RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-167

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1212600; Gene model (P.falciparum): PF3D7_1014200; Gene product: male gamete fusion factor HAP2, putative (HAP2; GCS1, Generative Cell Specific 1) Name tag: EGFP
Phenotype	Gametocyte/Gamete; Fertilization and ookinete;

Last modified: 28 January 2011, 14:06

*RMgm-167

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) : 18367645
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	RMgm-155
Other information parent line	A gfp-tagged version of hap2 is introduced into a mutant (RMgm-155) that lacks the endogeneous hap2 gene.
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The mutant parasite was generated by
Name PI/Researcher	Y. Liu; O. Billker
Name Group/Department	Division of Cell and Molecular Biology
Name Institute	Imperial College London
City	London
Country	United Kingdom
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Name of the mutant parasite
RMgm number	RMgm-167
Principal name	HAP2-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Gametocytes express the fusion protein HAP2-GFP. Complementation of a mutant containing a disrupted hap2 gene (RMgm-155) with the fusion gene hap2-gfp resulted in restoration of fertilisation, resulting in fertilisation rates that are comparable to wild type.
Fertilization and ookinete	Complementation of a mutant containing a disrupted hap2 gene (RMgm-155) with the fusion gene hap2-gfp resulted in normal ookinete production, comparable to wild type.
Oocyst	Not different from wild type
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a GFP-tagged form of HAP2 (GCS1,Generative Cell Specific 1) . The hap2-gfp gene is under the regulation of the endogenous hap2 gene promoter Protein (function) In Arabidopsis the male-specific sterility protein HAP2 has been identified by a genetic screen (HAP mutants: containing haploid-disrupting (hapless) mutations; Johnson M.A. et al., 2004, Genetics 168, 971-82) . A HAP2 family member called GCS1 (for generative cell-specific) was subsequently identified in a screen for lily genes whose transcripts were up-regulated in sperm (generative cells) . HAP2 is conserved and members were found in rice, Chlamydomonas, a red alga, a slime mold, Plasmodium falciparum, and Leishmania major. Phenotype analyses of mutants lacking expression of HAP2/GCS1 (RMgm-155, RMgm-156) indicate a role of HAP/GCS1 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. Male gametes are able to attach to female gametes and form tight prefusion membrane attachments but the membranes of the gametes do not merge or fuse. Phenotype Gametocytes express the fusion protein HAP2-GFP. The Complementation of a mutant containing a disrupted hap2 gene (RMgm-155) with the fusion gene hap2-gfp resulted in restoration of fertilisation, resulting in normal fertilisation rates and production of ookinetes, comparable to wild type. Phenotype analyses of mutants lacking expression of HAP2/GCS1 (RMgm-155, RMgm-156) indicate a role of HAP/GCS1 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. Male gametes are able to attach to female gametes and form tight prefusion membrane attachments but the membranes of the gametes do not merge or fuse. Additional information GenBank accession no. XM_671808. Other mutants RMgm-155: An independent mutant lacking expression of HAP2/GCS1. RMgm-156: A mutant lacking expression of HAP2/GCS1. RMgm-157: A mutant expressing a GFP-tagged form of HAP2/GCS1 RMgm-158: A mutant expressing GFP under the control of the hap2/gcs1 promoter RMgm-605: A mutant expressing a mutated form of HAP2/GCS1 lacking the HAP2-GCS1 (HG) domain RMgm-606: A mutant expressing a mutated form of HAP2/GCS1 lacking the entire C-terminal region downstream from the TM domain RMgm-607: A mutant expressing a mutated form of HAP2/GCS1 lacking the TM domain and the entire C-terminal region downstream from the TM domain

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1212600

Gene Model P. falciparum ortholog

PF3D7_1014200

Gene product

male gamete fusion factor HAP2, putative

Gene product: Alternative name

HAP2; GCS1, Generative Cell Specific 1

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Details of the genetic modification

Name of the tag

EGFP

Details of tagging

C-terminal

Additional remarks: tagging

A 4.2-kb fragment comprising 1.5 kb of upstream sequence and the complete hap2 genomic sequence, except the stop codon, was amplified by PCR from genomic DNA using primers 485 (5′-ATATGGTAC CACGCTACTTATATATAGTGATAACC-3′) and 482 (5′-ATA TGGGCCCTCGCAATGGGGGTATTTTACTTTTAC-3′), inserted into KpnI and ApaI restriction sites of vector p277, upstream of and in frame with an egfp gene, which was followed by 0.5 kb of 3′ untranslated region (UTR) derived from the Pbdhfr/ts gene. The vector was linearized in a unique EcoRV site in the putative hap2 promoter and introduced into a hap2 knockout mutant (RMgm-155).

Commercial source of tag-antibodies

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

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

WR99210

Selection (negative) procedure

Additional remarks genetic modification

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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