RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-913
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1356000; Gene model (P.falciparum): PF3D7_1342900; Gene product: AP2 domain transcription factor, putative (ApiAP2)
PhenotypeNo phenotype has been described
Last modified: 16 August 2013, 18:05
  *RMgm-913
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 3
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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_1356000
Gene Model P. falciparum ortholog PF3D7_1342900
Gene productAP2 domain transcription factor, putative
Gene product: Alternative nameApiAP2
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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GAACTCGTACTCCTTGGTGACGAAGCTTAGAAACCCGATATCCCAGTGGTAAATATAATC
TGAAAAAAAAACTTATAAATTTATGTAAAAATAGTAACCTTTGGAAACAACATTTTAAGT
TTATAGAATTTTTGATTATATATATTTTTTATTATGCATATGTTAAATATATGGATTACA
CATGTACATTATATTGTTCGTTAAAAATTAATAAATTTTATTTATTTTATTCGCATTTAT
ACTATGCTTAATTATGTTTTAATTTTTCATTTCTTGGAAAATGACGTTAGAAAAAAATAA
ATATATATATCGATTGTTTGCTTTGTGCGCATAAAATAAATGCGCCATTTGTGTTTTTAA
ATTTATTTGCATAGGATTATTAACACAAATATATATTTCACTATTTTACACACATATTTA
TAAGATTCCTATTCTCCAAAAATATATAATAAATAAATAAATAAATAATAAGATAATAAA
AGAAAATAAACAAAAACGAAAATAGAGGAACAAATTTTAATATATACAGTCAAAAAGCCT
GCTTCCTGTTTCCTCAGACATAAATTTCCATTTTTATTCACATATATTTTTATTAGTGAT
ATAATTAAATTTTTCTATTGTCATATATGATACCATTATATATATTAACAGTTATGTATA
TTTTTAAATAAATAGATAATTTATATTTTTCTGAAGATTTAGCTATATATAAATAATTAA
ATATAGCTTAACTTTTTTAAATGTAATTTTTTGCACCTGTGTACCTCTCCGCTTTGTATA
TTTCCTTCATTGTATATATATATTATTATTTATTTATATGTGTGTATATGTATTTTGTCA
TGCCGCGGAGGTCGACGATGCTTGTAGATGGCCCAGCTTAATTCTTTTCGAGCTCTTTAT
GCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTG
TGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTA
AATTTTCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACGCCATGTAA
ATCAAAAAATATATAAAAATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTAT
GCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATA
AAAAAATCCTCATATAATAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTT
TAATATAAAATAATATGTAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAA
ACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATAT
ATACTTTGTGTATAAATATATAAAAATATATATTTGCTTATAAATAAATAAAAAATTTTA
TAAAACATAGGGGGATCCATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAAC
ATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATAT
TTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGT
AAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTA
GTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTA
GATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGG
ATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTA
TTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTG
GAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAA
GGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATGCTAGCGGAGGAGGTGGATCT
GGTGGAGGTGGAAGTGCTAGCGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGT
ATGGACATTGCCTATGAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGC
GGATGTCTTATCAATAACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTT
CAAAAGGGATCCGCCACACTACATGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTA
GAGGGCAAAGTGTACAAAGATACCACTTTGTATACGACGCTGTCTCCATGCGACATGTGT
ACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTC
AAAAGTAAGGGCGAGAAATATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGAT
GAGAGGTGTAAAAAGATCATGAAACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAA
GATATTGGTGAGGCTTCGGAACCATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAA
TTGCTGGGTTTGTACACCATCATCAGAAATAAGAATACAACTAGACCTGATTTCATTTTC
TACTCCGATAGAATCATCAGATTGTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAA
AAGCAAATTGTGGAAACTGACACCAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAA
ATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGT
TGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCA
AAGTTATTCTACGAAAAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGAC
CCAATGCTGGCCACCGGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGT
GTTAAGCCAGAGAGAATTTACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAA
TACCATGCCGCCTTCCCAGAGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGAT
GAAAACAAGTATCTAGTTCCAGGGTTGGGTGACTTTGGTGACAGATACTACTGTGTTTAA
CTCGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAA
AATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAA
TTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGT
ATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTT
AGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATT
ATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTT
TTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAA
AACATATAAACACAAATGATGTTTTTTCCTTCAACCTTCAATTTCGGATCCACTAGCTCG
AGAAGCACATGGAGAGAAAGGAACAAGCAAATATAATTAAATGAAATAGATACTAAATTA
AATTAATAATTCGAAAAAAATGTTTATCTTAGTTTATATTTTTATTATTTTATTATTTTG
CTCTAATAAAAACAGTTACTATAATACTTTGGTTGTTAATACAAAATAAGTAAAACAATA
CGCATAAATCTATATAATTTTTTCAAACATATTATTTGGTCTATTTTTATTTCGTTACAT
TGATTTCTCTTTTTTAGTTATTCTACATCAATTCTCTTACATAGCACGTAGTACATTATT
ATTTTATATAATTTGTCGTTTAATCGTTTATTTAATTAATATAAATAATTATTCCATATA
ATTTTTTCTTTTAATTCGCTTTTATACTTTTCTATGTTTATCTTTATTTACTATATTATA
CATTTTCTTCATTCATTTAGTTTAATTATTTTTTTAAACCTAATATACGCATTATATTAG
ACATATAAAAATATATTAAGGGTGAAGAAACTGAATCCGATATATTTCCCCTTAATATAT
CTTGATAATGATATTGTTTTTTTATTCTCTTTATATTTATTTTTCATTTAATAAATGGTG
ATTTTTACACATTTATTTATTTATTATTTATTTTTTGTTATATTTCATATGCATATAATA
TGGAGGTGAATAATTTGAACATGTTTTATGTGTACATTTTTTATTTTAAAATATATTTTG
TTTATCTTTATAATTTTTTTCATGTTTTATTTTTGCACAATATTTCAATTGCGCTATTAA
TCAATAAAAATAATAAATGTAGTGAAATAGTAACTATTGTAGGTGTGTAGGCACCATAAG
AAATTAAGACTTAGCTAGCAAATCATATTTCAAGAGCTAAGATATCGCTGAGTGTCAATG
ACCAACCT
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) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThree unsuccessful attempts to disrupt the gene (2007; 2330, 2327).

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

The locus was 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::yfcu 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::yfcu selectable marker cassette used in this reaction was digested from pL0048 using restriction enzymes XhoI and NotI. pL0048 is available from The Leiden Malaria Research Group.

To remove the anchor-tags from the final KO construct, and to eliminate contaminating pL0048, 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 1GAACTCGTACTCCTTGGTGACGGGTACCTATATACATCAACGATGCATTC
Additional information primer 1L5999; 5’- ApiAP2 targeting region F (Asp718)
Sequence Primer 2CATCTACAAGCATCGTCGACCTCTTTTTCCCCTCTTCTTGTC
Additional information primer 2L6000; 5’- ApiAP2 targeting region R
Sequence Primer 3CCTTCAATTTCGGATCCACTAGCCAGAACCTCCTATTTTCC
Additional information primer 3L6001; 3’- ApiAP2 targeting region F
Sequence Primer 4AGGTTGGTCATTGACACTCAGCAGTACTGTTCACCATTGGGATCAG
Additional information primer 4L6002; 3’- ApiAP2 targeting region R (ScaI)
Sequence Primer 5GAACTCGTACTCCTTGGTGACG
Additional information primer 5L4661; anchor tag primer
Sequence Primer 6AGGTTGGTCATTGACACTCAGC
Additional information primer 6L4662: anchor tag primer