RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-807

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1310600; Gene model (P.falciparum): PF3D7_1446800; Gene product: heme detoxification protein (HDP)
Phenotype	No phenotype has been described

Last modified: 23 December 2016, 17:40

*RMgm-807

Successful modification	The 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	≥ 5
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 25941254
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line	676m1cl1 (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).
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Attempts to generate the mutant parasite were performed by
Name PI/Researcher	J. Lin; C.J. Janse; S.M. Khan
Name Group/Department	Leiden Malaria Research Group
Name Institute	Leiden University Medical Center
City	Leiden
Country	The Netherlands

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_1310600

Gene Model P. falciparum ortholog PF3D7_1446800

Gene product heme detoxification protein

Gene product: Alternative name HDP

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used (Linear) PCR construct double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

GAACTCGTACTCCTTGGTGACGGGTACCTTTGTGGAACTTAAAATTTGCATAAAAAATAT

TATTTTATTTATTTTTTTTGTGAAATAAATATACATATATAAATTATAATTCGAGTTTAA

ATATAAGAATTTAACCAATCTCATTTTAGTGTAAAACTAGTATATTTTATACCATTGTTT

TTTTTTTCTTTTTTTACAGCCACTATACTATTTTTATGCTGTTGCTGTCAATAATATTTT

ACATATAATAAGAGCAAACAATAACATGCCAATATTTTATATTTTTTGTGTTTAGTAAAA

ATGGAAAAATGCTTATGCTCGTAAAGTATCATTAATAAATAACCCATTACACGTTTGTAA

AAAATAACTATATTTTACCATCCATTCTAAAAAATAAAATAGCAAAATTAATAAATGTAC

TCACATAAATATAATTAAAAAATGTATATCTTGTTCAGACGGAACATTTGAAAAATATAT

CTGTGCGCATCCTTGCATTCATTTTTTTAAGTATGCTACTAGTGTTTGCGCATTTGTACG

CATATATGTCATATATGCAATATTTATGTGAATGCTTCCGAAAAATGAGGTCGACGATGC

TTGTAGATGAGTTAAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAAT

ATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCT

CAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTAT

GCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAAT

ATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCC

TTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAA

TATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAA

ATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCC

TTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATA

TAAAAATATATATTTGCTTATAAATAAATAAAAATTTTATAAAAATGGTTGGTTCGCTAA

ACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGC

CACCGCTCAGGAATGAATTCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAG

GTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATC

GACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAG

GTGCACATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAAT

TAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCA

TGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTG

ACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTG

TTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGA

ATGATTAATGTTCGTTTTTCTTATTTATATATTTATACCAATTGATTGTATTTATAACTG

TAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCT

AAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGC

TAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAA

ATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTAT

TATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTG

ATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAA

AAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGATCCACTAGCTTTTCA

CATCTTCAGTGGCTCATGTACAATGAAAAAAGAGAGGCCCCAGAAAAAATACCAACTTTA

GTAAACAACGACATAATTGTAAAAATAGAAGGGGAATTTAAAAATTGTGATCATTCTATA

TATTTAAATGAAGCAAAAATTATAAGACCCAATATGAAATGTCATAATGGCATAATTCAT

ATCATAGATAAGCCAATAATTTTTTAGTACTATTTAAATGAAGATTTTCCATATATTCAG

TAAATATAATTAAATAATCCAATTTTAATATAATTAATTTATTTAATTATTTTATATATA

TTTTTTATACAATCTTTATGTCCTTTATAGTGCCTACTGTATTTATTTTAAAGGAAATTA

AATTTTTCCTTTGGGAACTATATATGTTCATACATATGTTTATTATTACTAACTTTAATT

TATAAAACAAAAAAATGAAAAAAAAAAAAATAATAATAACAATATATAGTAAAAATAATA

ATGGAAAACACAAAATGCTGAATTATCAAATTCGGAAATTAAATATCAGATATTCCATGC

AGCAGGTACCGCTGAGTGTCAATGACCAACCT

Restriction sites to linearize plasmid

Partial or complete disruption of the gene Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite hdhfr

Promoter of the selectable marker eef1a

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification The unsuccessful attempts to delete this gene indicates an essential role for blood stage growth/multiplication.

Attempts to disrupt the P. falciparum hdp gene were also unsuccessful.

Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes.

In the first unsuccessful attempt to disrupt the gene described here (experiment 1748), 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 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.

See for more information on the 2-step anchor tagging PCR method, 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;30(16):2662-70

The experiment was repeated 4 more times without success: twice using the same PCR targeting construct (experiments 1778 and 2212) and two more times with a PCR targeting construct having the same targeting regions but with the positive-negative selectable marker (PCR1762, experiments 2208 and 2213). Experiment 2208 was done in the cl15cy1 background.

An independent unsuccessful attempt in the cl15cy1 background was done by the group of Roberta Spaccapelo (University of Perugia, Italy) using a circular, double cross-over targeting plasmid (pPhHDP). The 5' targeting region was amplified using primer pair R724/R725 (CCGGGCCCGCGGCAGCCACTATACTATTTTTATGCTG / GGGGATCGATCGGAAGCATTCACATAAATATTGC). The 3' targeting region was amplified using primer pair R726/R727 (GGGGGAATTCCTATTTAAATGAAGATTTTCCATATATTCAG / AAGGAAAAAAGCGGCCGCGGTTGAATCCTAATTCAGTATTGATTG)

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	GAACTCGTACTCCTTGGTGACGGGTACCAATTGGTGCCATAACCAG
Additional information primer 1	L5868; hdp 5’-targeting sequence, F (BamHI)
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCTCGATCAACCGATTTAGC
Additional information primer 2	L5869; hdp 5’-targeting sequence, R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGCACTTGCATGCTATTGTC
Additional information primer 3	L5870; hdp 3’-targeting sequence, F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCGGTACCCATGATGCCTTCATATGTG
Additional information primer 4	L5871; hdp 3’-targeting sequence, R (BamHI)
Sequence Primer 5	GAACTCGTACTCCTTGGTGACG
Additional information primer 5	L4661; anchor tag primer
Sequence Primer 6	AGGTTGGTCATTGACACTCAGC
Additional information primer 6	L4662: anchor tag primer

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