RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-933
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0808100; Gene model (P.falciparum): PF3D7_0317100; Gene product: 6-cysteine protein (B9)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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 Liver stage;
Last modified: 25 August 2016, 12:22
  *RMgm-933
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) : 24509910
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-29
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherT. Annoura; S.M. Khan; C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-933
Principal name1481cl4
Alternative nameΔb9-b
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
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNormal sporozoite production. Sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 PbΔb9 sporozoites none of the mice developed blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 PbΔb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period. Immunofluorescence analyses show that PbΔb9 parasites arrest early after invasion of hepatocytes. PbΔb9 sporozoites exhibit normal hepatocyte invasion, but at 24hpi most intra-cellular parasites had disappeared and only a few small parasites could be observed with a size that was similar to 5-10 hpi liver stages. Analysis of PbΔb9 parasites in the liver, using real-time in vivo imaging, confirmed the early growth-arrest observed in cultured hepatocytes.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of B9 (PBANKA_080810) and expresses the fusion protein GFP-Luciferase under the control of the constitutive eef1a promoter..

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. B9 transcripts are found in sporozoites. Evidence is presented for translational repression of b9 transcripts in P. berghei sporozoites. The B9 protein is found in (young) liver stages. For P. falciparum B9 evidence is presented for a location at the plasmalemma membrane of liver stages. P. falciparum and P. berghei mutants lacking expression of B9 abort development during development of young liver stages

Phenotype
Normal sporozoite production. Sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 PbΔb9 sporozoites none of the mice developed  blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 PbΔb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period. Immunofluorescence analyses show that PbΔb9 parasites arrest early after invasion of hepatocytes. PbΔb9 sporozoites exhibit normal hepatocyte invasion, but at 24hpi most intra-cellular parasites had disappeared and only a few small parasites could be observed with a size that was similar to 5-10 hpi liver stages. Analysis of PbΔb9 parasites in the liver, using real-time in vivo imaging, confirmed the early growth-arrest observed in cultured hepatocytes.
Combined, these analyses demonstrate that PbΔb9 has a critical role during early liver stage development, although a few liver stages can complete liver-stage development in the absence of B9. This phenotype is comparable to the phenotype of mutants lacking expression of the 6-Cys proteins P52 and P36.

Additional information
See also mutant RMgm-929 which expresses mCherry under the control of b9 regulatory sequences. No mCherry fluorescence signals were detected in blood-stages, oocysts and sporozoites, despite the presence of b9 transcripts in sporozoites. Strong fluorescence signals were detected in hepatocyte-culture 5 hours after the addition of mCherryb9 sporozoites. The non-fluorescent sporozoites indicate that b9 transcripts are translationally repressed and that the B9 protein is generated after a developmental switch to intrahepatic development. mCherry expression was observed both in intra-hepatic stages and in extracellular sporozoites that had been activated and  started to ‘round up’. Five hours post infection (hpi) fluorescence signals decreased; at 15hpi weak signals were detected in all parasites and no fluorescence was detected at 24hpi and 32hpi.

Supporting Figure S5
Generation of P. berghei mutants lacking expression of B9 (PbΔb9)

A. Schematic representation of the construct pL1439 targeting b9 for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the hdhfr::yfcu selection marker (SM). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). B. Schematic representation of the construct pL1499 targeting b9 for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the hdhfr selection marker (SM). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). The pL1499 construct was generated by an adapted ‘Anchor-tagging’ PCR-based method employing a 2-step PCR reaction. In the first PCR step two-flanking fragments of b9 were amplified from genomic DNA with the primers 4667/4557 (5’) and 4558/4668 (3’). Both primer 4557 and 4558 have 5’-terminal extensions homologues to the hdhfr selectable marker cassette (SM) obtained from plasmid pL0040 by digestion with restriction enzymes XhoI and NotI. Primers 4667 and 4668 have 5’-terminal overhang with an anchor-tag suitable for the second PCR step. In this step the fragments were annealed to either side of the SM with anchor-tag primers 4661/4662, resulting in the second PCR fragment. To remove the ‘anchor’, the second PCR fragment was digested with Asp718 and ScaI as primer 4667 contained an Asp718 restriction enzyme site and 4668 contained a ScaI site. C. Diagnostic PCRs (left) and Southern analyses of separated chromosomes (right) confirm the correct integration of the constructs in Δb9-a and Δb9-b. Primer pairs and amplicon sizes are shown in A. Δb9-a: selectable marker (M, primers 4698/4699); 5’-integration event (5’; primers 4288/L1858); 3’-interation event (3’; primers 4239/4289); ORF (O; primers 4737/4738). Δb9-b: selectable marker (M, primers L307C/3187); 5’-integration event (5’; primers 4288/4470); 3’-interation event (3’; primers 4471/4289); ORF (O; primers 4737/44738). For Southern analyses, pulsed field gel-separated chromosomes were hybridized with a 3’UTR pbdhfr probe. Mutant Δb9-a has been generated in the reference P. berghei ANKA line cl15cy1. Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 8 and the endogenous dhfr-ts gene located on chromosome 7. Mutant Δb9-b (right hand side) mutant has been generated in the reference P. berghei ANKA PbGFP-Luccon which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 8, the reporter GFP-Luccon construct on chromosome 3, and the endogenous dhfr-ts gene located on chromosome 7.

Other mutants
RMgm-932: mutant lacking expression of B9 in the WT background. B9 was disrupted by double cross-over homologous recombination using a plasmid DNA construct.
RMgm-934: mutant lacking expression of B9. The mutant does not contain a selectable marker.
RMgm-935: Triple knock-out mutant lacking expression of B9, P52 and P36
RMgm-936: P.yoelii 17XNL mutant lacking expression of B9 in the GFP-Luciferase expressing background.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0808100
Gene Model P. falciparum ortholog PF3D7_0317100
Gene product6-cysteine protein
Gene product: Alternative nameB9
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct 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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GAACTCGTACTCCTTGGTGACGGGTACCTAAATAACATGATGAAACGTCACTTTATAGAA
TAAGTCTTCAATATAAAACAAAAAAATGCATGTATTCAAAGGTCTCTAAAACAATGGAAA
TAAATAAATTTTTTTTCTGTCTTTTGTGCATGCAATTATTTTTCACTTTTATTAATCAGG
CTTATTTTGACAATAAAATATTCTCCAAATTGTTGGTTTTATACATTTGATATATACTAA
TCAATATTAAATATTATAAATAAATATATACACACATCAGTATATGAATATAAATAAATA
ACCTTCGTTAATTGCAAAAAGGTTGTCTTTTTCTCATATGTGTATGAGCATTTTTTTTTT
TACAATTAATAATTTGGCTATACAAAATTTAACAAAGCATGCATAAATGGTTGTACATTT
TTATTACATCTTTTTATTTGAAAAATAAACATGTTTCGTAAAATATAGTTTTAAAAATTA
CAACTGATAATTAGACACAACATCACAAATTTGTCTTTATAATATATTGATAGAAAAATC
AAAATATGAGCATAAATGTGAGCATGGATATGAGAAAAAGTTTTCTTATTTATTAAAAAA
ACAGTGAAAAAAAAAATACATATAATATATATTCAAAATTATATTACCCATTATATATGC
ACGAAGGATACATTAAAGTTTGGGCGCAGATAAAATAAAAAAAAATATCATACAATTTGA
CAATATAATAAAAAAAAAAAGTAAATTTTCAGAATGTGTTTAAATATTTTGTTAGTGTAT
TTATAGAGGGTAGAAGTAATGCATAGGAGGTCGACGATGCTTGTAGATGAGTTAAGCTTA
ATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTT
TTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTT
CATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATAT
AAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGC
ACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATT
ATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATG
TTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATA
TAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTT
TAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTA
TAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCC
CAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAATGAATTC
AGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATT
ATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATT
AATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGTGCACATTTTCTTTCCAGA
AGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATG
GTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTT
AAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATT
GATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAG
GAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAATGTTCGTTTTTC
TTATTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGT
GCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATT
TTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTT
TATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACA
TAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAA
TTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAAT
ATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAA
TGATGTTTTTTCCTTCAATTTCGGATCCACTAGGATATGCTTGAAATTCCTAGACATATT
ACAAATAATACTTCTTCTACACCCAATCTAGGTTCAAGAATAAACAATATAAATCGTAAA
TTATCAAAAAGAAATAATTATTTACCTAATAATATGAATGACGAAGATACAAAACATCAT
TATGATACTGATTCAGTTACTCAATTCGAATTTAATTTTGTCCAGGTTTTTATAGGTATA
ACTATATTCCATATTTTGATATGCTTTTTATAGAAATTGTAATAATATAAAAGAATGAGA
AATTCGAAAAAACAAATATATCTAGACATGAATAAAATGCTATTTCTTCCTCTAACAATT
ATGATACCAGTTTCCTATCTACCCTTTACATTTTAATCACCATTTTAAAATCCTTTAAAT
ATGTAAAAAATAAAACACCTCCCAAATAACAATGAGAATGCACACAGTAATTCAATAAAA
TTATTTAATTCCGTTGATCCATTCGCTTCTTTCAACCTTCGTTGTTTTCTCTTAAACGCG
ACATTATCTGCATACAATATATGCATTCATTTTTTGGTTATTATTTATGTATTTATTTAT
TTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTATTTCA
TTATTAACAATTTTGATTAAAACAATAATTGATAAAAAATAAAAGTATAATAAAATCGAT
ACACATATATATATTGAGTAAATTTTGTGACATGTAAAAGTATGCATTGCGAATATGAGT
ATTCTGTTCGTCGTGCTTACAAATAACACGATGTATGCAACCACAAGCGAGTACTGCTGA
GTGTCAATGACCAACCT
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe locus was successfully targeted using a linear construct that was generated using a 2-step, anchor tagging PCR method (for primer details see below).

The 5’- and 3’ targeting regions of the gene were PCR amplified from genomic DNA using primer pairs 1&2 and 3&4. Primers 2 and 3 have 5’-terminal extensions homologues to the hDHFR selectable marker cassette. Primers 1 and 4 both have a 5’-terminal overhang with an anchor-tag which serves as a primer site in the 2nd PCR reaction.

The target fragments from the first PCR reaction were annealed to either side of the selectable marker cassette by PCR with anchor-tag primers 5 and 6, resulting in the 2nd PCR product. The hDHFR selectable marker cassette used in this reaction was digested from pL0040 using restriction enzymes XhoI and NotI. pL0040 is available from The Leiden Malaria Research Group.

To remove the anchor-tags from the final KO construct, and to eliminate contaminating pL0040, the 2nd PCR product was digested with Asp718/ScaI and DpnI respectively. DpnI only cuts methylated plasmid DNA but not the PCR product.

For a more detailed understanding of the 2-step anchor tagging PCR method, please see the following publications:

J.W. Lin, S.M. Khan et al
A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites
PLoS One. 2011;6(12)

T. Annoura et al
Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates
Vaccine. 2012 Mar 30;30(16):2662-70
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 1GAACTCGTACTCCTTGGTGACGGGTACCTAAATAACATGATGAAACGTCAC
Additional information primer 14667 (Asp718); ∆b9 5' target F
Sequence Primer 2CATCTACAAGCATCGTCGACCTCTCTATGCATTACTTCTACCCTC
Additional information primer 24557; ∆b9 5' target R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGGATATGCTTGAAATTCCTAGAC
Additional information primer 34558; ∆b9 3' target F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCAGTACTCGCTTGTGGTTGCATACATC
Additional information primer 44668 (ScaI); ∆b9 3' target R
Sequence Primer 5GAACTCGTACTCCTTGGTGACG
Additional information primer 54661; Anhor tag for second pcr
Sequence Primer 6AGGTTGGTCATTGACACTCAGC
Additional information primer 64662; Anhor tag for second pcr

  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
61 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
121 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
181 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
241 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
301 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
361 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
421 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
541 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
661 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
721 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
781 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
841 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
901 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
961 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
1021 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
1081 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
1141 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
1261 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
1321 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
1381 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
1441 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
1501 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
1561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
1621 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
1681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
1741 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
1861 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
1921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
1981 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
2041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
2101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
2161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
2221 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
2281 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
2341 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
2401 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
2461 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
2521 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
2581 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
2641 ccgcgggtat atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct
2701 ataaataaat ttacagattt attttttaat acaaaagata tagatatacc agaaataaat
2761 gatcagttta aaggttttaa attctttatg acatcattta taaatcatgg atcatatcca
2821 ctaacaatag aatgtggtgt aacaaatggt ggaactagtt ataaaagagc aattatttta
2881 ttgcatgttc gaactgattt aaaagataga ccagtttcat tttgtgattt tcgaaaagga
2941 gaattatata attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa
3001 tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc aaaaaattgt
3061 tttacgcaag tatatgaaaa agggtatcta aatgacgcca ataaaattaa tactaaaaat
3121 gttattaact attcatttga aaatccagaa tatgcgctag ctggttytaa ttatacatta
3181 acaaaatcgt atcaatttga atgtcattgt gtagataaag aaacagaaca aattgtaaaa
3241 acggttttag tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg
3301 gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg catgcctgca
3361 gcccagctta attcttttcg agctctttat gcttaagttt acaatttaat attcatactt
3421 taagtatttt ttgtagtatc ctagatattg tgctttaaat gctcacccct caaagcacca
3481 gtaatatttt catccactga aataccatta aattttcaaa aaaatactat gcatataatg
3541 ttatacatat aaacataaaa cgccatgtaa atcaaaaaat atataaaaat atgtataaaa
3601 ataaatatgc actaaatata agctaattat gcataaaaat taaagtgccc tttattaact
3661 agtcgtaatt atttatattt ctatgttata aaaaaatcct catataataa tataattaat
3721 atatgtaatg ttttttttat tttataattt taatataaaa taatatgtaa attaattcaa
3781 aaaataaata taattgttgt gaaacaaaaa acgtaatttt ttcatttgcc ttcaaaattt
3841 aaatttattt taatatttcc taaaatatat atactttgtg tataaatata taaaaatata
3901 tatttgctta taaataaata aaaaatttta taaaacatag ggggatctat gagtaaagga
3961 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
4021 cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt
4081 aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc
4141 ggttatggtg ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc
4201 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg
4261 aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag
4321 ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac
4381 tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac
4441 ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
4501 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa
4561 tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta
4621 acagctgctg ggattacaca tggcatggat gaactataca aagggatcct ggctagccag
4681 tcgacctgca ggcatgcaag cttgcggccg atccaaatgg aagacgccaa aaacataaag
4741 aaaggcccgg cgccattcta tccgctggaa gatggaaccg ctggagagca actgcataag
4801 gctatgaaga gatacgccct ggttcctgga acaattgctt ttacagatgc acatatcgag
4861 gtgaacatca cgtacgcgga atacttcgaa atgtccgttc ggttggcaga agctatgaaa
4921 cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc
4981 tttatgccgg tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc gaacgacatt
5041 tataatgaac gtgaattgct caacagtatg aacatttcgc agcctaccgt agtgtttgtt
5101 tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa aattaccaat aatccagaaa
5161 attattatca tggattctaa aacggattac cagggatttc agtcgatgta cacgttcgtc
5221 acatctcatc tacctcccgg ttttaatgaa tacgattttg taccagagtc ctttgatcgt
5281 gacaaaacaa ttgcactgat aatgaattcc tctggatcta ctgggttacc taagggtgtg
5341 gcccttccgc atagaactgc ctgcgtcaga ttctcgcatg ccagagatcc tatttttggc
5401 aatcaaatca ttccggatac tgcgatttta agtgttgttc cattccatca cggttttgga
5461 atgtttacta cactcggata tttgatatgt ggatttcgag tcgtcttaat gtatagattt
5521 gaagaagagc tgtttttacg atcccttcag gattacaaaa ttcaaagtgc gttgctagta
5581 ccaaccctat tttcattctt cgccaaaagc actctgattg acaaatacga tttatctaat
5641 ttacacgaaa ttgcttctgg gggcgcacct ctttcgaaag aagtcgggga agcggttgca
5701 aaacgcttcc atcttccagg gatacgacaa ggatatgggc tcactgagac tacatcagct
5761 attctgatta cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt
5821 tttgaagcga aggttgtgga tctggatacc gggaaaacgc tgggcgttaa tcagagaggc
5881 gaattatgtg tcagaggacc tatgattatg tccggttatg taaacaatcc ggaagcgacc
5941 aacgccttga ttgacaagga tggatggcta cattctggag acatagctta ctgggacgaa
6001 gacgaacact tcttcatagt tgaccgcttg aagtctttaa ttaaatacaa aggatatcag
6061 gtggcccccg ctgaattgga atcgatattg ttacaacacc ccaacatctt cgacgcgggc
6121 gtggcaggtc ttcccgacga tgacgccggt gaacttcccg ccgccgttgt tgttttggag
6181 cacggaaaga cgatgacgga aaaagagatc gtggattacg tcgccagtca agtaacaacc
6241 gcgaaaaagt tgcgcggagg agttgtgttt gtggacgaag taccgaaagg tcttaccgga
6301 aaactcgacg caagaaaaat cagagagatc ctcataaagg ccaagaaggg cggaaagatc
6361 gccgtgtaat tctagaagat cccgtttttc ttacttatat atttatacca attgattgta
6421 tttataactg taaaaatgtg tatgttgtgt gcatattttt ttttgtgcat gcacatgcat
6481 gtaaatagct aaaattatga acattttatt ttttgttcag aaaaaaaaaa ctttacacac
6541 ataaaatggc tagtatgaat agccatattt tatataaatt aaatcctatg aatttatgac
6601 catattaaaa atttagatat ttatggaaca taatatgttt gaaacaataa gacaaaatta
6661 ttattattat tattattttt actgttataa ttatgttgtc tcttcaatga ttcataaata
6721 gttggacttg atttttaaaa tgtttataat atgattagca tagttaaata aaaaaagttg
6781 aaaaattaaa aaaaaacata taaacacaaa tgatgttttt tccttcaatt tcgggtaccg
6841 agctcgaatt ctcttgagcc cgttaatgaa atagatacaa ttcattcatg ttatatacat
6901 ctagaacata atctgaatat ggttcaagtt aaatgtccaa aaattataaa aagtgatgat
6961 atttttgatg gtaataccat aatagacacc aaggtaacat cacgaagtag tcaacaaaat
7021 aatttttatt tagaaaatac agatgttgaa ccagaagaaa tagagaaata taaaaatata
7081 gaatacatac cagaaaacga tgaagtaatg catctagaca aaaaagaaaa gctagatgat
7141 atattaccag gtgttatcat attagataaa aataaaatgt tcaaagaaaa aggacatttc
7201 acttttgtta ctccattaat tgtagaaaag gtattaatat taaaaatata ttgtgataat
7261 actaaaacaa taattaataa tatgaaaggg aaaaaaggta ttacagtaat aaggatttct
7321 caaaatacaa caaaaaataa attttatgga tgtgactttt caggtaattc taaaaaaaca
7381 ttttactatt ccaatgttta tgatttagaa aaaaaaaatg agttttgtga aatagaatta
7441 aaagaaaata tagtagttag cttaaattgt ccaactggta aaattaatcc aaaaaattgt
7501 tttagaaatg tatatataaa aagtaatatg aatgaacaaa caaccgaaaa tatagaaaat
7561 atatttaacg aaataaaagt tatagatgca gattatttta taaataattc atcaaccttt
7621 ttgatgattt ccaaaattac aaaaaaagag tttgattttt attgtacatg tgaagattat
7681 aaaaccaaaa atataggaac aatatatatt aaaaattatg aatatctaga ttcaaaacct
7741 aaatataaaa ataaacaaat ttcctatata gatgtagttc catacccgcg gggaaagggc
7801 g
Restriction sites to linearize plasmid ApaI, 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 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_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