RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1320
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene not Plasmodium: Luciferase
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 Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 31 October 2018, 15:00
  *RMgm-1320
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26208778
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-687
Other information parent lineGIMO-PbANKA (RMgm-687) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA is used for rapid introduction of transgenes free of drug-resistance genes (RMgm-687; PubMed: PMID: 22216235).
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The mutant parasite was generated by
Name PI/ResearcherPrado M, Heussler VT, Janse CJ
Name Group/DepartmentLeiden Malaria Research Group, Parasitology
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-1320
Principal name1868cl1
Alternative namemCherry(hsp70)+Luc(eef1α)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageStrong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)
Gametocyte/GameteStrong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)
Fertilization and ookineteStrong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)
OocystStrong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)
SporozoiteStrong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)
Liver stageStrong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)
Additional remarks phenotype

Mutant/mutation
This transgenic reporter line expresses  luciferase under the control of the eef1α (PBANKA_113330) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_071190) promoter. Both reporter cassetes are introduced into the silent 230p locus, using a single DNA construct. This transgenic line does not contain a drug-selectable marker (see Additional Information below). Compared to transgenic parasites that express a fusion of Luciferase with mCherry or GFP, this transgenic line that contains separate cassettes of mCherry and Luciferase, shows higher levels of luciferase activity as determined by measuring luminescence.

Protein (function)

Phenotype
Strong expression of mCherry and Luciferase in all life cycle stages. Both reporter proteins are expressed under constitutive promoters (eef1a and hsp70)

Additional information
This reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.

Other mutants

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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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GGCCGCTATTATTGTTCTGTACTTCTTTTGTGAATAGTATTTTTACATATGTACTGTTAA
TTTTAAAATGTGGAATGCCACGAACATATTTTTTCGAACAAAATATATATTTTTTGTGTC
GAAATACATAAATTTGTGTACCTCCTTTTTGTTGTATATATGAAAAAGGAATAAAGTGAA
TGGAGTCGAATTACTCTAGACCCTACAACTTGTATATCACTACTTTATAATACTATAATA
TTATTATTAACATTAAACTATAGAAGAAATATCAACAATACTTATGTATTTGTTCAAACT
TTATCAGTATAGTTTGGTAATCGCATATCATATATTTGTTATGTGTAATATTTTATGAAT
ATGCGTCTTTTTTTTTAACTATAAATAATTCAAAATAAAATAACATAATTAACATTTGCA
AGCATAATTAAGATGATTACTTTCACATTTCATCCAAAGTGGAAAATGACCACCACATTT
GATCGAAGCTTTTACCACTTTTGATGCATTAGCTCTGAAATGTATACAAGCTTTCATTTG
TTAATGTTACAAAGGGAAGTTTTTATTTGGGGGCACATTAAAAGTGAAAAAATTAAAAGT
TAAAAAAGTTAAAAAATGATCGGTGTAGTTGGTCATATAGACATGTTGTTATGCAAGTCT
TTCAAAACGTTCCTCATAAAATAAAAGGAGGGATCTAGAGAGCTTTCAATTCTCCAAACG
GTATTATTGGTATTTTCCGGTACCATCGAATTCCTGCAGCCCAGCTTAATTCTTTTCGAG
CTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTTGTAGTATCCT
AGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAA
TACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACG
CCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGCACTAAATATAAG
CTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTATTTATATTTCT
ATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATGTTTTTTTTATTT
TATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATATAATTGTTGTGA
AACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTA
AAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTATAAATAAATAAA
AAATTTTATAAAACATAGGGGGATCCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGG
CGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGA
GATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCA
CGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGC
TGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGG
TGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAAC
GTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGG
GGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCA
TGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATC
TACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAA
TTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGC
ATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCA
TTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTA
CACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGC
TGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTAT
TTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAA
TTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCC
ATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTA
CACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGA
AGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTG
TCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGA
TTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACT
TCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCG
CTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTC
TTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGA
CGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGT
TGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACG
CAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAAG
CGGCCGCCTGGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAA
CTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATA
GCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAAT
GGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTA
AAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTAT
TATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGAC
TTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATT
AAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGA
ATTCTCTTGAGCCCGTTAATGAAATAGATACAATTCATTCATGTTATATACATCTAGAAC
ATAATCTGAATATGGTTCAAGTTAAATGTCCAAAAATTATAAAAAGTGATGATATTTTTG
ATGGTAATACCATAATAGACACCAAGGTAACATCACGAAGTAGTCAACAAAATAATTTTT
ATTTAGAAAATACAGATGTTGAACCAGAAGAAATAGAGAAATATAAAAATATAGAATACA
TACCAGAAAACGATGAAGTAATGCATCTAGACAAAAAAGAAAAGCTAGATGATATATTAC
CAGGTGTTATCATATTAGATAAAAATAAAATGTTCAAAGAAAAAGGACATTTCACTTTTG
TTACTCCATTAATTGTAGAAAAGGTATTAATATTAAAAATATATTGTGATAATACTAAAA
CAATAATTAATAATATGAAAGGGAAAAAAGGTATTACAGTAATAAGGATTTCTCAAAATA
CAACAAAAAATAAATTTTATGGATGTGACTTTTCAGGTAATTCTAAAAAAACATTTTACT
ATTCCAATGTTTATGATTTAGAAAAAAAAAATGAGTTTTGTGAAATAGAATTAAAAGAAA
ATATAGTAGTTAGCTTAAATTGTCCAACTGGTAAAATTAATCCAAAAAATTGTTTTAGAA
ATGTATATATAAAAAGTAATATGAATGAACAAACAACCGAAAATATAGAAAATATATTTA
ACGAAATAAAAGTTATAGATGCAGATTATTTTATAAATAATTCATCAACCTTTTTGATGA
TTTCCAAAATTACAAAAAAAGAGTTTGATTTTTATTGTACATGTGAAGATTATAAAACCA
AAAATATAGGAACAATATATATTAAAAATTATGAATATCTAGATTCAAAACCTAAATATA
AAAATAAACAAATTTCCTATATAGATGTAGTTCCATACCCGCGGGGAAAGGGCGAATTCA
CTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC
CTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGC
CCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTT
ACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGAT
GCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT
TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGT
CAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTA
TTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGG
GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG
CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGT
ATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTT
GCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTG
GGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAA
CGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT
GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAG
TACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGT
GCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGA
CCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGT
TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTA
GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG
CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC
CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGT
ATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG
GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTG
ATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA
CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA
ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA
TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG
CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT
GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCAC
CACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG
GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCG
GATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGA
ACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCC
GAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACG
AGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC
AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTT
CCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC
GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGC
CCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGAC
AGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT
CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTG
AGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTCCGCGG
GTATATGGTAAAGAACCTACTAACACAATAAAATATTTAAATAATGTATTTCCTATAAAT
AAATTTACAGATTTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAATGATCAG
TTTAAAGGTTTTAAATTCTTTATGACATCATTTATAAATCATGGATCATATCCACTAACA
ATAGAATGTGGTGTAACAAATGGTGGAACTAGTTATAAAAGAGCAATTATTTTATTGCAT
GTTCGAACTGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTCGAAAAGGAGAATTA
TATAATTATTTGAATGCTTATACTGAAGGGGATGTATGCATAATAATTTCCAAATCAAAT
ACAAGTTTTGGTTTTAGATGCCCAGTAAATACAAAAAAAATGCCAAAAAATTGTTTTACG
CAAGTATATGAAAAAGGGTATCTAAATGACGCCAATAAAATTAATACTAAAAATGTTATT
AACTATTCATTTGAAAATCCAGAATATGCGCTAGCTGGTTYTAATTATACATTAACAAAA
TCGTATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAAACGGTT
TTAGTCAAATATGTAAATGAAGATGAAATATATGATTATAATGATTTTCCAATGGTGAAT
CACAAACCTATTATTGCACATCCAAATAAAACACATCAAGCTTGCATGCCGTAATATTTT
GTTGGTGAGCTTAAAAACTTTTAATATTGTTTGTGCAAAATTTTTGCTAAGAAAATAATT
TTATGAGTCATATTAGAAGTTCATTGAATTTGTTATATAAAATAAATTTTATATTCTGCC
TATTTTATGTACAACAAATTGCGGAATTGTACATAATATAATATGAAAAATATATGGCTT
TAAATTATAATTGCCAAATAAAATAGGGAGGGGCCCCCCCCAAGCCCTCAAAGGGTGCAT
ACTATGTTTATAATATATTATTATATAGCATTATATTAGCATTATATATATTTGTTATTT
TACAATACATTCATTTTGCGTGGGGCAATGCATTTGCGCTCTTTTTCAGTTCATATTTTT
GCAAATAATTGCACTTTCGGAAATTTGAAAAAATATAAAACCCATAAAAAGGTGGAATAC
AAAACATTTTTTGCATTTTCGAAGCATATAAATATGTATATATATTATGCATACTATATT
ATGACTATCCTTGCCAATAAAAAAAAAATATTATATAAAGGGGACCCCATATATAGGTAT
ATATAATAGCTGCTTTAACTAAGGGGCTAATAAATGGCCCCGTATTTTGTTTTTTTTATA
GCTAATAATAAAAATAAAAAAATAAAAAGTACGAACTTTAGTCCCATAAATTAAGAAGTA
TATATGCTTATAAAAAGGATATCAAATAAATTAATGTTAACTCAAATAAATTAATTTTCA
TGCAAAAAAATATTTAAAAAAGAATATATATATTAAGAAAAATAATTATAAATATTATTT
ACATATATATACCATAATATATACCCCCTTTTGATTATATATATATAATATTATATATTT
TTCTTTAGAGAAAAAATAAATAGTGTTATTTCCTATTATATATCGTAGTTAATTTTAATA
ATTTACAATACACTATATTTCTTATTTTATATAAAATATATTTATAATTAATTTTTTTAT
ATTATATGTATATATTTTAATTGCATTCTCCTTTTTTAATAAAAATTTTACCCAATTGTT
ATAGAGCTTGTTATTATTATATAATAACAATATAAGAATATTTTTTATATAACATTAAAA
AAAATTAACATATTTTTTATTTTTTTTAAATATTTATTCCAATTTATTTTTAACGGTCAA
ATTAAAGAAATATATAGATATATATTTATTGCTTAAATTATAAGTATATGTTTTTTTAAA
AAATTAAAGCCCAAAACATATTTCTTTGTATTATATACATTTTTGCATTTTACACTATTT
TGCCATAAGCACATTAAATATATATATATATGCTTGTAGCAAATTAATTAAATAAAATAA
TTACCAACATTATTATCCCAATAAATTACAATTACAATTAAAAAAAGGGATCCATGGTGA
GCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACA
TGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCT
ACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCT
GGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCG
ACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGA
ACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGT
TCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGA
AGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGA
AGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCA
AGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCA
AGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCG
AGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATCTAGAGCAATTTGC
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationConstruct pL1720 was made for generation of reporter line mCherry(hsp70)+Luc(eef1α) that expresses mCherry reporter under control of the hsp70 (PBANKA_071190) 5’- and 3’-regulatory sequences and luciferase under the control of the eef1α (PBANKA_113330) 5’-promoter/regulatory sequences and the pbdhfr/ts (PBANKA_071930) 3’-regulatory sequences. This construct was used to target the GIMO-ANKA mother line using the ‘gene insertion/marker out’ (GIMO transfection) procedure. Construct pL1720 was made as follows: first the selectable marker (SM) cassette from pL1661 (containing both 5’hsp70 and 3’hsp70 sequences, tgdhfr/ts SM, and 230p targeting regions) was removed by digesting the plasmid with SnaBI (Roche, 10997480001) and SbfI (New England Biolabs; R06425) followed by treatment of the DNA fragment with Klenow (Roche, 11008404001) and religation resulting in a plasmid that contains only the 5’hsp70-3’hsp70 sequences and the 230p (PBANKA_030600) targeting regions; the mCherry coding sequences (excised from pL0047 which contains the 5’pbeef1α-mCherry-3’pbdhfr cassette) was then introduced into this plasmid using sites BamHI (Roche, 10220612001) and NotI (Roche, 11014706001). Next a luciferase expression cassette (5’pbeef1a-LucIAV-3’pbdhfr) excised from pL1098 using KpnI (Roche 11198939001) and AflII (Roche, 11198939001) was introduced into this vector using the same restriction sites. This final construct, pL1720, therefore contains the 5’hsp70-mCherry-’hsp70 and 5’pbeef1a-LucIAV-3’pbdhfr expression cassettes and 230p targeting sequences but no SM. This construct was linearized by digesting with SacII (Roche, 11117807001) before transfection. The linear DNA construct was introduced into GIMO-ANKA parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (Sigma, F7129-5G) in the drinking water of mice. Negative selection results in selection of parasites where the hdhfr-yfcu SM in the 230p locus is replaced by the mCherry reporter-cassette.
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
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3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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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GGCCGCTATTATTGTTCTGTACTTCTTTTGTGAATAGTATTTTTACATATGTACTGTTAA
TTTTAAAATGTGGAATGCCACGAACATATTTTTTCGAACAAAATATATATTTTTTGTGTC
GAAATACATAAATTTGTGTACCTCCTTTTTGTTGTATATATGAAAAAGGAATAAAGTGAA
TGGAGTCGAATTACTCTAGACCCTACAACTTGTATATCACTACTTTATAATACTATAATA
TTATTATTAACATTAAACTATAGAAGAAATATCAACAATACTTATGTATTTGTTCAAACT
TTATCAGTATAGTTTGGTAATCGCATATCATATATTTGTTATGTGTAATATTTTATGAAT
ATGCGTCTTTTTTTTTAACTATAAATAATTCAAAATAAAATAACATAATTAACATTTGCA
AGCATAATTAAGATGATTACTTTCACATTTCATCCAAAGTGGAAAATGACCACCACATTT
GATCGAAGCTTTTACCACTTTTGATGCATTAGCTCTGAAATGTATACAAGCTTTCATTTG
TTAATGTTACAAAGGGAAGTTTTTATTTGGGGGCACATTAAAAGTGAAAAAATTAAAAGT
TAAAAAAGTTAAAAAATGATCGGTGTAGTTGGTCATATAGACATGTTGTTATGCAAGTCT
TTCAAAACGTTCCTCATAAAATAAAAGGAGGGATCTAGAGAGCTTTCAATTCTCCAAACG
GTATTATTGGTATTTTCCGGTACCATCGAATTCCTGCAGCCCAGCTTAATTCTTTTCGAG
CTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTTGTAGTATCCT
AGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAA
TACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACG
CCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGCACTAAATATAAG
CTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTATTTATATTTCT
ATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATGTTTTTTTTATTT
TATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATATAATTGTTGTGA
AACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTA
AAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTATAAATAAATAAA
AAATTTTATAAAACATAGGGGGATCCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGG
CGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGA
GATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCA
CGTACGCGGAATACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGC
TGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGG
TGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAAC
GTGAATTGCTCAACAGTATGAACATTTCGCAGCCTACCGTAGTGTTTGTTTCCAAAAAGG
GGTTGCAAAAAATTTTGAACGTGCAAAAAAAATTACCAATAATCCAGAAAATTATTATCA
TGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATC
TACCTCCCGGTTTTAATGAATACGATTTTGTACCAGAGTCCTTTGATCGTGACAAAACAA
TTGCACTGATAATGAATTCCTCTGGATCTACTGGGTTACCTAAGGGTGTGGCCCTTCCGC
ATAGAACTGCCTGCGTCAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCA
TTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTA
CACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGC
TGTTTTTACGATCCCTTCAGGATTACAAAATTCAAAGTGCGTTGCTAGTACCAACCCTAT
TTTCATTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAA
TTGCTTCTGGGGGCGCACCTCTTTCGAAAGAAGTCGGGGAAGCGGTTGCAAAACGCTTCC
ATCTTCCAGGGATACGACAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTA
CACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGA
AGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAGAGAGGCGAATTATGTG
TCAGAGGACCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGA
TTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACT
TCTTCATAGTTGACCGCTTGAAGTCTTTAATTAAATACAAAGGATATCAGGTGGCCCCCG
CTGAATTGGAATCGATATTGTTACAACACCCCAACATCTTCGACGCGGGCGTGGCAGGTC
TTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGA
CGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGT
TGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACG
CAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAAG
CGGCCGCCTGGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAA
CTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATA
GCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAAT
GGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTA
AAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTAT
TATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGAC
TTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATT
AAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGA
ATTCTCTTGAGCCCGTTAATGAAATAGATACAATTCATTCATGTTATATACATCTAGAAC
ATAATCTGAATATGGTTCAAGTTAAATGTCCAAAAATTATAAAAAGTGATGATATTTTTG
ATGGTAATACCATAATAGACACCAAGGTAACATCACGAAGTAGTCAACAAAATAATTTTT
ATTTAGAAAATACAGATGTTGAACCAGAAGAAATAGAGAAATATAAAAATATAGAATACA
TACCAGAAAACGATGAAGTAATGCATCTAGACAAAAAAGAAAAGCTAGATGATATATTAC
CAGGTGTTATCATATTAGATAAAAATAAAATGTTCAAAGAAAAAGGACATTTCACTTTTG
TTACTCCATTAATTGTAGAAAAGGTATTAATATTAAAAATATATTGTGATAATACTAAAA
CAATAATTAATAATATGAAAGGGAAAAAAGGTATTACAGTAATAAGGATTTCTCAAAATA
CAACAAAAAATAAATTTTATGGATGTGACTTTTCAGGTAATTCTAAAAAAACATTTTACT
ATTCCAATGTTTATGATTTAGAAAAAAAAAATGAGTTTTGTGAAATAGAATTAAAAGAAA
ATATAGTAGTTAGCTTAAATTGTCCAACTGGTAAAATTAATCCAAAAAATTGTTTTAGAA
ATGTATATATAAAAAGTAATATGAATGAACAAACAACCGAAAATATAGAAAATATATTTA
ACGAAATAAAAGTTATAGATGCAGATTATTTTATAAATAATTCATCAACCTTTTTGATGA
TTTCCAAAATTACAAAAAAAGAGTTTGATTTTTATTGTACATGTGAAGATTATAAAACCA
AAAATATAGGAACAATATATATTAAAAATTATGAATATCTAGATTCAAAACCTAAATATA
AAAATAAACAAATTTCCTATATAGATGTAGTTCCATACCCGCGGGGAAAGGGCGAATTCA
CTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGC
CTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGC
CCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTT
ACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGAT
GCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCT
TGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGT
CAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTA
TTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGG
GGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCG
CTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGT
ATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTT
GCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTG
GGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAA
CGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT
GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAG
TACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGT
GCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGA
CCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGT
TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTA
GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGG
CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC
CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGT
ATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACG
GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTG
ATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAA
CTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA
ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA
TCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCG
CTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT
GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCAC
CACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTG
GCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCG
GATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGA
ACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCC
GAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACG
AGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTC
TGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCC
AGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTT
CCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC
GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGC
CCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGAC
AGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT
CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTG
AGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTCCGCGG
GTATATGGTAAAGAACCTACTAACACAATAAAATATTTAAATAATGTATTTCCTATAAAT
AAATTTACAGATTTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAATGATCAG
TTTAAAGGTTTTAAATTCTTTATGACATCATTTATAAATCATGGATCATATCCACTAACA
ATAGAATGTGGTGTAACAAATGGTGGAACTAGTTATAAAAGAGCAATTATTTTATTGCAT
GTTCGAACTGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTCGAAAAGGAGAATTA
TATAATTATTTGAATGCTTATACTGAAGGGGATGTATGCATAATAATTTCCAAATCAAAT
ACAAGTTTTGGTTTTAGATGCCCAGTAAATACAAAAAAAATGCCAAAAAATTGTTTTACG
CAAGTATATGAAAAAGGGTATCTAAATGACGCCAATAAAATTAATACTAAAAATGTTATT
AACTATTCATTTGAAAATCCAGAATATGCGCTAGCTGGTTYTAATTATACATTAACAAAA
TCGTATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAAACGGTT
TTAGTCAAATATGTAAATGAAGATGAAATATATGATTATAATGATTTTCCAATGGTGAAT
CACAAACCTATTATTGCACATCCAAATAAAACACATCAAGCTTGCATGCCGTAATATTTT
GTTGGTGAGCTTAAAAACTTTTAATATTGTTTGTGCAAAATTTTTGCTAAGAAAATAATT
TTATGAGTCATATTAGAAGTTCATTGAATTTGTTATATAAAATAAATTTTATATTCTGCC
TATTTTATGTACAACAAATTGCGGAATTGTACATAATATAATATGAAAAATATATGGCTT
TAAATTATAATTGCCAAATAAAATAGGGAGGGGCCCCCCCCAAGCCCTCAAAGGGTGCAT
ACTATGTTTATAATATATTATTATATAGCATTATATTAGCATTATATATATTTGTTATTT
TACAATACATTCATTTTGCGTGGGGCAATGCATTTGCGCTCTTTTTCAGTTCATATTTTT
GCAAATAATTGCACTTTCGGAAATTTGAAAAAATATAAAACCCATAAAAAGGTGGAATAC
AAAACATTTTTTGCATTTTCGAAGCATATAAATATGTATATATATTATGCATACTATATT
ATGACTATCCTTGCCAATAAAAAAAAAATATTATATAAAGGGGACCCCATATATAGGTAT
ATATAATAGCTGCTTTAACTAAGGGGCTAATAAATGGCCCCGTATTTTGTTTTTTTTATA
GCTAATAATAAAAATAAAAAAATAAAAAGTACGAACTTTAGTCCCATAAATTAAGAAGTA
TATATGCTTATAAAAAGGATATCAAATAAATTAATGTTAACTCAAATAAATTAATTTTCA
TGCAAAAAAATATTTAAAAAAGAATATATATATTAAGAAAAATAATTATAAATATTATTT
ACATATATATACCATAATATATACCCCCTTTTGATTATATATATATAATATTATATATTT
TTCTTTAGAGAAAAAATAAATAGTGTTATTTCCTATTATATATCGTAGTTAATTTTAATA
ATTTACAATACACTATATTTCTTATTTTATATAAAATATATTTATAATTAATTTTTTTAT
ATTATATGTATATATTTTAATTGCATTCTCCTTTTTTAATAAAAATTTTACCCAATTGTT
ATAGAGCTTGTTATTATTATATAATAACAATATAAGAATATTTTTTATATAACATTAAAA
AAAATTAACATATTTTTTATTTTTTTTAAATATTTATTCCAATTTATTTTTAACGGTCAA
ATTAAAGAAATATATAGATATATATTTATTGCTTAAATTATAAGTATATGTTTTTTTAAA
AAATTAAAGCCCAAAACATATTTCTTTGTATTATATACATTTTTGCATTTTACACTATTT
TGCCATAAGCACATTAAATATATATATATATGCTTGTAGCAAATTAATTAAATAAAATAA
TTACCAACATTATTATCCCAATAAATTACAATTACAATTAAAAAAAGGGATCCATGGTGA
GCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACA
TGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCT
ACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCT
GGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCG
ACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGA
ACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGT
TCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGA
AGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGA
AGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCA
AGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCA
AGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCG
AGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATCTAGAGCAATTTGC
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationConstruct pL1720 was made for generation of reporter line mCherry(hsp70)+Luc(eef1α) that expresses mCherry reporter under control of the hsp70 (PBANKA_071190) 5’- and 3’-regulatory sequences and luciferase under the control of the eef1α (PBANKA_113330) 5’-promoter/regulatory sequences and the pbdhfr/ts (PBANKA_071930) 3’-regulatory sequences. This construct was used to target the GIMO-ANKA mother line using the ‘gene insertion/marker out’ (GIMO transfection) procedure. Construct pL1720 was made as follows: first the selectable marker (SM) cassette from pL1661 (containing both 5’hsp70 and 3’hsp70 sequences, tgdhfr/ts SM, and 230p targeting regions) was removed by digesting the plasmid with SnaBI (Roche, 10997480001) and SbfI (New England Biolabs; R06425) followed by treatment of the DNA fragment with Klenow (Roche, 11008404001) and religation resulting in a plasmid that contains only the 5’hsp70-3’hsp70 sequences and the 230p (PBANKA_030600) targeting regions; the mCherry coding sequences (excised from pL0047 which contains the 5’pbeef1α-mCherry-3’pbdhfr cassette) was then introduced into this plasmid using sites BamHI (Roche, 10220612001) and NotI (Roche, 11014706001). Next a luciferase expression cassette (5’pbeef1a-LucIAV-3’pbdhfr) excised from pL1098 using KpnI (Roche 11198939001) and AflII (Roche, 11198939001) was introduced into this vector using the same restriction sites. This final construct, pL1720, therefore contains the 5’hsp70-mCherry-’hsp70 and 5’pbeef1a-LucIAV-3’pbdhfr expression cassettes and 230p targeting sequences but no SM. This construct was linearized by digesting with SacII (Roche, 11117807001) before transfection. The linear DNA construct was introduced into GIMO-ANKA parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (Sigma, F7129-5G) in the drinking water of mice. Negative selection results in selection of parasites where the hdhfr-yfcu SM in the 230p locus is replaced by the mCherry reporter-cassette.
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
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Other details transgene
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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
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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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren