RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-117

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1035200; Gene model (P.falciparum): PF3D7_1407000; Gene product: LCCL domain-containing protein \| scavenger receptor-like protein (PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3) Name tag: EGFP
Phenotype	Gametocyte/Gamete;

Last modified: 18 February 2009, 12:57

*RMgm-117

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene tagging
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 18452513
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	V. Carter; J.T. Dessens
Name Group/Department	Department of Infectious and Tropical Diseases
Name Institute	London School of Hygiene and Tropical Medicine
City	London
Country	United Kingdom
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Name of the mutant parasite
RMgm number	RMgm-117
Principal name	PbSR/EGFP
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	Expression of the PbSR/EGFP protein in female gametocytes (no expression in asexual blood stages and in male gametocytes). Analysis of purified gametocytes by Western blot using an anti-GFP antibody failed to detect the full-length PbSR-EGFP (predicted to be 175 kDa in size), but gave rise to a band of 27 kDa corresponding to monomeric GFP. This indicated that the C-terminal GFP tag was efficiently cleaved off the full-length PbSR protein. For this reason an additional mutant (RMgm 116; mCherry/PbSR/EGFP) has been generated which, in addition to the C-terminal GFP tag, contained an N-terminal red fluorescent protein (mCherry) tag.
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a GFP-tagged version of PbSR (PbSR (P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; LCCL domain containing protein CCp3). The endogenous pbsr gene is replaced with a c-terminal gfp-tagged version of pbsr (see also the additional remarks under 'Phenotype'). Protein (function) The pbsr gene is a member of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's. PbSR contains four LCCL domains; a LH2 (lipoxygenase homology 2) domain; two SR (scavenger receptor cysteine-rich) domains and a PTX/LamG (pentraxin/laminin-G) domain related to concanavalin A-like module. Phenotype analyses of mutants lacking expression of PbSR (RMgm-113, RMgm-114) indicate a role of PbSR in the formation of sporozoites in the oocyst. Phenotype Expression of the PbSR/EGFP protein in female gametocytes (no expression in asexual blood stages and in male gametocytes). Analysis of purified gametocytes by Western blot using an anti-GFP antibody failed to detect the full-length PbSR-EGFP (predicted to be 175 kDa in size), but gave rise to a band of 27 kDa corresponding to monomeric GFP. This indicated that the C-terminal GFP tag was efficiently cleaved off the full-length PbSR protein. For this reason an additional mutant (RMgm-116; mCherry/PbSR/EGFP) has been generated which, in addition to the C-terminal GFP tag, contained an N-terminal red fluorescent protein (mCherry) tag. Additional information Other mutants RMgm-113: A mutant lacking PbSR RMgm-114: A mutant lacking PbSR RMgm-115: A mutant containing a mutated form of PbSR RMgm-116: A mutant expressing a GFP- and mCherry-tagged version of PbSR

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1035200

Gene Model P. falciparum ortholog

PF3D7_1407000

Gene product

LCCL domain-containing protein | scavenger receptor-like protein

Gene product: Alternative name

PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3

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Details of the genetic modification

Name of the tag

EGFP

Details of tagging

C-terminal

Additional remarks: tagging

Full length expression of PbSR containing a c-terminal green fluorescent protein (GFP) tag

Commercial source of tag-antibodies

Anti GFP (Invitrogen 33-2600)

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

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Additional remarks selection procedure

The endogeneous pbsr gene was replaced with a c-terminal gfp-tagged version of pbsr.

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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