RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-818
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1040100; Gene model (P.falciparum): PF3D7_1401800; Gene product: choline kinase (CK)
PhenotypeNo phenotype has been described
Last modified: 23 December 2016, 17:46
  *RMgm-818
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 4
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25941254
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).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherJ. Lin; C.J. Janse; S.M. Khan
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1040100
Gene Model P. falciparum ortholog PF3D7_1401800
Gene productcholine kinase
Gene product: Alternative nameCK
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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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GAACTCGTACTCCTTGGTGACGGGTACCAATATTTAGATCTTGTACAATTATAATTCTTC
TTAAAAAATTATAGAAAAAATATAATAAATTGGGTGCAATTAATATATTGTAGCATTGGC
ATGCATCAATATATATAACACCATAAAAATAATTTTTCTTTCCTTAAATCAAAAAATATT
ATTTGACTAATATATTTTTGTTTTGTTTTGCAAATTTTACCACATTAAAATTTCGACACA
TAACATATTCGTCATTTAATTGACTTTCGTTATTTCTTTTGTGTTGTGTGCTTCTTTAAG
AATAAATTAAGACTGTTTGATACAACAATAATATAAATCATAAGATATATAGGTGCATAC
CCATATATATATTTGTTTATTTATATGTATATGATTTTATATTGTCCTATCTTTAATCGT
GAATGTGAATAATATCCCTATTTTGTATTACTTTTGCTTTCTATATTGCAATTTGTTTGC
ATTTTTATATTTAAATATTGTAAATTACATGCCTATAATAATACAACACACAAAAAAATA
CATATCAAAATAAAAAAATCTCAAGTGAGGTCGACGATGCTTGTAGATGAGTTAAGCTTA
ATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTT
TTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTT
CATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATAT
AAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGC
ACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATT
ATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATG
TTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATA
TAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTT
TAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTA
TAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCC
CAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAATGAATTC
AGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATT
ATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATT
AATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGTGCACATTTTCTTTCCAGA
AGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATG
GTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTT
AAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATT
GATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAG
GAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAATGTTCGTTTTTC
TTATTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGT
GCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATT
TTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTT
TATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACA
TAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAA
TTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAAT
ATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAA
TGATGTTTTTTCCTTCAATTTCGGATCCACTAGTCTATTGATTATTCTACAGACACTTAT
CCTTTTTATGAAATAAATAAAAATCACTATATATCACATGAAAATAGAAAATTGTTTATT
AATGAATATTTATCAATTTATTTAGGAAAGTCACAAATACCGTATGATCAAAAAATCGTC
GACTCGATTTTAGATGCAATTGAAATCCACGCATTAGGTGCTAACTTATTATGGGGGTTC
TGGTCGATTATTCGAGGTTATCAAGTAAAATGCTATAATGAATTTGATTTTTTCCTATAT
GCACAAGATCGATTTAAACTGTATGATGAACAAAAGAAATATTTACTTTCAAAGAATCTT
ATTCCAGACTATGATTAAATATATAATAAACAACTTATAAAAAACAAATTATAGCATATA
TACTAAAAATTGAATGTGCTTGTTTCTCCTCCAACTAAAAAACAAATATATTATATATAT
ATTTACTACCTATTAGCTAAGGATTAAATGAGCTTGAATGGAACATTAATTAAACATTTA
TTTAATGTATTATCAGCAGCATTGGGGCAAAATGACTTAAATATCGTAAAATTTGCACTT
TCAAACATTTTGAATTATATAGTACTGCTGAGTGTCAATGACCAACCT
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSeveral unsuccessful attempts to disrupt the gene (1527, 1609, 1649, 1682).

The unsuccessful attempts indicate an essential role during blood stage development/multiplication.


The locus was unsuccessfully targeted 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.

For a more detailed understanding of the 2-step anchor tagging PCR method, please see the following publications:

J.W. Lin, S.M. Khan et al
A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites
PLoS One. 2011;6(12)

T. Annoura et al
Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates
Vaccine. 2012 Mar 30;30(16):2662-70

Attempts to disrupt the gene were carried out in both the 676m1cl1 and 1037m1f1mocl1 background (RMgm-32)
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 1GAACTCGTACTCCTTGGTGACGGGTACCAATATTTAGATCTTGTACAATTATAATTC
Additional information primer 1L5193; 5’- ck targeting region F (Asp718)
Sequence Primer 2CATCTACAAGCATCGTCGACCTCACTTGAGATTTTTTTATTTTGATATG
Additional information primer 2L5194; 5’- ck targeting region R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGTCTATTGATTATTCTACAGACAC
Additional information primer 3L5195; 3’- ck targeting region F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCAGTACTATATAATTCAAAATGTTTGAAAGTG
Additional information primer 4L5196; 3’- ck targeting region R (ScaI)
Sequence Primer 5GAACTCGTACTCCTTGGTGACG
Additional information primer 5L4661; anchor tag primer
Sequence Primer 6AGGTTGGTCATTGACACTCAGC
Additional information primer 6L4662: anchor tag primer