RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-669

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PBANKA_0719300; Gene model (P.falciparum): PF3D7_0417200; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase (dhfr/ts) Details mutation: The dhfr-ts gene of P. berghei replaced with the wild type dhfr-ts gene of P. vivax
Transgene	Transgene not Plasmodium: GFP (gfp-mu3) Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a) 3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts) Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype	No phenotype has been described

Last modified: 11 December 2011, 12:01

*RMgm-669

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 21981896
MR4 number
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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 507cl1 (RMgm-7)
Other information parent line	P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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The mutant parasite was generated by
Name PI/Researcher	V. Somsak; S. Kamchonwongpaisan
Name Group/Department	Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering
Name Institute	National Science and Technology Development Agency (NSTDA)
City	Pathumthani
Country	Thailand
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Name of the mutant parasite
RMgm number	RMgm-669
Principal name	PbPvDTcl4
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation In the mutant the dhfr-ts gene of P. berghei replaced with the wild type dhfr-ts gene of P. vivax. Wild-type and mutant P. vivax dhfr-ts genes were obtained from Ubolsree Leartsakulpanich, BIOTEC, Thailand (Leartsakulpanich U. et al., 2002, Mol. Biochem Parasitol. 119, 63-73). No details are provided on the sequence of (the primers of) these genes. Phenotype Blood stages showed a normal growth in mice and the drug-sensitivity of blood stages to pyrimethamine, chloroquine and artesunate were comparable to wild type P. berghei parasites (as shown in the standard 4-day suppressive test). Additional information Other mutants RMgm-670: a mutant in which the dhfr-ts gene of P. berghei is replaced with a pyrimethamine-resistant copy of the dhfr-ts gene of P. vivax.

Mutated: Mutant parasite with a mutated gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0719300

Gene Model P. falciparum ortholog

PF3D7_0417200

Gene product

bifunctional dihydrofolate reductase-thymidylate synthase

Gene product: Alternative name

dhfr/ts

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Details of the genetic modification

Short description of the mutation

The dhfr-ts gene of P. berghei replaced with the wild type dhfr-ts gene of P. vivax

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

hdhfr/yfcu

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

5-fluorocytosine (5-FC)

Additional remarks genetic modification

Plasmodium berghei transfection plasmids were constructed from plasmids pL0017 and pL0035 (MR4). In order to generate a P. berghei parasite line stably expressing wild-type PvDHFR-TS enzyme, transfection plasmids were modified to consist of two expression cassettes. The first cassette has the wild-type Pvdhfr-ts gene flanked by 5' and 3'UTR Pbdhfr-ts gene sequences. The second cassette is a drug selection cassette containing a fusion gene of positive (human dihydrofolate reductase; hdhfr) and negative (yeast yfcu) selectable markers under the control of 5' flanking region of P. berghei elongation factor 1α-a (Pbef1α-a) and 3'UTR sequence of Pbdhfr-ts gene.

The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and fusion gene of human dhfr (hdhfr) and yeast yfcu as positive and negative selectable markers respectively. After double homologous recombination, the drug selectable markers are excised by single homologous recombination via the Pbdhfr-ts 3'UTR repeated sequence elements, whilst retaining the wild-type Pvdhfr-ts gene.

Additional remarks selection procedure

For selection of P. berghei harbouring wild-type Pvdhfr-ts, a two-step drug selection procedure was performed. First, transgenic parasites with integrated wild-type Pvdhfr-ts were selected with pyrimethamine. Subsequently, negative selection was performed to select for parasites which had excised the positive-negative drug selection cassette in order to obtain parasites that have only wild-type Pvdhfr-ts gene integrated to the P. berghei genome.

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 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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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 name GFP (gfp-mu3)

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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

   51  tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta

  101  atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg

  151  aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg

  201  tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg

  251  catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga

  301  cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc

  351  cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga

  401  gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata

  451  ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg

  501  aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca

  551  tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt

  601  atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt

  651  ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg

  701  ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac

  751  agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat

  801  gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg

  851  ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg

  901  gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt

  951  aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca

 1001  acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg

 1051  cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct

 1101  gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa

 1151  tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct

 1201  tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc

 1251  acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg

 1301  gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat

 1351  ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac

 1401  tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta

 1451  agcattggta actgtcagac caagtttact catatatact ttagattgat

 1501  ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga

 1551  taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt

 1601  cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg

 1651  cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt

 1701  ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct

 1751  tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta

 1801  ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct

 1851  aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg

 1901  ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga

 1951  acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga

 2001  actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag

 2051  ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag

 2101  cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt

 2151  cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag

 2201  gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc

 2251  ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc

 2301  tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc

 2351  gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa

 2401  gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta

 2451  atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca

 2501  acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac

 2551  tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt

 2601  tcacacagga aacagctatg accatgatta cgccaagctt ccgcgggtat

 2651  atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct

 2701  ataaataaat ttacagattt attttttaat acaaaagata tagatatacc

 2751  agaaataaat gatcagttta aaggttttaa attctttatg acatcattta

 2801  taaatcatgg atcatatcca ctaacaatag aatgtggtgt aacaaatggt

 2851  ggaactagtt ataaaagagc aattatttta ttgcatgttc gaactgattt

 2901  aaaagataga ccagtttcat tttgtgattt tcgaaaagga gaattatata

 2951  attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa

 3001  tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc

 3051  aaaaaattgt tttacgcaag tatatgaaaa agggtatcta aatgacgcca

 3101  ataaaattaa tactaaaaat gttattaact attcatttga aaatccagaa

 3151  tatgcgctag ctggttytaa ttatacatta acaaaatcgt atcaatttga

 3201  atgtcattgt gtagataaag aaacagaaca aattgtaaaa acggttttag

 3251  tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg

 3301  gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg

 3351  catgcctgca gcccagctta attcttttcg agctctttat gcttaagttt

 3401  acaatttaat attcatactt taagtatttt ttgtagtatc ctagatattg

 3451  tgctttaaat gctcacccct caaagcacca gtaatatttt catccactga

 3501  aataccatta aattttcaaa aaaatactat gcatataatg ttatacatat

 3551  aaacataaaa cgccatgtaa atcaaaaaat atataaaaat atgtataaaa

 3601  ataaatatgc actaaatata agctaattat gcataaaaat taaagtgccc

 3651  tttattaact agtcgtaatt atttatattt ctatgttata aaaaaatcct

 3701  catataataa tataattaat atatgtaatg ttttttttat tttataattt

 3751  taatataaaa taatatgtaa attaattcaa aaaataaata taattgttgt

 3801  gaaacaaaaa acgtaatttt ttcatttgcc ttcaaaattt aaatttattt

 3851  taatatttcc taaaatatat atactttgtg tataaatata taaaaatata

 3901  tatttgctta taaataaata aaaaatttta taaaacatag ggggatccat

 3951  gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat

 4001  tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa

 4051  ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg

 4101  aaaactacct gttccatggc caacacttgt cactactttc ggttatggtg

 4151  ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc

 4201  aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa

 4251  agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata

 4301  cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga

 4351  aacattcttg gacacaaatt ggaatacaac tataactcac acaatgtata

 4401  catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta

 4451  gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa

 4501  aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct

 4551  gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca

 4601  tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat

 4651  gaactataca aataaatgga tcccgttttt cttacttata tatttatacc

 4701  aattgattgt atttataact gtaaaaatgt gtatgttgtg tgcatatttt

 4751  tttttgtgca tgcacatgca tgtaaatagc taaaattatg aacattttat

 4801  tttttgttca gaaaaaaaaa actttacaca cataaaatgg ctagtatgaa

 4851  tagccatatt ttatataaat taaatcctat gaatttatga ccatattaaa

 4901  aatttagata tttatggaac ataatatgtt tgaaacaata agacaaaatt

 4951  attattatta ttattatttt tactgttata attatgttgt ctcttcaatg

 5001  attcataaat agttggactt gatttttaaa atgtttataa tatgattagc

 5051  atagttaaat aaaaaaagtt gaaaaattaa aaaaaaacat ataaacacaa

 5101  atgatgtttt ttccttcaat ttcgggtacc gagctcgaat tctcttgagc

 5151  ccgttaatga aatagataca attcattcat gttatataca tctagaacat

 5201  aatctgaata tggttcaagt taaatgtcca aaaattataa aaagtgatga

 5251  tatttttgat ggtaatacca taatagacac caaggtaaca tcacgaagta

 5301  gtcaacaaaa taatttttat ttagaaaata cagatgttga accagaagaa

 5351  atagagaaat ataaaaatat agaatacata ccagaaaacg atgaagtaat

 5401  gcatctagac aaaaaagaaa agctagatga tatattacca ggtgttatca

 5451  tattagataa aaataaaatg ttcaaagaaa aaggacattt cacttttgtt

 5501  actccattaa ttgtagaaaa ggtattaata ttaaaaatat attgtgataa

 5551  tactaaaaca ataattaata atatgaaagg gaaaaaaggt attacagtaa

 5601  taaggatttc tcaaaataca acaaaaaata aattttatgg atgtgacttt

 5651  tcaggtaatt ctaaaaaaac attttactat tccaatgttt atgatttaga

 5701  aaaaaaaaat gagttttgtg aaatagaatt aaaagaaaat atagtagtta

 5751  gcttaaattg tccaactggt aaaattaatc caaaaaattg ttttagaaat

 5801  gtatatataa aaagtaatat gaatgaacaa acaaccgaaa atatagaaaa

 5851  tatatttaac gaaataaaag ttatagatgc agattatttt ataaataatt

 5901  catcaacctt tttgatgatt tccaaaatta caaaaaaaga gtttgatttt

 5951  tattgtacat gtgaagatta taaaaccaaa aatataggaa caatatatat

 6001  taaaaattat gaatatctag attcaaaacc taaatataaa aataaacaaa

 6051  tttcctatat agatgtagtt ccatacccgc ggggaaaggg cg"

Restriction sites to linearize plasmid KspI (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 gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.

Additional remarks selection procedure This reporter mutant expressing GFP 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	5'-cccaagcttccgcgggtatatggtaaagaacctactaacac
Additional information primer 1	primer L1345: 0.7kb 5'region of PBANKA_030600 (230p gene)
Sequence Primer 2	5'-cccaagcttgatgtgttttatttggatgtgc
Additional information primer 2	primer L1346: 0.7kb 5'region of PBANKA_030600 (230p gene)
Sequence Primer 3	5'-ccggaattctcttgagcccgttaatg
Additional information primer 3	primer L1347: 1kb 3'region of PBANKA_030600 (230p gene)
Sequence Primer 4	5'-tccccgcgggtatggaactacatctatatagg
Additional information primer 4	primer L1348: 1kb 3'region of PBANKA_030600 (230p gene)

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