RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-385
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0208000; Gene model (P.falciparum): PF3D7_0105300; Gene product: cyclase-associated protein (C-CAP, C-terminal actin binding domain)
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 Oocyst; Sporozoite; Liver stage;
Last modified: 5 February 2010, 19:55
  *RMgm-385
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) : 20083609
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherM. Hliscs; K. Matuschewski; H. Schüler
Name Group/DepartmentDepartment of Parasitology
Name InstituteHeidelberg University School of Medicine
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-385
Principal namec-cap(-)
Alternative name
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
OocystFluorescence microscopy revealed that c-cap(-) oocysts were markedly reduced in size and numbers as compared to the wild tye. c-cap(-) parasites consistently formed slightly fewer oocysts, i.e. on average 110 (± 42) oocysts per infected mosquito as compared to an average of 213 (± 64) oocysts per infected mosquito for wild type. Up until day 6 oocyst development was comparable between c-cap(-) and wildtype, indicating that initial events in sporogony, including increase of parasite mass and the first few mitotic replications, were unaffected in the mutant. By day 8 c-cap(-) oocysts were significantly smaller in size and arrested their development, when compared to the linear size increase in wildtype oocysts. Quantification of oocyst diameters revealed that c-cap(-) occysts typically reached an average size of 15 μm, corresponding to day 8 wild type oocysts. However, few c-cap(-) oocysts reached sizes comparable to wild type. In mutant oocysts only few nuclei corresponding to 4-5 rounds of nuclear divisions and no sporozoites were detected. No formation of sporozoites inside the oocysts.
SporozoiteNo formation of sporozoites within the oocyst. No salivary gland sporozoites
Liver stageNo formation of sporozoites within the oocyst. No salivary gland sporozoites. Infected mosquitoes are not able to infect mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of C-CAP (C-terminal actin binding domain; adenylate cyclase-associated protein, putative).

Protein (function)
Cyclase associated proteins (CAP) are involved in the turnover of actin cytoskeletal structures in part through binding to monomeric actin (G-actin). The Plasmodium protein consists of the conserved actin binding domain and lacks the adenylate cyclase binding domain and the WH2 domain.

Phenotype
Phenotype analyses indicate an essential role of C-CAP during oocyst development. c-cap(-)  oocysts were markedly reduced in size and numbers. No formation of sporozoites within the oocyst and no salivary gland sporozoites.

Additional information
rt-PCR analysis showed transcription in ookinetes, sporozoites and was most abundant in blood stage merozoites.

In the same study a mutant parasite (RMgm-386) was generated in which the P. berghei c-cap was replaced with the c-cap ortholog of Cryptosporidium parvum. This mutant produced mature oocysts, oocyst-derived sporozoites and salivary gland sporozoites that were infective to mice. These results show that the C. parvum C-CAP protein can complement the function of P. berghei C-CAP.
In the paper evidence is presented that purified recombinant C. parvum C-CAP protein binds actin monomers and prevents actin polymerization.

The sequences of the primers used to PCR-amplify the target regions for gene disruption by homologous recombination are not provided.

Other mutants
RMgm-386: A mutant in which the P. berghei c-cap is replaced with the c-cap ortholog of Cryptosporidium parvum


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0208000
Gene Model P. falciparum ortholog PF3D7_0105300
Gene productcyclase-associated protein
Gene product: Alternative nameC-CAP, C-terminal actin binding domain
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid KpnI, SacII
Partial or complete disruption of the geneUnknown
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor gene deletion via the replacement strategy, a 5’ and a 3’ fragment of PbC-CAP were amplified by PCR from P. berghei genomic DNA. For the 949 bp 5` fragment primers CAP5`for and CAP5`rev, and for the 614 bp 3` fragment primers CAP3`for and CAP3`rev were used respectively.
The sequences of these primers are not provided.
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 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Click to view information
Click to hide information
Plasmid/construct sequence
Click to view information
Click to hide information
AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT
AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC
GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT
CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC
TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA
CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC
ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA
CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT
TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG
TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT
ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT
GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA
CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC
GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC
CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG
GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA
TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC
GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT
GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG
CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT
TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC
TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT
CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC
ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC
TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT
TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG
ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC
AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA
CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG
GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTA
GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA
CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG
TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACG
CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG
CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC
CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG
TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT
GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA
GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG
CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG
CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA
ATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTT
CCGCGGGTATATGGTAAAGAACCTACTAACACAATAAAATATTTAAATAATGTATTTCCT
ATAAATAAATTTACAGATTTATTTTTTAATACAAAAGATATAGATATACCAGAAATAAAT
GATCAGTTTAAAGGTTTTAAATTCTTTATGACATCATTTATAAATCATGGATCATATCCA
CTAACAATAGAATGTGGTGTAACAAATGGTGGAACTAGTTATAAAAGAGCAATTATTTTA
TTGCATGTTCGAACTGATTTAAAAGATAGACCAGTTTCATTTTGTGATTTTCGAAAAGGA
GAATTATATAATTATTTGAATGCTTATACTGAAGGGGATGTATGCATAATAATTTCCAAA
TCAAATACAAGTTTTGGTTTTAGATGCCCAGTAAATACAAAAAAAATGCCAAAAAATTGT
TTTACGCAAGTATATGAAAAAGGGTATCTAAATGACGCCAATAAAATTAATACTAAAAAT
GTTATTAACTATTCATTTGAAAATCCAGAATATGCGCTAGCTGGTTYTAATTATACATTA
ACAAAATCGTATCAATTTGAATGTCATTGTGTAGATAAAGAAACAGAACAAATTGTAAAA
ACGGTTTTAGTCAAATATGTAAATGAAGATGAAATATATGATTATAATGATTTTCCAATG
GTGAATCACAAACCTATTATTGCACATCCAAATAAAACACATCAAGCTTGCATGCCTGCA
GCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTT
TAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCA
GTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATG
TTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAA
ATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACT
AGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAAT
ATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAA
AAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTT
AAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATA
TATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGAGTAAAGGA
GAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGG
CACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT
AAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTC
GGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTC
AAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGG
AACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAG
TTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAAC
TATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAAC
TTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAA
AATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAA
TCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTA
ACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAATGGATCCCGTTTTT
CTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTG
TGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTAT
TTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATT
TTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAAC
ATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATA
ATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAA
TATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAA
ATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGAATTCTCTTGAGCCCGTTAATGA
AATAGATACAATTCATTCATGTTATATACATCTAGAACATAATCTGAATATGGTTCAAGT
TAAATGTCCAAAAATTATAAAAAGTGATGATATTTTTGATGGTAATACCATAATAGACAC
CAAGGTAACATCACGAAGTAGTCAACAAAATAATTTTTATTTAGAAAATACAGATGTTGA
ACCAGAAGAAATAGAGAAATATAAAAATATAGAATACATACCAGAAAACGATGAAGTAAT
GCATCTAGACAAAAAAGAAAAGCTAGATGATATATTACCAGGTGTTATCATATTAGATAA
AAATAAAATGTTCAAAGAAAAAGGACATTTCACTTTTGTTACTCCATTAATTGTAGAAAA
GGTATTAATATTAAAAATATATTGTGATAATACTAAAACAATAATTAATAATATGAAAGG
GAAAAAAGGTATTACAGTAATAAGGATTTCTCAAAATACAACAAAAAATAAATTTTATGG
ATGTGACTTTTCAGGTAATTCTAAAAAAACATTTTACTATTCCAATGTTTATGATTTAGA
AAAAAAAAATGAGTTTTGTGAAATAGAATTAAAAGAAAATATAGTAGTTAGCTTAAATTG
TCCAACTGGTAAAATTAATCCAAAAAATTGTTTTAGAAATGTATATATAAAAAGTAATAT
GAATGAACAAACAACCGAAAATATAGAAAATATATTTAACGAAATAAAAGTTATAGATGC
AGATTATTTTATAAATAATTCATCAACCTTTTTGATGATTTCCAAAATTACAAAAAAAGA
GTTTGATTTTTATTGTACATGTGAAGATTATAAAACCAAAAATATAGGAACAATATATAT
TAAAAATTATGAATATCTAGATTCAAAACCTAAATATAAAAATAAACAAATTTCCTATAT
AGATGTAGTTCCATACCCGCGGGGAAAGGGCG
Restriction sites to linearize plasmid KspI (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 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 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 15'-cccaagcttccgcgggtatatggtaaagaacctactaacac
Additional information primer 1primer L1345: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 25'-cccaagcttgatgtgttttatttggatgtgc
Additional information primer 2primer L1346: 0.7kb 5'region of PB000214.00.0 (230p gene)
Sequence Primer 35'-ccggaattctcttgagcccgttaatg
Additional information primer 3primer L1347: 1kb 3'region of PB000214.00.0 (230p gene)
Sequence Primer 45'-tccccgcgggtatggaactacatctatatagg
Additional information primer 4primer L1348: 1kb 3'region of PB000214.00.0 (230p gene)