RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-272

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0304900; Gene model (P.falciparum): PF3D7_0207500; Gene product: serine repeat antigen 6 (PbSERA3) Name tag: TAP
Phenotype	Asexual bloodstage; Liver stage;

Last modified: 2 August 2011, 10:54

*RMgm-272

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) : 18419771
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	A. Schmidt-Christensen, V.T. Heussler
Name Group/Department	Bernhard Nocht Institute for Tropical Medicine
Name Institute	Bernhard Nocht Institute for Tropical Medicine
City	Hamburg
Country	Germany
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Name of the mutant parasite
RMgm number	RMgm-272
Principal name	PbSERA3-TAP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Expression (and processing) of PbSERA3-TAP in blood stages
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Expression (and processing) of PbSERA3-TAP in late liver stages
Additional remarks phenotype	Mutant/mutation The mutant expresses in addition to the endogenous SERA3 (the ortholog of P. falciparum SERA6) a TAP-tagged form of SERA3 under control of its own 5'-UTR and the dhfr/ts 3'-UTR. Protein (function) Plasmodium species possesses SERAs of two major groups, specified as 'cysteine-type SERAs' and 'serine-type SERAs'. Serine-type SERAs contain an active site serine residue instead of the canonical cysteine residue. The genomes of different Plasmodium species contain different numbers of SERA genes. P. falciparum possesses nine SERA protease genes whereas the P. berghei genome contains five. P. falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. In P. falciparum mutants have been generated lacking expression of SERA1, 2, 3, 4 , 7, 8 and 9 without a distinct phenotype in the blood stages (MvCoubrie J.E. et al, 2007, Infection and Immunity, 75:5565-74). SERA genes of P. berghei are arranged in a tandem cluster that contains two serine-type SERAs (PbSERA1, -2) and three cysteine-type SERAs (PbSERA3, -4 and -5). Sequence homology and syntenic organisation indicate that PbSERA3, -4 (PB000352.01.0) and -5 (PB000649.01.0) are orthologs of P. falciparum SERA6, -7, -8. Phenotype The mutants has been used to analyse processing of SERA3 in blood and liver stages by Western blotting and to analyse the location of SERA3 by IFA analysis. Processed PbSERA3 has been detected by Western blot analysis in cell extracts of schizont-infected cells and merozoite-infected detached hepatic cells. Immunofluorescence assays revealed that PbSERA3 leaks into the host cell cytoplasm during merozoite development, where it might contribute to host cell death or activate host cell proteases that execute cell death. Expression of the additional tagged SERA protease had no effect on parasite development throughout the life cycle. Additional information In the paper RT-PCR data is presented on transcription of the five SERA genes of P. berghei. PbSERA1-4 are expressed both in blood and liver stages. PbSERA5 is expressed in oocysts and sporozoites. Other mutants RMgm-102: A mutant lacking expression of SERA8 (PB000649.01.0; PFB0325c), a cysteine-type SERA homologue (serine repeat antigen 8, SERA-8; ECP1, egress cysteine protease 1; PbSERA5) RMgm-271: A mutant expressing GFP under the control of the 5'-UTR of PbSERA3 (an ortholog of P. falciparum SERA6). RMgm-365: A mutant expressing the endogenous SERA1 protein fused to the mCherry red fluorescent protein RMgm-366: A mutant expressing the endogenous SERA2 protein fused to the mCherry red fluorescent protein RMgm-367: A knock-out mutant lacking expression of SERA1 RMgm-368: A knock-out mutant lacking expression of SERA2 RMgm-369: A double knock-out mutant lacking expression of SERA1 and SERA2

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0304900

Gene Model P. falciparum ortholog

PF3D7_0207500

Gene product

serine repeat antigen 6

Gene product: Alternative name

PbSERA3

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Details of the genetic modification

Name of the tag

TAP

Details of tagging

C-terminal

Additional remarks: tagging

The P. berghei TAP transfection plasmid pL0032, for homologous recombination into the genome, was obtained from MR4 (www.MR4.org). The 5' UTR and the complete PbSERA3 ORF targeting sequence were obtained by amplification of P. berghei gDNA using the primer set: SERA3-TAP/for (5'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3') and SERA3-TAP/rev (5'-CATGCCATGGACATAACAGAAGAGACATTTGTTTTTTCC-3'). The resulting fragment was cloned into the NcoI/XbaI cloning sites of pL0032 in frame with the TAP-tag coding sequence. The plasmid was linearized for transfection using the unique restriction site XcmI.
The TAP-tag was visualised by Western analysis and IFA using human gamma-globulins (anti-TAP, Sigma).

Commercial source of tag-antibodies

human gamma-globulins (anti-TAP, Sigma).

Type of plasmid/construct

Plasmid single 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

The P. berghei TAP transfection plasmid pL0032, for homologous recombination into the genome, was obtained from MR4 (www.MR4.org). The 5' UTR and the complete PbSERA3 ORF targeting sequence were obtained by amplification of P. berghei gDNA using the primer set: SERA3-TAP/for (5'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3') and SERA3-TAP/rev (5'-CATGCCATGGACATAACAGAAGAGACATTTGTTTTTTCC-3'). The resulting fragment was cloned into the NcoI/XbaI cloning sites of pL0032 in frame with the TAP-tag coding sequence. The plasmid was linearized for transfection using the unique restriction site XcmI.
Integration of the construct into the SERA3 locus results in the presence of a TAP-tagged copy of SERA3 in addition to the endogenous SERA3 gene. The TAP-tagged form of SERA3 is under control of its own 5'-UTR region and the dhfr/ts 3'-UTR.

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	5'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3'
Additional information primer 1	SERA3-TAP/for
Sequence Primer 2	5'-CATGCCATGGACATAACAGAAGAGACATTTGTTTTTTCC-3'
Additional information primer 2	SERA3-TAP/rev
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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