RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-804

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1137000; Gene model (P.falciparum): PF3D7_1360800; Gene product: falcilysin (FLN; bergheilysin; BLN)
Phenotype	No phenotype has been described

Last modified: 23 December 2016, 17:39

*RMgm-804

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	3
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_1137000

Gene Model P. falciparum ortholog PF3D7_1360800

Gene product falcilysin

Gene product: Alternative name FLN; bergheilysin; BLN

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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

GAACTCGTACTCCTTGGTGACGGGTACCCATTAATATGCTAAGCATTACACATATTTTAT

ATCTTTAAGGAAATTTTTATGCTTTTATAGATTCACAGAAACTGATAAATAAGCATATAT

AAATAAATATCAATTGGGGTATTTATGATTTGTTGGGCAAAATCCGTTCACTATTGCTTA

ATTTAGTGAATTTAAAAAATCCATAATTCCCCTCAAAAAAAAATGTAAATGCCATGCAGA

AAATAAAAAAAAACGATAAAAAATATAGTTCCAACTTTAATTTAAAGGCATGCAATTTGA

ATAAAAAAACTTATAAGAAATAATCGAAAAAAAAAATTGTAATTAGCTAGTAATAAATAA

AAATAATTAATAGTGACGAAAATAAATAATATAATTGCAGATGAAATGAACAAATAGTAA

AAATAAATAATAATGAAAAAATATAATAAAAATATACCCAATTCATAATCGTGAAGATAA

TCATTTAAGAAAGCCACATTTTATTTGTAAAAATAACAGGTTGAATATGTACATATAGTA

CATACGCATATAATACATAATATAGCTATAATTGATATCGGGTACGTAATATCACGTAAC

GGAAAACCCACAATTGCATTCATATCGTTAATCCATACATACATTTGCTTGCAGTATCAT

GCATTCACATGTAAATAATAAATAAATAAATATATATATATATATATATATATATTAAAT

ATATGTTTTAATGCGTACTTCATATAGAAATTTACGTCGTATTTAAAGATACATATAGTA

AAAGTCAAGTGAATTATGTGAAGAGAGGTCGACGATGCTTGTAGATGAGTTAAGCTTAAT

TCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAAGTATTTTTT

GTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCA

TCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTATACATATAA

ACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATAAATATGCAC

TAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTAT

TTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATATGTAATGTT

TTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAAATAAATATA

ATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTA

ATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATATTTGCTTATA

AATAAATAAAAATTTTATAAAAATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCA

GAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAATGAATTCAG

ATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTAT

GGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAA

TTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGTGCACATTTTCTTTCCAGAAG

TCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGT

CTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAA

ACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGA

TTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGA

GAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAATGTTCGTTTTTCTT

ATTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGC

ATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTT

TTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTA

TATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATA

ATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATT

ATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATAT

GATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATG

ATGTTTTTTCCTTCAATTTCGGATCCACTAGACAATTGATAGACCTAGAAGAGGTGTTGA

ATTGAGCAAACTTTCATTTTCACGAATTATATCAAATGAAACTGAACAAGATAGAATTGA

ATTCAGAAATAGAGTTATGAATACTAAAAAAGAAGACTTTTATAAATTTGCAGATTTATT

AGAAAAAAAAGTTAAAGAATTTGAAAAAAATGTTGTCATCATAACTAGTAAAGAAAAGGC

AAATGAATATATAAATAATGTTGATAACGATTTTAAAAAAATACTTATCGAATAAGTTTT

TGTTCACTCCTTTTTTACATATAAACAATGCATATACAATTTACCTGTATTTAGCACATT

TTTGAGACATTTTTAATTTATGTGCGAACTGTAGTTTTACTGTGTATATAAATTATAATA

AAATTGCGTCAATTTTATGCTAAAATTATATTCTTTCATTTTTGTAAATGAACCTAACTC

AAACGCATACATAATATACGCGATCAAGCGAATGTATAATTTGCATTTAAACAGTAAATT

TAATTTCATTATTATTTAAGCATTATAATTTTGTAAAAGGAACAATATAAATATTTGTTT

ATGAATTTTATGCTCTAAAAAAATAAATGAGTGACATAATAATAGTTGTCAATTTGCATT

TTCAAAAAAAATAGTTTACTAATTAAAATTACACATGTGTAGATATAACATATATTGCAC

ATACAATTTGGTTTATTAAAAAAAAATATATATCCGCCCAGTTGCATTTGCGCGTAATAA

TAATTACAAATTTTTATGAAGAGTATCTCATTGTATGCAACAGTACTGCTGAGTGTCAAT

GACCAACCT

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 multiple, unsuccessful attempts to disrupt PBANKA_113700 indicate an essential role for asexual blood stages.
Also attempts to disrupt the P. falciparum ortholog (PF3D7_1360800) have been unsuccessful (Ponpuak M et al., 2007, Mol. Microbiol.).

Falcilysin (FLN) is a zinc metalloprotease thought to degrade globin peptides in the acidic vacuole of the human malaria parasite Plasmodium falciparum. The enzyme has been found to have acidic or neutral pH optima on different peptides and to have an additional location outside the food vacuole. Evidence has been presented for an apicoplast location of falcilysin (Ponpuak M et al., 2007, Mol. Microbiol.). Also attempts to disrupt P. falciparum falcilysin were insuccessful.

In the first unsuccessful attempt to disrupt the gene described here (experiment 1502), 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 homologous 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 without success using a circular, double cross-over targeting plasmid with identical target regions (pL1557, experiment 1543). A third independent unsuccessful attempt was done by the group of Roberta Spaccapelo (University of Perugia, Italy) using a circular, double cross-over targeting plasmid (pLTgLysin). The 5' targeting region was amplified using primer pair R447/R448 (CCGGGCCCGCGGAAATATAGTTCCAACTTTAATTTAAAGG / CCATCGATTTATTATACTGCACATATATAAAAAAATGC). The 3' targeting region was amplified using primer pair R449/R450 (GGAATTCGTTTTTGTTCACTCCTTTTTTACATATAAAC / CGGGATCCACAATGAGATACTCTTCATAAAAATTTG).

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	GAACTCGTACTCCTTGGTGACGGGTACCCATTAATATGCTAAGCATTACAC
Additional information primer 1	L5101; bln5’-targeting sequence, F (Asp718I)
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCTCTTCACATAATTCACTTGAC
Additional information primer 2	L5102; bln5’-targeting sequence, R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGACAATTGATAGACCTAGAAGAG
Additional information primer 3	L5103; bln3’-targeting sequence, F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCAGTACTGTTGCATACAATGAGATACTC
Additional information primer 4	L5104; bln3’-targeting sequence, R (ScaI)
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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