RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1339
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4, up-regulated in infective sporozoites; ETRAMP10.3)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (UIS4, up-regulated in infective sporozoites; ETRAMP10.3)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Sporozoite; Liver stage;
Last modified: 25 October 2015, 14:12
  *RMgm-1339
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) : 26271010
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 GIMO-PbANKA (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 line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
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The mutant parasite was generated by
Name PI/ResearcherSalman AM; Khan SM; Janse CJ
Name Group/DepartmentDepartment of Parasitology
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-1339
Principal name2204cl1 (or cl5)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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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
SporozoiteNo expression in oocysts.(Strong) mCherry expression in salivary gland sporozoites
Liver stagemCherry expression in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses mCherry under control of the UIS4 promoter.

The mutant does not contain a drug-selectable marker. The mutant has been generated and selected using the GIMO transfection method ('gene insertion/marker out') of transfection of GIMO mother lines that contain the hdhfr::yfcu selectable marker into the silent 230p locus.

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

Protein (function)

Phenotype
mCherry expression in salivary gland sporozoites and developing liver stages. No expression in oocysts.

Additional information
Mutants cloned in Leiden (cl1,2) and in Baltimore (cl5)


A. Schematic representation showing the introduction of the mCherry-expression cassette into the neutral (230p) locus of the P. berghei GIMOPbANKA mother line by GIMO-transfection. Construct pL1937 contains the 5’uis4-mCherry-3’uis4 cassette and 230p targeting sequences. This construct is integrated into the modified P. berghei 230p locus that contains the hdhfr::yfcu selectable marker cassette (hatched box) by double cross-over homologous recombination at the target regions (grey boxes). Negative selection with 5-FC selects for the transgenic parasite line (line 2204) that has the mCherry-expression cassette introduced into the 230p locus and the hdhfr::yfcu selectable marker cassette removed.

B. mCherry expression in mosquito stages of the transgenic parasite lines, mCherry@Pbuis4 (2204) and mCherry@Pbef1 (a parasite line expressing mCherry under the control of the constitutive eef1 promoter) in oocysts (17 days) in mosquito midguts (MG) and sporozoites (23 days) in salivary glands (SG).
In mCherry@Pbuis4 parasites mCherry-expression is detected only in SG sporozoites. In mCherry@Pbef1parasites mCherry is expressed both in oocysts and sporozoites. Bright field (BF).

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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GGCCGCATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGC
TTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGC
GAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCC
CTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTG
AAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGG
GAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTG
CAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGC
CCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAG
GACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTAC
GACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTAC
AACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAG
TACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATCT
GATATCTATAATTCATTATGAGTAGTGTAATTCAGAAAGAGAACAAATTGTTTTATGATA
AATATATTTATAAAATAAAAAGTAAGAGAAAAGGAAAAATAATGTTATAGAGAAAAAAAG
GGAAAACGTTATAAAAAAATAGCATCTTAACTATTATTATTTTACTTTATGTGTGTATAA
AACTTTTACAATATATTTTTTTATTTTGAACGTATATAAAAATATTTCCTTTAATTTGTA
GTGTGCAAATAATTTATAAAAATAATTTTTTTAAATATAAATATATTGTCTCGAAAAGAA
ATGAAAATGGATTCTAGCAAATAAACAAGTCTGGCTTCGAAAAGAAATATAAGGCAAAAA
TTATATATAATATATTTGAAAATTATATTTGTGAGAGTGTGTATTATTTTTGTTTGAAAG
ATAATTTTATGGGTTTTTGAGAGTGATTTTTAGTGTTTTATTTGTTTTTTTTTAATAATT
TGTAAGTTTTGTCAATGTAGAGAGAATTATACTTCATAACGTATTATTTGAACGATTAAA
AAATATTTTTTTTAAACAGTGAAATATAAATATGAATGGAAGCAGCCGTATATGCAAACT
TTTTTACATATGTTGTATGCATAACATGCGTTTTTATTTTTTACGAAGTATACATAATTC
CCTTTAAAATATAACTTTATATAATTTGTTTTTATTGTAATGGATATAGTATTGTATAAA
TTTTATATTTATTTCCTAAAGACTTTTTCATAAAAATTGTTGGTGTTCCAGATATATTAT
TTTTAAAAGTCAATATATCAAGTTGTTATATTGTCTTAGAGTTCAAAAGTTTGTCCTAAT
TGAATTTTTTATAACTTTATAATATTTATTGTTTTACTTTGGACCATTAAGTATATATAA
GTAGTAACTTATTAAAATAATAAATTATATGTATCATAATTCATGGCTTTATAGATTCTG
CATTAACCTTAAATATGAAAAACACAAAAATGATGACAAGCATTAAAGCGAAAGGTACCG
AGCTCGAATTCTCTTGAGCCCGTTAATGAAATAGATACAATTCATTCATGTTATATACAT
CTAGAACATAATCTGAATATGGTTCAAGTTAAATGTCCAAAAATTATAAAAAGTGATGAT
ATTTTTGATGGTAATACCATAATAGACACCAAGGTAACATCACGAAGTAGTCAACAAAAT
AATTTTTATTTAGAAAATACAGATGTTGAACCAGAAGAAATAGAGAAATATAAAAATATA
GAATACATACCAGAAAACGATGAAGTAATGCATCTAGACAAAAAAGAAAAGCTAGATGAT
ATATTACCAGGTGTTATCATATTAGATAAAAATAAAATGTTCAAAGAAAAAGGACATTTC
ACTTTTGTTACTCCATTAATTGTAGAAAAGGTATTAATATTAAAAATATATTGTGATAAT
ACTAAAACAATAATTAATAATATGAAAGGGAAAAAAGGTATTACAGTAATAAGGATTTCT
CAAAATACAACAAAAAATAAATTTTATGGATGTGACTTTTCAGGTAATTCTAAAAAAACA
TTTTACTATTCCAATGTTTATGATTTAGAAAAAAAAAATGAGTTTTGTGAAATAGAATTA
AAAGAAAATATAGTAGTTAGCTTAAATTGTCCAACTGGTAAAATTAATCCAAAAAATTGT
TTTAGAAATGTATATATAAAAAGTAATATGAATGAACAAACAACCGAAAATATAGAAAAT
ATATTTAACGAAATAAAAGTTATAGATGCAGATTATTTTATAAATAATTCATCAACCTTT
TTGATGATTTCCAAAATTACAAAAAAAGAGTTTGATTTTTATTGTACATGTGAAGATTAT
AAAACCAAAAATATAGGAACAATATATATTAAAAATTATGAATATCTAGATTCAAAACCT
AAATATAAAAATAAACAAATTTCCTATATAGATGTAGTTCCATACCCGCGGGGAAAGGGC
GAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT
TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCAC
CGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTT
TCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTG
CTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTG
ACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTG
CATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGAT
ACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCAC
TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT
GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAG
TATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCC
TGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGC
ACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCC
CGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATC
CCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT
GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT
ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT
CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCT
TGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT
GCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC
TTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCG
CTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC
TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTA
CACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGC
CTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA
TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT
GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGAT
CAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAA
ACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAA
GGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTT
AGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTT
ACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA
GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT
GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCAC
GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGA
GCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG
CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAA
AAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACAT
GTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC
TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGA
AGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTG
GCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTA
GCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGG
AATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCT
TCCGCGGGTATATGGTAAAGAACCTACTAACACAATAAAATATTTAAATAATGTATTTCC
TATAAATAAATTTACAGATTTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAA
TGATCAGTTTAAAGGTTTTAAATTCTTTATGACATCATTTATAAATCATGGATCATATCC
ACTAACAATAGAATGTGGTGTAACAAATGGTGGAACTAGTTATAAAAGAGCAATTATTTT
ATTGCATGTTCGAACTGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTCGAAAAGG
AGAATTATATAATTATTTGAATGCTTATACTGAAGGGGATGTATGCATAATAATTTCCAA
ATCAAATACAAGTTTTGGTTTTAGATGCCCAGTAAATACAAAAAAAATGCCAAAAAATTG
TTTTACGCAAGTATATGAAAAAGGGTATCTAAATGACGCCAATAAAATTAATACTAAAAA
TGTTATTAACTATTCATTTGAAAATCCAGAATATGCGCTAGCTGGTTYTAATTATACATT
AACAAAATCGTATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAA
AACGGTTTTAGTCAAATATGTAAATGAAGATGAAATATATGATTATAATGATTTTCCAAT
GGTGAATCACAAACCTATTATTGCACATCCAAATAAAACACATCAAGCTTGCATGCCTGC
AGGGTGATAGTGTAGATTTTTTTGTTTGACCATTGCATCATTGTCTTTTATTCATCGATT
TTATTATGTTATATATATTTTTTTAAATATCATAATATTTTTTCAATATAGCTTCTTTGA
GCAAATACTGAACAATAAGGAATGCTTCTATGCATCCGTGGATGTATATGTACGTAATAA
AATAATTTTTTGTCCATAAAAATATCTTTAACAGCTGTTATACAAAAGCATTGAATTGGC
TGATACATTTTTGTCGTAAATAAAATAAATGAATTAATGGTACAAAATTACAATAAAAAG
GAAGCGAATTTATTGCATTTTGTTTAATTTATGTCCTTTCCATTTTATTAATACAATTAT
GGTACTAGTAATTCGTATTAAACATATATTTTGCATGTACCAAATGTTTGGGAAATAAGC
ATATGGATCCCACTAAATTTAATAATATTCTGCGATTTTTCTTATATTTACTATTAAATA
TAATGGATTCATTTTTTGATGCATGCAATTTTTTCTTTTAATGTATTAATTAGTGTGACA
ATTTTGAAAAACTTTACTCATTTATTTATTTTCCTTATTATATTGGATTTATAATACCAG
TATTATTATATAAACCACATTAAATAATTTGTAATTTATTCGAGGGTATCAAAAAAAACA
TATACAAAATGCATATATCCACATATGGCTCATTATAGGGTGAATAAAAATGGGAACAGC
AGATTAATTATTATATCATGAAAGTAATGAAAAAATTTAAATTATATGGATATATACATA
TATAATATGTTAAAAAATAATAATAAATAAATAAAATGTATTATAAATCTTATAAAATAA
GTTTTGTTGTGAATAATCCACGAAATATGCCATAAATAGACACTGAACAAATTAGTGGTT
CTTAATATTATTTTGGATACATGCGGATATTATCATTGTCAGATGATTTATTTTTTGTTA
TTTTTAAATTATAAATATTCATATTTTATAGTCCTCAAAAAATAGGATGTTTTATTTTTT
TATAGCTATATTTTATGGTTGATCCTTTCCTTTTATGGTGTTTCATAAAAATTTTATTGA
GCTATATACAATCCAATAAAAAAAGGCAATTGAATTTTGAAATATATTAAACTATTTTTA
TAATAAAATAAATATAATTTTTAAATAATGTTATATTATATATAATTTTTATCTTTACAC
AGAAATTTTTTTTTAGAGTCCAATATATATAATTAGTTATATATATAGAGATATACACTA
CATACACCACCATAAATAATTATAAGGAAAACCAGTTATTTTAATTTTAACTGAAGAAAT
TAAATAAATATATAAAAAAGAAAAGAACAAAAAATAAAACGACAGCAACCTTAAAATTTT
ATTATTATTACATATTTATACATAAAAAAAATAAAATACTTAGACAAAAAATAATAAATA
TTATAGATCGATATTAGCATACATATATACCTTTCAGCACATAATTATTACGTCTGC
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 modificationDNA constructs for generation of the transgenic reporter line expressing mCherry is based on the standard GIMO DNA construct pL0043 (Lin, Annoura et al. 2011). This construct contains the 5’ and 3’ targeting sequences (TRs) for the 230p locus as well as a multiple-cloning site for integration of transgene-expression cassettes. This construct integrates by double cross-over homologous recombination and replaces the positive-negative selectable marker (hdhfr::yfcu; SM) cassette with the transgene-expression cassette in the 230p locus in P. berghei parasites of GIMOPbANKA. The mCherry expression cassette contains mCherry flanked by the 5’ and 3’UTR (promoter and transcription terminator) sequences of P. berghei uis4 (PBANKA_050120) which were amplified from P. berghei WT genomic DNA. The 5'UTR was amplified with primer pair 7169 (tatcctgcaggGTGATAGTGTAGATTTTTTTGTTTGAC) and 7170 (ataagaatgcggccgcAGACGTAATAATTATGTGCTGAAAGG). The 3'UTR was amplified with primer pair 7171 (cggatatcTATAATTCATTATGAGTAGTGTAATTCAG) and 7172 (ggccggtaccTTTCGCTTTAATGCTTGTCATC)

The mCherry-expression cassette was cloned in pL0043 resulting in plasmid pL1937.
Additional remarks selection procedureThis reporter mutant expressing mCherry 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_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites; ETRAMP10.3
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_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites; ETRAMP10.3
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