RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-815

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_0833100; Gene model (P.falciparum): PF3D7_0932300; Gene product: M18 aspartyl aminopeptidase (M18AAP; DAP)
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	No phenotype has been described

Last modified: 23 December 2016, 17:44

*RMgm-815

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-815
Principal name	2060cl1
Alternative name	∆dap
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
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 M18AAP (DAP) Protein (function) Plasmodium falciparum (PfM18AAP)is expressed by all intra-erythrocytic stages. Like the human aspartyl aminopeptidase, the metallo-exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate.PfM18AAP is expressed in the cytosol. Phenotype Blood stages showed a normal growth/multiplication in mice (and produced normal levels of hemozoin). 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_0833100

Gene Model P. falciparum ortholog PF3D7_0932300

Gene product M18 aspartyl aminopeptidase

Gene product: Alternative name M18AAP; DAP

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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

GAACTCGTACTCCTTGGTGACGTCGCGATGTTGAAGGCATAATAAAACAGCAGCTATTGG

TTATTTATTATATGAAAAAAAATATATATAGTGCCGAAACTTTCTACAAAAATCGAACAG

AAAGAGGATTTATAAATATTAACAAAATAGGTGTAGTAATAAATTGTATCGATAGTATAT

ACACTTATTTATGAAGCAACGTAGAGTATTCATTCATTAAAACATTTAATATTCTATTCT

TTTTACAATCCATGGGCTTATTATAATGATTTGCCACATGTTTCATACCAATTAAAATAA

TTTAAAATGATTAAAGTAAATTTTATTTTATTTTATTTTATTTTAGTTATTTTTTTCTAT

TCTTAGTACACCAAACATATATACAGTATGTATGTACATTTTCGATAAGCCACTGATAAA

AAAATAAAAATATCGATTTTATTTACTCTTGTTTTTTTATGCATTATAATATATGCCTCA

AATTTTTATATTCTATTATTATTTTGTATATATATGCATATATAATATCCAAAGTATAGA

AAACAAAAATATTTGAATTATCAAAAATGATAAAAAAGCATATGACTGGAAATATGCCAT

TTCACTAAAATACAAACTAGTTTTATACATTTTTTTATATTTCCAACATATCTATAAAAA

AAATAATATTATAATTTTTTTTTTATTTTAATTTAATATGATAAAATAGCTAGCTTGTTA

ATGCGATTTGTATCATATGTTATAAGTATTTTTATTTTATAAAAATATTTGAAATATACA

CATACAAAATCCACAACATAGTGTAAGTGTAGGCATATCGTATGTATATGAGGTCGACGA

TGCTTGTAGATGGCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTA

ATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCC

CTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACT

ATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAA

ATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGC

CCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAAT

AATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGT

AAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTG

CCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATA

TATAAAAATATATATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCC

ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAAC

GGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACA

ACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCC

ATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTC

AAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTT

ACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCT

GTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATG

CAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTG

CCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTT

GAAGTATATGAGAAGAATGATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGTGCT

AGCGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAG

GAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAAC

AAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACA

CTACATGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAA

GATACCACTTTGTATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATG

TATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAA

TATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAGATC

ATGAAACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCTTCG

GAACCATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACC

ATCATCAGAAATAAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATC

AGATTGTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACT

GACACCAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCCATT

GTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGTATC

GGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAAAAA

TTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACCGGT

GGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATT

TACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCA

GAGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTT

CCAGGGTTGGGTGACTTTGGTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTTCTT

ACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGC

ATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTT

TTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTA

TATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATA

ATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATT

ATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATAT

GATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATG

ATGTTTTTTCCTTCAACCTTCAATTTCGGATCCACTAGCAGTCAAATTATAAGTACAAAG

GAAAATCCACACACATTGGCATTATATAGAAATTGAGAAAAAATTATACGCACACCCCCC

CAAAAAAATGTTTATGTACTAATTCAAGGAATCATATCACTACAATGAAATACTTGTGAT

ATAATAACATTATATTTCGTACATTTTTTAATATTCATTTAAATTTATCTCTAATATATA

AATGTCGGCATTTCCGCAATAAGCCACATTTTTTTTTACATATGATTTTATTATTTATAC

ATTTTCAACAAATCTGATAAATAAAAATGAGCAATATTCAGCACTTTTTGTGTCTTTTAA

TATATGATAAATAGAACTATAAACGAAAAAAATTAATTTTAAATATACTTTTTCATTTTT

CTTAAACAACGCAGATTAAGTTCCCTTTTATTTTATTCACTAAAAACGCTTTTAAAATTT

TGCACATGTGTATTATGTTTCATGCATAATTTTTATGGAATATCAACATTCTTCTTTTTT

AATAAAACCTTATTATACCATAATGAATAAAATGTATAAAAAAAAGCTTTATACTTATGC

AATATAAATTATATGTAAAAAAATAATGTGCATATGCGCATGCATATACATATGAATAAT

GTGACCAAAAAAACATGACATCCATAAAATTGGAAAGTGGCCATGGTATTGTTATATAAT

TGTCAATGTTTAATGCAATATTTTTCTAATTACAGATAGGGAAAATAAATAATACACACA

TATATATGCATACATCCATGCAATATGCATTTTAATGTCGCGAGCTGAGTGTCAATGACC

AACCT

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	L6971; dap 5’-targeting sequence, F (NruI)
Additional information primer 1	GAACTCGTACTCCTTGGTGACGTCGCGATGTTGAAGGCATAATAAAACAG
Sequence Primer 2	L6972; dap 5’-targeting sequence, R
Additional information primer 2	CATCTACAAGCATCGTCGACCTCATATACATACGATATGCCTACAC
Sequence Primer 3	L6973; dap 3’-targeting sequence, F
Additional information primer 3	CCTTCAATTTCGGATCCACTAGCAGTCAAATTATAAGTACAAAGG
Sequence Primer 4	L6974; dap 3’-targeting sequence, R (NruI)
Additional information primer 4	AGGTTGGTCATTGACACTCAGCTCGCGACATTAAAATGCATATTGCATGG
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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