Summary

RMgm-828
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0208900; Gene model (P.falciparum): PF3D7_0104200; Gene product: StAR-related lipid transfer protein
PhenotypeNo phenotype has been described
Last modified: 16 August 2013, 18:58
  *RMgm-828
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 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_0208900
Gene Model P. falciparum ortholog PF3D7_0104200
Gene productStAR-related lipid transfer protein
Gene product: Alternative name
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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GAACTCGTACTCCTTGGTGACGGGTACCAATTGGTGCCATAACCAGAACTAATATGTCTA
AAAATTAAATTTGTGTTTAAAACAATTTTTTCATACTAAAAAAAATAAATGAAAATAGAA
AATTACTAATATAGAAAAAATATAACTTTTTTCTGTTTCATTAATTTGAAGATTTAATAG
CACATTTTTTTGAATTATTTTATTTATTCTATTATAATCATATTTTTATTTTTCCAATGT
ACTTTTAAATAAAAATTTTTATTTTTGTTGTCATAATTTACAAAAAAACTTTTAGCGTTT
TTTCGATTTTCATTTCATAAATTAAATCGTTTATTTCTTTTTTTTCTATTCTACTTATTT
TATGTGCATTTTTTTACTACGATTTTATTGTTAATTGTTTGTTTTATTTAGTGCTTAATG
CAGTTACTATAAACTATTTATGGATCCTTTTTTTTTCATAATTTCTGTGGATATTATATA
GTTGTATAATGTTAACTCTCTTATATATGTGCATGTAATAATTAAACTTTATTTTCTATA
ATAATAGAAAAAAAAGTATATATTTTTTGTAGCTAAATCGGTTGATCGAGAGGTCGACGA
TGCTTGTAGATGAGTTAAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTT
AATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACC
CCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATAC
TATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAA
AATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTG
CCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAA
TAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATG
TAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTT
GCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAAT
ATATAAAAATATATATTTGCTTATAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGC
TAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCT
GGCCACCGCTCAGGAATGAATTCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAG
AAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGA
ATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCAC
AAGGTGCACATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAG
AATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAG
CCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAA
GTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAG
GTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGA
AGAATGATTAATGTTCGTTTTTCTTATTTATATATTTATACCAATTGATTGTATTTATAA
CTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATA
GCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAAT
GGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTA
AAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTAT
TATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGAC
TTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATT
AAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGATCCACTAGCACT
TGCATGCTATTGTCTTTATAATATTTTGTTTCAATATTTAATTTTTGTACATTACTTTCT
TGAGTTGTTTTCTTGTTTTATTTTTTTTAAGTATTTCAACGTTTTAATACAATCAACTTT
ATGTTTCAATTTTAAAATATAATTTAATTAACTCAAAGGTACAGTTATTAAAAAATGTCG
AAAAATATATGAACAATTCATAAAATAAAAGGTTAGCTAGCTAAAATGAAAAAAAAATAT
AGAAAGTTCACGCATTTACACATAAACATATAGATTGAACTGTTTTCACTTTTGATTTTT
TGCAGACGTCATTCCTATACTTACAGACTATTACTACATTATTTTATTTTTATTGGTTCT
TTATTATAAACATGGATGGTTTAGACTCAAACGAAAAAACACGAAAAAAATTATTAATTA
AAACTAAAAATAGTAAAGTATTATATTTATATATAGCTTAAGTTTTATTAGAGTTTTCAA
AAAAAGAAGGAAAATAATAAATTTCAAAAAAAAAAAATAAATGAATAAAAATACAAATCG
GTATCACTTCACATATGAAGGCATCATGGGTACCGCTGAGTGTCAATGACCAACCT
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 (1779; 1893).

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

One more unsuccessful attempt to disrupt the gene was carried out using a second linear construct that was generated using a 2-step, anchor tagging PCR method. This construct (PCR1774) used the same targeting regions but differed by having the hdhfr::yfcu selectable marker cassette. The attempt was made in the 1037m1f1cl1 (RMgm-32) background.
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 1GAACTCGTACTCCTTGGTGACGGGTACCAATTGGTGCCATAACCAG
Additional information primer 1L5868; 5’-PBANKA_020890 targeting region F (Asp718)
Sequence Primer 2CATCTACAAGCATCGTCGACCTCTCGATCAACCGATTTAGC
Additional information primer 2L5867; 5’-PBANKA_020890 targeting region R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGCACTTGCATGCTATTGTC
Additional information primer 3L5870; 3’-PBANKA_020890 targeting region F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCGGTACCCATGATGCCTTCATATGTG
Additional information primer 4L5871; 3’-PBANKA_020890 targeting region R (Asp718)
Sequence Primer 5GAACTCGTACTCCTTGGTGACG
Additional information primer 5L4661; anchor tag primer
Sequence Primer 6AGGTTGGTCATTGACACTCAGC
Additional information primer 6L4662: anchor tag primer