Summary

RMgm-32
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1
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; Oocyst; Sporozoite; Liver stage;
Last modified: 8 October 2015, 11:38
  *RMgm-32
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20019192
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherB. Franke-Fayard; C.J. Janse; A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-32
Principal name1037cl1
Alternative name1037m1f1mocl1; 1037m1f1m1cl1; PbGFP-Luc(ama1); PbGFP-Lucama1; wt-GFP-Luc(schiz); ANKA-GFP-Luc(schiz)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGFP-Luciferase expression in mature schizonts and young ring forms.
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot tested
OocystGFP-Luciferase expression in maturing (late-stage) oocysts
SporozoiteGFP-Luciferase expression in salivary gland sporozoites
Liver stageGFP-Luciferase expression in maturing liver stages (liver merozoites)
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP-luciferase under the control of the schizont-specific ama-1 promoter.

Phenotype
GFP-Luciferase is expressed in maturing schizonts and is still present in young ringforms (0-4 hours after invasion), carried over from the schizonts. No expression of GFP-luciferase in gametocytes/gametes.

Unpublished observations: GFP-Luciferase expression in maturing (late-stage) oocysts. (Relatively strong) GFP-Luciferase expression in salivary gland sporozoites and GFP-Luciferase expression in maturing liver stages (liver merozoites).

Additional information
This mutant has been used to analyse CD36-mediated sequestration of the schizont stage of P. berghei in live animals (or in isolated organs) by real time in vivo imaging (by measurement of bioluminescence) (Franke-Fayard et al., 2005, Proc Natl Sci USA, 102, 11468-73). These studies indicate that the schizont of P. berghei mainly sequester/accumulate in small capillaries of adipose tissue, lungs and the spleen. 

In comparison with mutants expressing only GFP under the same promoter (ama1 promoter), the GFP-fluorescence intensity of this reporter mutant is lower as a result of the GFP-Luciferase fusion.

This mutant does not contain a drug-selectable marker. This mutant, which has been selected by FACS sorting (based on GFP-fluorescence), can therefore be used for the introduction of additional genetic modifications using vectors that contain drug-selectable markers.

This mutant is used for in vitro antimalarial drug screening in mice using the ITDL assay (Franke-Fayard et al., 2008, Int. J. Parasitol. 38, 1651-62). The in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC(50) values comparable to those obtained with standard in vitro drug-susceptibility assays. See also the luciferase expressing mutant RMgm-29 which is used for in vivo antimalarial drug screening.

Other mutants
Mutant RMgm-30 expressing GFP-Luciferase under the control of the ama-1 promoter containing a drug selectable marker (the pyrimethamine resistant tg-dhfr gene).


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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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1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
121 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
241 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
361 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
721 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
841 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
961 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
1081 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
1321 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
1441 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
1561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
1681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
1921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
2041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
2161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
2281 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
2401 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
2521 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
2641 ccgcgggtat atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct ataaataaat ttacagattt attttttaat acaaaagata tagatatacc agaaataaat
2761 gatcagttta aaggttttaa attctttatg acatcattta taaatcatgg atcatatcca ctaacaatag aatgtggtgt aacaaatggt ggaactagtt ataaaagagc aattatttta
2881 ttgcatgttc gaactgattt aaaagataga ccagtttcat tttgtgattt tcgaaaagga gaattatata attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa
3001 tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc aaaaaattgt tttacgcaag tatatgaaaa agggtatcta aatgacgcca ataaaattaa tactaaaaat
3121 gttattaact attcatttga aaatccagaa tatgcgctag ctggttytaa ttatacatta acaaaatcgt atcaatttga atgtcattgt gtagataaag aaacagaaca aattgtaaaa
3241 acggttttag tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg catgcctgca
3361 ggaattcgat atcgaattcc catggaacca aaataacata ttaatgaatt ttcttttatt gtgaaaatgt tcttatttgt tttaaaaaat caaagtaaat ttaattcctt taattgtgct
3481 aaatattgtg tttgtataac tataatgaca tttcctatgc ttttttaata ttatatataa ttttttgtgt tttttcaaaa aaaacgtgta tttgttttta tttgagtgta caatttgcat
3601 agtgagttta aagtgtttga aaattattta atttataatt ttcataaaac gtttaatcat ttatcaaaat tgcggttgca atatagacag aaaatattaa cttaattttc attgttttgt
3721 aaaacatttt cattattttt tttaacaaaa ttaaagtaaa tgagatggta aattatttta attataacca tttgtttaat tttttttatt ttattactat tatttttttt cttttgtgct
3841 gcgcaattaa aaagagaatt aagggtgcac caatatattt aagcaatctt ttgcgacaca caagaaaaaa tataacatca acatatttaa aataatggaa attttttaat aatttattct
3961 atatatattt aatatatgta aaaaaactaa tattgtaata taattaaata aaacttgaaa ttgatataat atgtatatat attgcatgtt tagctttaaa taatatttaa gtattgattt
4081 tctttacaca caaaaacatt tgccttttta tgtgaggatt attgtttctt tatttatatt gctatgaatt tattcttagt ataaatttat tttttttcgg ctttgaaaat aattttatat
4201 atataaaaag aaaaaatatg gaaaataaaa ttcataaatc atatttgaca catttcaatt tttttttctt atttcacagc tagccatata ataatatata tgtgtctatg ggttcaaata
4321 gcttaatttc aatgtgcgtg attttttttt ataaaaaaac taattttatg tttggttatg catgcaactg ttataaaata aaaattgtaa aatttgtttt tttgtacaca tatttgtact
4441 ttttttaatg cacatgaaaa aagaaaggaa aaaaaagggg gaaaaaaaag gaaaaaaaaa gggaaaaaag ggaaaaaacg tacatctacg cattgttatt tagcaataaa atagagaaca
4561 aatctataaa tatataatgt gtacaacaaa ttaatttaat ttatattttt actgtaattt cataataatt ttcattttta cattttaatg tttttttaat tggaatataa ttcaaaatga
4681 ttttgacaca ttttacttat agaagaaggc tatataatta tataagtgtg tatatataag tattgtatgg taattgtttt ataaattaaa tttttaaaaa acattattct attttataat
4801 ttgtaaatta aaaatattaa taaaataaaa cgatataaaa ggatccatga gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
4921 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccatggcca acacttgtca ctactttcgg
5041 ttatggtgtt caatgctttg cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag atgacgggaa
5161 ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg aatacaacta
5281 taactcacac aatgtataca tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg aagatggaag cgttcaacta gcagaccatt atcaacaaaa
5401 tactccaatt ggcgatggcc ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg agtttgtaac
5521 agctgctggg attacacatg gcatggatga actatacaaa gggatcctgg ctagccagtc gacctgcagg catgcaagct tgcggccgat ccaaatggaa gacgccaaaa acataaagaa
5641 aggcccggcg ccattctatc cgctggaaga tggaaccgct ggagagcaac tgcataaggc tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac atatcgaggt
5761 gaacatcacg tacgcggaat acttcgaaat gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc ttcaattctt
5881 tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa catttcgcag cctaccgtag tgtttgtttc
6001 caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaaa ttaccaataa tccagaaaat tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca cgttcgtcac
6121 atctcatcta cctcccggtt ttaatgaata cgattttgta ccagagtcct ttgatcgtga caaaacaatt gcactgataa tgaattcctc tggatctact gggttaccta agggtgtggc
6241 ccttccgcat agaactgcct gcgtcagatt ctcgcatgcc agagatccta tttttggcaa tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg gttttggaat
6361 gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt atagatttga agaagagctg tttttacgat cccttcagga ttacaaaatt caaagtgcgt tgctagtacc
6481 aaccctattt tcattcttcg ccaaaagcac tctgattgac aaatacgatt tatctaattt acacgaaatt gcttctgggg gcgcacctct ttcgaaagaa gtcggggaag cggttgcaaa
6601 acgcttccat cttccaggga tacgacaagg atatgggctc actgagacta catcagctat tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg ttccattttt
6721 tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc agagaggcga attatgtgtc agaggaccta tgattatgtc cggttatgta aacaatccgg aagcgaccaa
6841 cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact gggacgaaga cgaacacttc ttcatagttg accgcttgaa gtctttaatt aaatacaaag gatatcaggt
6961 ggcccccgct gaattggaat cgatattgtt acaacacccc aacatcttcg acgcgggcgt ggcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg ttttggagca
7081 cggaaagacg atgacggaaa aagagatcgt ggattacgtc gccagtcaag taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa
7201 actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg gaaagatcgc cgtgtaattc tagaagatcc cgtttttctt acttatatat ttataccaat tgattgtatt
7321 tataactgta aaaatgtgta tgttgtgtgc atattttttt ttgtgcatgc acatgcatgt aaatagctaa aattatgaac attttatttt ttgttcagaa aaaaaaaact ttacacacat
7441 aaaatggcta gtatgaatag ccatatttta tataaattaa atcctatgaa tttatgacca tattaaaaat ttagatattt atggaacata atatgtttga aacaataaga caaaattatt
7561 attattatta ttatttttac tgttataatt atgttgtctc ttcaatgatt cataaatagt tggacttgat ttttaaaatg tttataatat gattagcata gttaaataaa aaaagttgaa
7681 aaattaaaaa aaaacatata aacacaaatg atgttttttc cttcaatttc gggtaccgag ctcgaattct cttgagcccg ttaatgaaat agatacaatt cattcatgtt atatacatct
7801 agaacataat ctgaatatgg ttcaagttaa atgtccaaaa attataaaaa gtgatgatat ttttgatggt aataccataa tagacaccaa ggtaacatca cgaagtagtc aacaaaataa
7921 tttttattta gaaaatacag atgttgaacc agaagaaata gagaaatata aaaatataga atacatacca gaaaacgatg aagtaatgca tctagacaaa aaagaaaagc tagatgatat
8041 attaccaggt gttatcatat tagataaaaa taaaatgttc aaagaaaaag gacatttcac ttttgttact ccattaattg tagaaaaggt attaatatta aaaatatatt gtgataatac
8161 taaaacaata attaataata tgaaagggaa aaaaggtatt acagtaataa ggatttctca aaatacaaca aaaaataaat tttatggatg tgacttttca ggtaattcta aaaaaacatt
8281 ttactattcc aatgtttatg atttagaaaa aaaaaatgag ttttgtgaaa tagaattaaa agaaaatata gtagttagct taaattgtcc aactggtaaa attaatccaa aaaattgttt
8401 tagaaatgta tatataaaaa gtaatatgaa tgaacaaaca accgaaaata tagaaaatat atttaacgaa ataaaagtta tagatgcaga ttattttata aataattcat caaccttttt
8521 gatgatttcc aaaattacaa aaaaagagtt tgatttttat tgtacatgtg aagattataa aaccaaaaat ataggaacaa tatatattaa aaattatgaa tatctagatt caaaacctaa
8641 atataaaaat aaacaaattt cctatataga tgtagttcca tacccgcggg gaaagggcg
Restriction sites to linearize plasmid KspI (SacII)
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markerama-1
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative name
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