTCTAAAACGGGAGGTACACAAG-3') and 8-CTSP/D (5'-GCGCGCACCCCGAAGATGCAGAAT-3'), digested with MluI and BssHII, and cloned into MluI-digested pCN-CTRP to give pCTSN-CTRP.

Finally, a 949 bp fragment of the ctrp 3’ UTR was amplified with the primers 17-C3’/U (5’-GGCCGTTGACTGAGGTAAAGGCATCCCTT-3’) and 18C3’/D (5’-GGCCGTTGACGAATGCTCATATGCGTGTG-3’), digested with HincII and cloned into HincII digested pCTSN-CTRP to obtain pCTSN-CTRP/DHR

The plasmid backbone was excised with NheI and SbfI and and the linear construct was used for transfection.
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