RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1140
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0902100; Gene model (P.falciparum): PF3D7_1147000; Gene product: sporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein
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 November 2014, 12:46
  *RMgm-1140
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) : 25407681
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 P. berghei ANKA 676m1cl1 (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).
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The mutant parasite was generated by
Name PI/ResearcherSchaijk B van; Janse CJ; Khan SM; Sauerwein RW
Name Group/DepartmentLeiden Malaria Research Group, Department of Parasitology
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-1140
Principal namePbΔslarp
Alternative name1839cl3
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
SporozoiteNot different from wild type
Liver stageNormal numbers of motile sporozoites are formed. Sporozoites show normal traversal and invasion of hepatocytes in vitro. Development of liver stages is completely aborted soon after invasion of sporozoites. Intravenous injection of up to 500.000 PbΔslarp sporozoites never led to full development of parasites in the liver as assayed by in vivo imaging of parasite liver loads or analysis of blood stage infection.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SLARP and expresses the fusion protein GFP-Luciferase under the control of the constitutive eef1a promoter

Protein (function)
SLARP is a conserved Plasmodium gene that is specifically expressed at pre-erythrocytic stages and is required for parasite development in the liver. SLARP-deficient sporozoites injected into susceptible mice cannot convert into blood stages in vivo.The SLARP protein is expressed in sporozoites and in early liver stages and is involved in the regulation of gene expression.

Phenotype
Normal numbers of motile sporozoites are formed. Sporozoites show normal traversal and invasion of hepatocytes in vitro. Development of liver stages is completely aborted soon after invasion of sporozoites. Intravenous injection of up to 500.000 PbΔslarp sporozoites never led to full development of parasites in the liver as assayed by in vivo imaging of parasite liver loads or analysis of blood stage infection.

See also the  link slarp for other mutants lacking expression of SLARP

Additional information

Other mutants:
See the  link slarp for other mutants lacking expression of SLARP or expressing fluorescently-tagged forms of SLARP

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0902100
Gene Model P. falciparum ortholog PF3D7_1147000
Gene productsporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein
Gene product: Alternative name
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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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GAACTCGTACTCCTTGGTGACGGGTACCGGGAGTCAAAAACGGTATGCAAAGCTAAATAA
GCATGTGCAATATGAAAATGACAATGCTCGATAAGAAAACATTAAATTCCATGAAATTGT
CTTTACACAATTATTTATCCATAAATATTTATATATTTCATTATTAGCAATTACAGTTTC
AATATATGTGTATCTTAAGTATTCTACAATATAACTTTTGTATGTATGCATGTATGTAAG
AATATATATATATATATTCATATAGAAAAATACTCATTTTTTTAAGCAAAAAAAATGATA
TGGATATTCATTCAAAAAATATATATACATGCATATATTGACATGTATTCATTGTGAACA
AATAAATCGTATGTTGTATATAATTAAAATAAATATCTCTATATATAGATATTTTTATCT
TTAACAATCCCTTTTTTAATATTAATAAATATGAGTTCATCTTTTTGCACGCTACATTAT
ACAATGAACTTTTCCTTTTTTTATTTTTTCAACATTTCCTTTTTCAATTTACACTTGTAT
AATATATTTATGCCATCATACTAATTTACATGCATATTATATATATATTCATAATATGCA
TACTTGTATTTATGAATATAAATATATACATATTCTGCTAAAAACATATAAATATTTATG
ATAACATGCCATATTTTAATTAAATATCAATAAAATAAATAATAGTTGTTAATATGTTTT
GTTCTATTACACATCCTAGATATACAACTTTCATCAAGAAAAATATTAAAATAAATAATA
ATAAATAAATATAATTTTATAATATTTACAAAATAAATTTTACATTTTTTTTCTTTGATA
CCGCTTTTCTTATATATTGATTCACTTTTACCTATATTTAATACATTTACACCTCCACGT
ATGTTTTTATTTGATTTTTTTCTTTTTTTTCAATTAGTATATATTATACATATTATTACA
TGCTTTATATAGTAAGATTTTTAAATAACAATAAAAAATCATATTCTTATAATCATACGA
AAATTAAACCCTTTATGATCCTAAGCAAATTAAGAATCTTCCCTTTACCTTTGAAAACCA
CAATTAAATAGAAGGCACCCCTAATAGACACGCATAAAAATAGAGAGAAAAATATATATA
ATATAAGAACAATCTCAAAATATATTTTATACATACTCAATTGCAAAAGAAAATATAAAG
CCTACGTGGGCATGTACTATAGGAGGGGTACCGAGGTCGACGATGCTTGTAGATGGCCCA
GCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGT
ATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAAT
ATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATA
CATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAA
TATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCG
TAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATG
TAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAAT
AAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATT
TATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTT
GCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGTTGGTTCGCTAAA
CTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCC
ACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGG
TAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCG
ACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGG
AGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATT
AGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCAT
GAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGA
CACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGT
TCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAA
TGATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGTGCTAGCGTGACAGGGGGAAT
GGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGGCCTTAGGTTA
CAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGTTCT
CGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTC
CACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGTATAC
GACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTG
TGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTAGAGG
TCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTATCGA
TGAAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCTTCGGAACCATTTAAGAACGT
CTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAATAAGAA
TACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTGAAGA
AGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAAACTT
CGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATC
GATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCA
AAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACCAGAGGATATATC
TGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAGTGCTATCATGGC
TACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTTCTTAAACCTAAT
CTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAGAATTGTTAC
TGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTTGGGTGACTT
TGGTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTTCTTACTTATATATTTATACC
AATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCA
TGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAA
ACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTAT
GAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATA
AGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATG
ATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAAT
AAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAAC
CTTCAATTTCGGATCCACTAGCTCGAGCGGCATGTTAGGAGCACGAAACCATATTCAACG
TTTATTGGCATTTGCTAAACACGGAAAAATATAAATATATACAAAACATAATATTGTTTT
TAGATAATAATTTATACACAAGAGCAGCATGGCTATTAAAAAAAGGATATAATATGGATG
ATTTTGAAGTTCAAAAGGCAGAAAGAAAATTAATAAAACTAATGCTTTTAGTATTTAAAT
TTACTTATGATCATAAAAATGACAATTCGAAATATGAACATATAAAGGCTTTTCTATATC
CATTAAGGAGCTCCCATATAAATTTTGAAATAATGAATCGATTCTTATTTTATTAAACTG
TTTTTGTACATATTATTTCCCCATCTTCATAAGCATACACATTCGTGTGAATATATATAT
ATATAGAATTATTTTTTTAAACTAATCTATATTCATTTTTTTAATTTAGACAATTTTCTA
TACGTCGTTACATGATTTTCTTAGATATTTTCATATTCAAAATTTTCAATATATGTGAAA
TATACTCATGCATATATCCTAAATATTACATGCTTGTACATACGCGTACTATTGCCTTTC
TTTCATTAAATATTTTTGTAGCATTTAATAGCATTTAAAAAAGTAAAAAAAACGCATATA
TATGTTTGTATGCAATTCCCCCTTATTTTATTCATCGAATTTAAAATAATATGTATATGC
AAAATATATAATAATAAATAAATATATTCATCACTATAGACCAGTTATAATTTCATGTCC
AATGTTTAAGTACACCGCTCATATTTTTATAATCCTCAATAAAAAAGAAAACAAGTGGAT
TCCCACAATTTTAGAGTACTGCTGAGTGTCAATGACCAACCT
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe mutant was generated by disruption of the gene 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.

Using this PCR-based targeting construct (pL1740) the mutant PbΔslarp-a (1839cl3) was generated in the PbGFP-Luccon reference line using standard methods of transfection and positive selection with pyrimethamine.
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 1GAACTCGTACTCCTTGGTGACGGGTACCGGGAGTCAAAAACGGTATGC
Additional information primer 15960 (Asp718); ∆slarp 5' target F
Sequence Primer 2CATCTACAAGCATCGTCGACCTCTCCTATAGTACATGCCCACG
Additional information primer 25961; ∆slarp 5' target R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGCATGTTAGGAGCACGAAACC
Additional information primer 35962; ∆slarp 3' target F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCAGTACTCTAAAATTGTGGGAATCCACTTG
Additional information primer 45963 (ScaI); ∆slarp 3' target R
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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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
Plasmid/construct sequence
Restriction sites to linearize plasmid
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 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.
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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 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
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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
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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