Summary

RMgm-403
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1023400; Gene model (P.falciparum): PF3D7_1419800; Gene product: glutathione reductase (GR)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite;
Last modified: 4 July 2010, 10:48
  *RMgm-403
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20573956
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherR. Pastrana-Mena; B. Franke-Fayard; C.J. Janse; A.E. Serrano
Name Group/DepartmentDepartment of Microbiology
Name InstituteUniversity of Puerto Rico-School of Medicine
CitySan Juan
CountryPuerto Rico, USA
Name of the mutant parasite
RMgm numberRMgm-403
Principal name1195cl1
Alternative nameΔgr3
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
OocystMutant oocysts numbers in Anopheles stephensi, counted at day 10-12 post infection, were slightly lower than those produced by wild type parasites and the size of mutant oocysts at day 10-12 was significantly smaller compared to the size of wild type oocysts with a maximum size that is comparable to immature 6-8 days wt oocysts.
The small oocysts that are present at day 10-12 showed a highly vacuolated cytoplasm and the absence of sporozoite formation. No sporozoites were observed in A. stephensi salivary glands at day 21-22 post infection
SporozoiteThe small oocysts that are present at day 10-12 showed a highly vacuolated cytoplasm and the absence of sporozoite formation. No sporozoites were observed in A. stephensi salivary glands at day 21-22 post infection
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of glutathione reductase (GR).
In this mutant, Δgr3,  a ~700 bp region of the gr gene was replaced with the hdhfr/yfcu selectable marker cassette. The mutant does not contain a drug selectable marker which has been removed by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC)(Braks et al., 2006, Nucleic Acids Res 34, e39).
In the paper two addtional mutants (Δgr1, Δgr2) are described in which a ~700 bp region of the gr gene was replaced with the tgdhfr//ts selectable marker cassette.
See RMgm-404 for a mutant (Δgr4) lacking the complete ORF of the gr gene.

Protein (function)
Plasmodium species have a fully functional glutathione (GSH) redox system. GSH is synthesized by the sequential action of gamma-glutamylcysteine synthase (γ-GCS) and GSH synthase; both of the genes encoding these enzymes are present in the Plasmodium. In addition, GSH is  generated by recycling GSSG back to GSH via glutathione reductase (GR). De novo synthesis of GSH is not essential for survival of  P. berghei blood stages. Blood stages of parasites lacking the enzyme γ-GCS (Δγ-gcs; RMgm-204) showed only a minor reduction in growth rate compared to wild type parasites.  In contrast, Δγ-gcs parasites show a complete block in oocyst development and were unable to produce infectious sporozoites. These results indicate that de novo synthesis of GSH is pivotal for development in the mosquito.

Phenotype
The phenotype analyses indicate that GR is not essential for blood stage development. In contrast, a dramatic effect of the lack of GR expression was observed on development of the parasites in the mosquito. Infection of mosquitoes resulted in reduced numbers of stunted oocysts that did not produce sporozoites.

Through disruption of the gene encoding γ-GCS it has been shown that de novo GSH synthesis is not critical for P. berghei blood stage multiplication but is essential for oocyst development. The phenotype analyses of mutant parasites lacking expression of GR confirms that GSH metabolism is critical for the mosquito oocyst stage.  

Attempts to generate parasites lacking GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful (see 'Additional Information'). This demonstrates that the maintenance of cytosolic GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or GSSG reduction by Plasmodium GR.

Additional information
Parasites lacking GR showed the same sensitivity to methylene blue and Eosin B as wild type parasites demonstrating that these compounds target other molecules than GR.

In order to examine whether blood stage parasites can survive without de novo synthesis of GSH and without a functional GR attempts were performed to disrupt the γ-gcs in a mutant lacking GR expression. For these experiments the ∆gr3 mutant was generated which lacks a drug-selectable marker to enable us to take advantage of the γ-gcs disruption construct which contains the tgdhfr selectable marker (RMgm-204). The drug selectable marker, a fusion of the hdhfr and yfcu genes, was removed from the genome by negative selection with 5-fluorocytosine. Three attempts to disrupt the γ-gcs gene using two different constructs (pL1217, pL1223) in the ∆gr3 mutant were unsuccessful whereas wild type parasites were readily transfected with the same constructs (RMgm-204). In addition, attempts were performed to disrupt the gr gene in γ-gcs mutants (∆γ-gcs1,2; RMgm-204) with construct a construct containing the hdhfr as a selectable marker, allowing selection of mutants using treatment with WR99210. In 5 attempts it was not possible to select parasites with both the γ-gcs and the gr genes disrupted. These results indicate that the maintenance of cytosolic GSH levels needed for blood stage survival requires either de novo GSH synthesis or the formation of GSH through reduction of GSSG by a Plasmodium GR. These results also strongly suggest that blood stage parasites cannot depend solely on host-derived GSH and GR for their GSH metabolism.

Other mutants
RMgm-404: An independent  mutant lacking expression of gluthatione reductase (GR). In this mutant the complete gr gene was replaced by the hdhfr/yfcu selectable marker.
RMgm-204: A mutant lacking expression of gamma-glutamylcysteine synthetase (γ-GCS).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1023400
Gene Model P. falciparum ortholog PF3D7_1419800
Gene productglutathione reductase
Gene product: Alternative nameGR
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT
AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC
GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT
CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC
TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA
CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC
ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA
CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT
TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG
TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT
GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC
GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC
CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG
GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA
TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC
GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG
CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC
TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT
CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC
ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC
TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT
TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG
ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC
AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA
CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG
GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTA
GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA
CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG
TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACG
CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC
CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT
GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA
GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG
CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG
CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA
ATTGTGAGCGGTAAACAATTTCACACAGGAAACAGCTTGACCATGATTACGCCAAGCTTC
GCTAGTTTATATACACGTGGATAAAATAAAGTACATTTTATGCATTTCATAAATGTATAT
ATATTCTTCACGCTTTTCGTAAATTCAGGAATAAGGCAAAAGTTGCTTTGGTCGAAAAGT
CCTATTTAGGAGGAACTTGCGTAAATGTAGGATGTGTTCCAAAAAAGGTATAAAAATAAA
ATTCGTTTTTTAAATTATGGGAAAAAAAAATATAATATTAATAAGGTGTAAAATAAAGGT
GGAATTTTCGACTCACTAGTACATTAAAGAGTTTGTAAAAAAAAAAATTTTGTATATATG
TACAACTATTTATACATTTTTTTCATACACTTTTTATATCATAGATAATGTTTAATGCCG
CATCAATTCATGATATTTTACAAAACTCAAGACATTATGGATTTGATACCAGATTTACAT
TCAATCTTCCACAATTAGTAGAAAGGAGAGATAAATATATCAGAAGATTGAATGATATAT
ATCGAAATAATTTAAAGAATGATAATGTTGAAGTTTATGAAGGAACTGCTAGCCTTTTAA
ATGAGCGTAAAGTTCTTATAAAAAGTAAAAATAAATCAGAAAATGATGAAAACAATAATG
AAATAATAGAAGGAAAAAATATATTAATAGCAGTTGGGAATACCCCAATTTTCCCAACAG
TTAAAGGCGTGGAACATACAATTTCGAGTGATGAATTTTTTGATATTAAAGAAGCTAAAA
GAATAGGAATAATCGGAAGTGGATATATAGCTGTTGAATTAATTAATGTAATAAAAAGAT
TAGGAATAGAATCATATATTTTTGCAAGAGGAAAAAGATTGTTAAGAAAATTTGATGAAT
CTATTGTAAATGAATTAGAAAATGATATGAAAAAAAATAATATAAATATTATAACAATGG
CAAATGTAGAAGAAATAGAAAAAGTTCATGCCGCGGTGGCGGCCGCTCTAGCTTTGATCC
CGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTA
TGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAAC
ATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAG
CCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTT
ATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTAC
TGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATG
TTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATA
AACACAAATGATGTTTTTTCCTTCAATTTCGATTGATAATTCCTGCAGCCCAGCTTAATT
CTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTTG
TAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCAT
CCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATATAAA
CATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGCACT
AAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGAACTAGTCGTA
ATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTA
ATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAA
ATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTA
TTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGC
TTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGTTGGTTCGCTAAACT
GCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCAC
CGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTA
AACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGAC
CTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAG
CTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAG
CAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGA
ATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACA
CGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTC
TCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATG
ATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGTGCTAGCGTGACAGGGGGAATGG
CAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGGCCTTAGGTTACA
AAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGTTCTCG
GTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTCCA
CTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGTATACGA
CGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTG
TTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTAGAGGTC
ACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTATCGATG
AAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCTTCGGAACCATTTAAGAACGTCT
ACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAATAAGAATA
CAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTGAAGAAG
GTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAAACTTCG
AAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGA
TGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAA
GGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACCAGAGGATATATCTG
AAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAGTGCTATCATGGCTA
CAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTTCTTAAACCTAATCT
GTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAGAATTGTTACTG
GTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTTGGGTGACTTTG
GTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTTCTTACTTATATATTTATACCAA
TTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATG
CACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAAC
TTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGA
ATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAG
ACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGAT
TCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAA
AAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTT
CGGGTACCGTTGCTATAAATGCGGGGCGATTATTAGCTGATAGAATTTTTTTAAATAAAA
CAAGAAAAACAAATTATAGTCTTATTCCAACTGTTATATTTTCACACCCACCTATAGGAA
CAATAGGTTTATCTGAAGAAGAAGCAATAAATATATATGGAAAGGAAAATGTTAAAATAT
ATGAATCCAAATTTACAAACTTATTTTTTTCTGTATATGATATAGAACCAAGTCAAAAAG
AAAAAACTTATATTAAATTAGTATGTGTTGGAAAAGAGGAGTTAATTAAAGGATTACATA
TAATAGGATTAAATGCGGATGAAATCATACAAGGTTTTGCAGTAGCATTAAAAATGAATG
CGACAAAAAAAGATTTTGATGAGACAATTCCAATTCATCCAACAGCTGCTGAAGAACTTG
TAACTTTACATCCATGGATGAAGTAAATATAAAAAATAAATTGCGCATATTTTAATTCCT
TAAACTACTTTCTGGTGATGTATCATTATTATACATCCATTATTTATTCTCATGTTTGGG
AACACATAAATGTGGGAAAATAGCTAATCATTTGTATTACTCTTAAATAGGTATATTATA
TTCCTTCAAAATATATTTGTGTTTTCTTAAAATAAGATGACAAAATAAGGGATATGATCA
AAGAAGGGATATCTGATCACCCGGGGCGGCCGCG
Restriction sites to linearize plasmid HindIII, EcoRI, ScaI
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption A ~700 bp region of the gr gene was replaced with the hdhfr/yfcu selectable marker cassette
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThe mutant does not contain a drug selectable marker which has been removed by the negative selection procedure using the negative selectable marker yfcu (yeast cytosine deaminase and uridyl-phosphoribosyltransferase) and the drug 5-fluorocytosine (5-FC)( (Braks et al., 2006, Nucleic Acids Res 34, e39).
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 1gggAAGCTTCGCTAGTTTATATACACGTGG
Additional information primer 1L3742 (HindIII); 5'gr targeting region
Sequence Primer 2tccCCGCGGCATGAACTTTTTCTATTTCTTCTAC
Additional information primer 2L3743 (SacII); 5'gr targeting region
Sequence Primer 3cggGGTACCGTTGCTATAAATGCGGGGCGATTATTAGCTG
Additional information primer 3L3680 (EcoRV); 3'gr targeting region
Sequence Primer 4ccgGATATCCCTTCTTTGATCATATCCCTTATTTTGTC
Additional information primer 4L3681 (KpnI); 3'gr targeting region
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
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 KspI (SacII)
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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