RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-814

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_1309900; Gene model (P.falciparum): PF3D7_1446200; Gene product: M17 leucyl aminopeptidase (LAP; M17LAP; A-M17)
Transgene	Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV) Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1 (ama-1) 3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts) Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype	Asexual bloodstage;

Last modified: 1 April 2024, 19:00

*RMgm-814

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 25941254
MR4 number
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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 1037m1f1mocl1 (RMgm-32)
Other information parent line	P. 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).
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The mutant parasite was generated 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
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Name of the mutant parasite
RMgm number	RMgm-814
Principal name	2112cl3(2nd); 2112cl4(2nd)
Alternative name	∆lap
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Strongly reduced growth rate of blood stages (normal levels of hemozin production)
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of LAP (M17LAP) Protein (function) In P. falciparum the metalloenzyme aminopeptidase leucyl aminopeptidase (PfLAP) is expressed in all stages in the intra-erythrocytic developmental cycle with highest expression in trophozoites. The enzyme was located to the cytosolic compartment of the parasites at all developmental stages, including segregated merozoites. Phenotype Strongly reduced growth rate of blood stages (normal levels of hemozin production). (Only mutant clones from a single transfection experiment were analysed; in additional transfection experiments no mutants could be selected, probably due to the strongly reduced growth of blood stages). In a paper published in 2024 (https://doi.org/10.1101/2023.10.24.563045) mutant parasites lacking expression of LAP were analyzed during mosquito development. Mosquito development was comparable to wild type parasites. Additional information Figure: Generation of P. berghei mutants ∆app (RMgm-813), ∆lap (RMgm-814) and ∆dap (RMgm-815). (primer sequences can either be found in Lin et al. (2015). J. Exp. Med. or obtained from the Leiden Malaria Research Group) (A) Schematic representation of the gene-deletion constructs targeting the ORF of genes expressing aminopeptidase P (app), leucyl aminopeptidase (lap) and aspartyl aminopeptidase (dap) by double cross-over homologous recombination and the wt gene loci before and after disruption. The constructs contain a drug-selectable marker cassette (SM; black box) and gene target regions (hatched boxes). Primer positions (arrows) for diagnostic PCRs are shown (see Table S4 for primer sequences and expected product sizes). (B) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm correct disruption of app in ∆app-a and ∆app-b. Northern analysis of blood-stage mRNA (right) confirms the absence of the app transcripts in the ∆app mutants. The following primers were used for diagnostic PCRs: 5' integration (5’ in): L7107/L4770; 3' integration (3’ in): L4771/L7108; SM (hdfhr::yfcuI): L4698/L4699; app ORF: L7109/L7110. Separated chromosomes of ∆app-a and ∆app-b were hybridized using a 3’UTR pbdhfr probe that recognizes the construct integrated into the app locus on chromosome 13, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. Northern blot was hybridized using a PCR probe recognizing the app ORF (primers L7109/L7110) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (C) Diagnostic PCR (left) and Southern analysis of separated chromosomes (center) confirm correct disruption of lap in mutant ∆lap. Northern analysis of blood-stage mRNA (right) confirms the absence of lap transcripts in the ∆lap mutant. The following primers were used for diagnostic PCRs: 5’ in, L6967/L4770; 3’ in, L4771/L6968; SM (hdfhr::yfcu), L4698/L4699; lap ORF, L6969/L6970. Separated chromosomes were hybridized using a 3’UTR pbdhfr probe that recognizes the construct integrated into lap on chromosome 13, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. Northern blot was hybridized using a PCR probe recognizing lap ORF (primers L6969/L6970) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (D) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm correct disruption of dap in ∆dap. Northern analysis of blood-stage mRNA (right) confirms the absence of the lap transcripts in the ∆dap mutant. The following primers were used for diagnostic PCRs: 5’ in, L6975/L4770; 3’ in, L4771/L6976; SM (hdfhr::yfcu), L4698/L4699; dap ORF, L6977/L6978. Separated chromosomes were hybridized using a 3’UTR pbdhfr probe that recognizes the construct integrated into dap on chromosome 8, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. For Northern blot was hybridized using a PCR probe recognizing dap ORF (primers L6977/L6978) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). See Table S4 for primers used for generation of probes. Other mutants

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_1309900

Gene Model P. falciparum ortholog PF3D7_1446200

Gene product M17 leucyl aminopeptidase

Gene product: Alternative name LAP; M17LAP; A-M17

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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

GAACTCGTACTCCTTGGTGACGTCGCGAGGATTAAGAGATGATCGTAGTGTGCTTAATGA

AAAGTATATTATTTATAGTGATTTTTATATTTATTTATTATTTATCATTTATTTTTTTCG

ATTTTATTTATGTTTTTTTCTCTACAAAATTATGGGTAGATTGTTTAGATTAAAAGCAAA

TTAAAATAAATTTAATTAACTAATGCCACCTGGATTATATAGACTTTTTTTTTTTTTTTT

TTTTTTATAATGCGCATTACTAATTTTATTTAGCCCCTGTTATTTTTTATACTTATCATA

ATTTATGTGCGCTATATATAGCAAAAAAATAAATAATATTATAAATATATTTATATAATA

TATAAAGGCGTTTTTTTTATTCGCGCCAAATTGCGTACACTCTTTTAAAAATAAAAGGAA

AAAAAATATATATATATTGTAATCCTTTAATAGTGTATGCATATTTGAACGCACTATTTT

GTATACTTATTTTTATGGAAATTTATTATATTAGGGATGGACCTCTCTCATATGTGAAGT

GGTTGGCTATGTATTGATGCACATATGGAAATATAATTATTTAAATTGGTAAATGTTCAA

AAGGTAGATAAACAAATAAAAAAATAAATAAATATTTTCCTATGCCATATAACGTACTCC

TTCCCCGCTAGCATCCATTGTGTTAATTTATTTTACGAGCATTGCTTCATATAATTATTA

TATGCATATATTGTGGAACTCAAAATATGTGTTAATATATAACAACCACGCTTTTTTTCA

TTTCCCCGTAAAATCGCCAATTCTACGTTTTACCTTATTTTTTAAACCCAAAAATAGATA

ATTAAATATATGCAAAGTTGCGTATTTTCAATTTGTGCATAATAGAGGTCGACGATGCTT

GTAGATGGCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATT

CATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAA

AGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCA

TATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATG

TATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTT

ATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATAT

AATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATT

AATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTC

AAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAA

AAATATATATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGT

TGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGA

CCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTC

TTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCC

TGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGA

ACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGA

ACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTA

TAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGA

CTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGA

ATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGT

ATATGAGAAGAATGATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGTGCTAGCGT

GACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGC

GGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGA

CGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACA

TGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATAC

CACTTTGTATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGG

TATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTT

ACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAA

ACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCTTCGGAACC

ATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCAT

CAGAAATAAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATT

GTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACAC

CAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAG

AGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATCGGTAA

AATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAATTACC

AGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGTGGTAG

TGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTTACTT

CTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGT

CAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGG

GTTGGGTGACTTTGGTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTTCTTACTTA

TATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATT

TTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTT

CAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAA

ATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATG

TTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTT

GTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTA

GCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTT

TTTTCCTTCAACCTTCAATTTCGGATCCACTAGCAAAGTGGTAGTTTTTGTTATATCTAT

TATGTATGTTATGAAAAGTGGTGAAATATATTTTTTCATTTTTAAAGTGAATATAGTTAT

GCATTTATATTAATTTCCAAATCAGAAATTTTATAATTTAGGCGTTTAATTATTAGCTTA

TTGTATTCGAAATTCATTTTAATATTTAGTAGACAATTTAGTACTTTGTTAAAATTTTCA

TATAATACATTTTCGTATTTGTGAACATTTGCATCTCCATTTTCTTATATTTGTAAAAAT

ATATATGGATATATTTTCGACTATACTAAATTTGTCTTTTAACTTAATTTATAGCCAAAC

TTTGAAAATGTCATATTTCCAACTATATTAATAATGCATAGAAATTTTAAGAGAAAACTG

GTAAATAGGTTTTACAACTATGTTTTGTATATATAAATATATATGATAAATGTAGTTCTT

TTATTTGTATAAAAGTTTTTCTATAATTGAATTGTCATTAGGAATATAAGTTATTTTCTC

AAAGGCTGATATGCATATTTGTGCAATATATTTTGTTTTTATAAAAAAATATATTTTTAT

TTATATATGTTTTTAGGTTTGTGAAAGATTCAATTGAAAAGATCAAATTTGAATTTATCT

ATTTTATATTTTTTCCTTTAATTAATTATATCATAATAGTTTCACATATATTTTGTTTTC

CTTAGTATATATATTATACCGTTATTTTAACAACCCCTCTATTTATTTCTCTTATTTTTT

TTCATATTTGTGTTAACAATTTTGTAATTTTTTAAAATATGATTAGATTGAAATGCACAC

AAAAATGAAAAAAAAAATAAATGCACAAATATTTATGCATGAGCATAGCTATATTTTCAG

TTTTTTTTTCATTGTTGTGTATCGCGAGCTGAGTGTCAATGACCAACCT

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/yfcu

Promoter of the selectable marker eef1a

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification 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.

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

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	L6963; lap 5’-targeting sequence, F (NruI)
Additional information primer 1	GAACTCGTACTCCTTGGTGACGTCGCGAGGATTAAGAGATGATCGTAGTG
Sequence Primer 2	L6964; lap 5’-targeting sequence, R
Additional information primer 2	CATCTACAAGCATCGTCGACCTCTATTATGCACAAATTGAAAATACG
Sequence Primer 3	L6965; lap 3’-targeting sequence, F
Additional information primer 3	CCTTCAATTTCGGATCCACTAGCAAAGTGGTAGTTTTTGTTATATC
Sequence Primer 4	L6966; lap 3’-targeting sequence, R (NruI)
Additional information primer 4	AGGTTGGTCATTGACACTCAGCTCGCGATACACAACAATGAAAAAAAAACTG
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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Transgene: Mutant parasite expressing a transgene

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Type and details of transgene

Is the transgene Plasmodium derived

Transgene: not Plasmodium

Transgene name

A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

KspI (SacII)

Selectable marker used to select the mutant parasite

gfp (FACS)

Promoter of the selectable marker

ama-1

Selection (positive) procedure

FACS (flowsorting)

Selection (negative) procedure

Additional remarks genetic modification

The GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.

Additional remarks selection procedure

This reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.

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Other details transgene

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Promoter

Gene Model of Parasite

PBANKA_0915000

Gene Model P. falciparum ortholog

PF3D7_1133400

Gene product

apical membrane antigen 1

Gene product: Alternative name

ama-1

Primer information details of the primers used for amplification of the promoter sequence Click to view information

Primer information details of the primers used for amplification of the promoter sequence Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2

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3'-UTR

Gene Model of Parasite

PBANKA_0719300

Gene product

bifunctional dihydrofolate reductase-thymidylate synthase, putative

Gene product: Alternative name

dhfr/ts

Primer information details of the primers used for amplification the 3'-UTR sequences Click to view information

Primer information details of the primers used for amplification the 3'-UTR sequences Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2

Insertion/Replacement locus

Replacement / Insertion

Replacement locus

Gene Model of Parasite

PBANKA_0306000

Gene product

6-cysteine protein

Gene product: Alternative name

230p

Primer information details of the primers used for amplification of the target sequences Click to view information

Primer information details of the primers used for amplification of the target sequences Click to hide information

Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4

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