RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-820
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0923700; Gene model (P.falciparum): PF3D7_1124600; Gene product: ethanolamine kinase (EK)
PhenotypeNo phenotype has been described
Last modified: 16 August 2013, 18:39
  *RMgm-820
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 2
Reference (PubMed-PMID number) Not published (yet)
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 1037m1f1mocl1 (RMgm-32)
Other information parent lineP. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 20019192).
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_0923700
Gene Model P. falciparum ortholog PF3D7_1124600
Gene productethanolamine kinase
Gene product: Alternative nameEK
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPCR 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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GAACTCGTACTCCTTGGTGACGGGTACCGCATCATTTCCCCTTATCGTTTTATTTTTTAA
TAATATTTTCTTATCAAAGAATAACAAAGTATTATTATTGATCATTTGTGAGTTTGAAAA
AAATAAATTTATGAGTCAAATACTTAATGGGTATATGCATATATATATGCTAATTTTCGT
AAAATAAAATAACTTAAAAAAAATATACCCTATATGCAAATAAGCACACATTAAACATTT
ACACAAATTAATGAAAAAAAAATATAATTTGAATTTCAAAAAAAACATATAAAATTTAGT
TTATATTGCTATATATTTTTATTATCCATTTATTTCGATTAAAAAGGATAATATGCGTAT
TTAAGTGTGCATATATAATTTGTTAAATTTTTTGGAAATTATAAATAAGAAAATTCCAAT
TTCTATACTCCGATATGCATCGATTTATACATGTGTACATATGGATGAATTCATAGTAAT
TATATTTGTTTTATTACCATTGAAATTATTTAGATAGTAAATCGTTTTATTCACACGTAT
ATATTACTTTATTTAAATTTTTTTTTTCGCATCCTAGTAGCACTTTATTATCCCTCTGAA
ATTGCATTACCGTTGAGGTCGACGATGCTTGTAGATGAGTTAAGCTTAATTCTTTTCGAG
CTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTTGTAGTATCCT
AGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAA
TACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACG
CCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGCACTAAATATAAG
CTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTATTTATATTTCT
ATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATGTTTTTTTTATTT
TATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATATAATTGTTGTGA
AACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTA
AAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTATAAATAAATAAA
AATTTTATAAAAATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGC
ATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAATGAATTCAGATATTTCCAG
AGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAG
ACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTC
AGCAGAGAACTCAAGGAACCTCCACAAGGTGCACATTTTCTTTCCAGAAGTCTAGATGAT
GCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTT
GGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTG
ACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAA
TATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATT
AAGTACAAATTTGAAGTATATGAGAAGAATGATTAATGTTCGTTTTTCTTATTTATATAT
TTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTTTTT
TTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCAGAA
AAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAATTAA
ATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTTTGA
AACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGTCTC
TTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGCATA
GTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTTTTC
CTTCAATTTCGGATCCACTAGTCCCAACGTTATAATTTACTGAGTTCAATTATAAAATAT
TTTGTAAAATTAAAGAATCGTTTTAACATATTCTATGAGGAAAACAACTTTGATTTTTTT
TTTTTTTTCAACTAATTTTTTTCATTATTAAGAAATTTTTAATATTATTATGGTATTTAT
TACTAATTAGTTTTGTTTTGTCTTTTTTTTTTTTTTTAACATTATTTCTTGTGAAAAACA
TTATGAATAAGTTTTATAAAAATATAATTCGTTTTCCTACTCTAAAGGAAGCAAAAATAA
CTGTCGATAAGCAAATCAGTTTAAAATGGGTCAGTTTTAAATAAGCCAGTTTTAAATAAA
CCTGTTTTAAATATACTAGTTTTAAATAAACTCGCGAATTTGCCTTTACAGAGATCAATA
ATATTTCTATAAGAAAAAAAAAGTTTAAGATACTTTTTAATTATTCATGATGAATATGTG
TTATCTTAGTTTATGATTAGAGGTAGTTCCTTTTTGTCAAAAACTTTTGAATAATAATTT
TTTTCATTTTTAATAAAATTTTCATTTATTTCATTCATATAGGATTGATAATATTGAGCT
AGTTCATTTGGCGAGTACTGCTGAGTGTCAATGACCAACCT
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 (1673; 1695).

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
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 1GAACTCGTACTCCTTGGTGACGGGTACCGCATCATTTCCCCTTATCG
Additional information primer 1L5794; 5’- ek targeting region F (Asp718)
Sequence Primer 2CATCTACAAGCATCGTCGACCTCAACGGTAATGCAATTTCAG
Additional information primer 2L5795; 5’- ek targeting region R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGTCCCAACGTTATAATTTACTG
Additional information primer 3L5796; 3’- ek targeting region F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCAGTACTCGCCAAATGAACTAGCTC
Additional information primer 4L5797; 3’- ek targeting region R (Asp718)
Sequence Primer 5GAACTCGTACTCCTTGGTGACG
Additional information primer 5L4661; anchor tag primer
Sequence Primer 6AGGTTGGTCATTGACACTCAGC
Additional information primer 6L4662: anchor tag primer