RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-29

Malaria parasite	P. berghei
Genotype
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	Asexual bloodstage; Gametocyte/Gamete; Oocyst; Sporozoite; Liver stage;

Last modified: 6 January 2012, 17:49

*RMgm-29

Successful modification	The 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) : 16242190
MR4 number	MRA-868
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA cl15cy1
Other information parent line	A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/Researcher	B. Franke-Fayard; C.J. Janse; A.P. Waters
Name Group/Department	Leiden Malaria Research Group
Name Institute	Leiden University Medical Center
City	Leiden
Country	The Netherlands
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Name of the mutant parasite
RMgm number	RMgm-29
Principal name	676m1cl1
Alternative name	PbGFP-LUC(con); PbGFP-LUCcon; ANKA-GFP-Luccon; ANKA-GFP-Luc(con)
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	GFP-Luciferase expression in all stages; increase of GFP-Luciferase expression during development from ring form into mature trophozoite/immature schizont; decrease of GFP-Luciferase expression in mature schizonts. Strong GFP-Luciferase expression in female gametocytes
Gametocyte/Gamete	GFP-Luciferase expression in female and male gametocytes. Strong GFP-Luciferase expression in female gametocytes. Higher expression in females, compared to males.
Fertilization and ookinete	Not tested
Oocyst	Strong GFP-Luciferase expression in maturing oocysts
Sporozoite	(Weak) GFP-Luciferase expression in salivary gland sporozoites
Liver stage	Strong GFP-Luciferase expression in maturing liver stages
Additional remarks phenotype	Mutant/mutation This mutant expresses GFP-luciferase under the control of the constitutive eef1a promoter. Phenotype GFP-Luciferase is expressed in all life cycle stages and expression of the transgene has no influence on parasite viability and infectivity. Additional information This mutant has been used to analyse parasite distribution and parasite load in live animals (or in isolated organs) by real time in vivo imaging (by measurement of bioluminescence). In comparison with mutants expressing only GFP under the same promoter (eef1a promoter), the GFP-fluorescence intensity of this reporter mutant is lower as a result of the GFP-Luciferase fusion. Because all blood stages express luciferase, this reporter mutant is less optimal for the analysis of the dynamics of schizont sequestration. Mutants are available that express GFP-Luciferase under the control of the schizont specific ama-1 promoter (RMgm-30; RMgm-32). These mutants are more suitable for the analysis of schizont sequestration. This mutant does not contain a drug-selectable marker. This mutant, that has been selected by FACS sorting, can therefore be used for the introduction of additional genetic modifications using vectors that contain drug-selectable markers. A mutant (RMgm-28) expressing GFP-Luciferase under the control of the eefa promoter exists that contains a drug selectable marker (the pyrimethamine resistant tgdhfr gene). This mutant is used for in vivo antimalarial drug screening in mice using the IVDL assay (Franke-Fayard et al., 2008, Int. J. Parasitol. 38, 1651-62). The in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1a promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. See also the luciferase expressing mutant RMgm-32, which is used for in vitro antimalarial drug screening. This mutant is used for visualisation and quantitative analysis of liver stage by real time imaging ( Franke-Fayard et al., 2009, Plos One 18;4(11):e7881).

Transgene: Mutant parasite expressing a transgene

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Type and details of transgene

Is the transgene Plasmodium derived Transgene: not Plasmodium

Transgene name A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

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 parasite gfp (FACS)

Promoter of the selectable marker eef1a

Selection (positive) procedure FACS (flowsorting)

Selection (negative) procedure No

Additional remarks genetic modification The GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.

Additional remarks selection procedure This 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.

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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 product elongation factor 1-alpha

Gene product: Alternative name eef1a

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 product bifunctional dihydrofolate reductase-thymidylate synthase, putative

Gene product: Alternative name dhfr/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 / Insertion Replacement locus

Gene Model of Parasite PBANKA_0306000

Gene product 6-cysteine protein

Gene product: Alternative name 230p

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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