RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-623

Malaria parasite	P. berghei
Genotype
Mutated	Gene 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 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) : 21729699
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	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).
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The mutant parasite was generated by
Name PI/Researcher	C. Ramakrishnan; R.E. Sinden
Name Group/Department	Division of Cell and Molecular Biology, Sir Alexander Fleming Building
Name Institute	Imperial College London, South Kensington Campus
City	London
Country	UK
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Name of the mutant parasite
RMgm number	RMgm-623
Principal name	ΔA6
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	Ookinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts.
Oocyst	Ookinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts.
Sporozoite	Not tested
Liver stage	Not 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

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0412900

Gene Model P. falciparum ortholog

PF3D7_0315200

Gene product

circumsporozoite- and TRAP-related protein

Gene product: Alternative name

CTRP; circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP)

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Details of the genetic modification

Short description of the mutation

Mutated ctrp lacking the six A-domains

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

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

NheI, SbfI

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

A 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

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