RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-770
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS; CSP)
Details mutation: Endogenous csp replaced by csp of P. galinaceum with the repeat region of P. bergehi csp
Phenotype Sporozoite; Liver stage;
Last modified: 22 September 2012, 19:09
  *RMgm-770
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) : 22393411
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. Aldrich; R. Spaccapelo
Name Group/DepartmentDepartment of Experimental Medicine
Name InstituteUniversity of Perugia
CityPerugia
CountryItaly
Name of the mutant parasite
RMgm numberRMgm-770
Principal namePgCS/PbRR clones 4 and 5
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNormal numbers of oocysts and midgut sporozoites (at day 14 post infection). Strongly reduced numbers of salivary gland sporozoites at day 21 p.i. Salivary gland sporozoites are unable to infect mice after intravenous inoculation
Liver stageSalivary gland sporozoites are unable to infect mice after intravenous inoculation
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei csp (circumsporozoite protein) gene is replaced by the P. gallinaceum csp  of which the repeat region was replaced by the  repeat region of P. berghei csp

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Normal numbers of oocysts and midgut sporozoites (at day 14 post infection). Strongly reduced numbers of salivary gland sporozoites at day 21 p.i. Sporozoites are not infectious to mice. This phenotype is similar to the phenotype of mutants in which the complete P. berghei csp is replaced with the complete P. gallinaceum csp
These observations indicate that the replacement of the PgCS repeat region with the homologous PbCS repeat region is not sufficient to rescue salivary gland invasion. In addition, the replacement of the PgCS repeat region with the homologous
PbCS repeat region is unable to rescue infectivity of sporozoites to mice.

Additional information
Staining of midgut sporozoites with anti-CSP antibodies showed normal expression, location. processing and release of the mutated CSP

Figure 1



Figure 1.
Schematic representation of the CS proteins present in each of the four transgenic P. berghei parasite lines generated.
PbCSDHFR parasites carry the wildtype PbCS coding sequence (white boxes, RI and RII indicated with light and dark green respectively)
PgCSSX parasites carry the full PgCS coding sequence (grey boxes, RI and RII indicated with light and dark blue respectively), but with the SpeI (S) and XhoI (X) restriction endonuclease sites inserted on either side of the repeat region.
PgCS/PbRR parasites contain the PgCS N-terminal and C-terminal regions (grey boxes) and the PbCS repeat region (white box).
PbCS/PgCT parasites carry the PbCS N-terminal and repeat regions (white boxes) and the PgCS C-terminal region (grey box).

Other mutants
RMgm-769: A mutant with endogenous P. berghei csp gene is replaced by the P. gallinaceum csp with introduced SpeI and XhoI sites flanking the repeat region.
RMgm-771: A mutant with the endogenous csp gene replaced by a chimeric csp gene: the C-terminal region of P. galinaceum csp and the N-terminal and repeat regions of P. berghei csp.


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCS; CSP
Details of the genetic modification
Short description of the mutationEndogenous csp replaced by csp of P. galinaceum with the repeat region of P. bergehi csp
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 ApaI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe targeting construct pPgCS/PbRR, generated in this study contains the following structural elements: (i) the first 1,130 nucleotides of the PbCS 5’UTR sequence immediately upstream of the start codon; (ii) a chimeric form of the CS coding sequence and (iii) the first 1,150 nucleotides of the PbCS 3’ UTR sequence immediately downstream of its stop codon, in which the T. gondii dihydrofolate reductase/thymidylate synthase (TgDHFR/TS) drug selectable marker cassette (5,150 bp) was inserted at its HindIII site. The constructs was generated from pPgCSSX (RMgm-769) by replacing the PgCS repeat region in pPgCSSX with the PbCS repeat region.

The 1.02 kb PbCS gene was amplified from P. berghei genomic DNA (Anka strain, clone 2.34) and sequenced. Forward and reverse primers were designed to amplify the PbCS repeat region from this sequence, introducing the SpeI and XhoI sites at either end of the repeat region (Spefor 5’-TAATAATAAATTGAAACAACCAACTAGTCCACCACCACCAAACCCAAATG-
3’ (SpeI is underlined) and XhoRev 5’-CCGCTCGAGGATATAAGAATCGTCATTATTATTATTTTTGTTATTG-
3’ (XhoI is underlined)). The PgCS repeat region was then replaced with the PbCS repeat region, via SpeI and XhoI digestions.

The targeting construct was designed to direct the double cross-over event between the 1.13 kb sequence of the PbCS 5’ untranslated region (UTR) and the 0.85 kb sequence of the PbCS 3’ UTR in the linearized plasmid and their corresponding sequences in the PbCS locus. Plasmids pPgCSSX was digested with ApaI to release the 8.6 kb targeting insert from the plasmid backbone.
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