RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1159

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_0401200; Gene model (P.falciparum): PF3D7_0302600; Gene product: ABC transporter B family member 4, putative (MDR4 (multidrug resistance protein 4); ABCB4)
Phenotype	No phenotype has been described

Last modified: 20 March 2016, 14:31

*RMgm-1159

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	4
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 26991313
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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	Rijpma SR, Janse CJ, Franke-Fayard BM
Name Group/Department	Leiden Malaria Research Group
Name Institute	Leiden University Medical Center (LUMC)
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_0401200

Gene Model P. falciparum ortholog PF3D7_0302600

Gene product ABC transporter B family member 4, putative

Gene product: Alternative name MDR4 (multidrug resistance protein 4); ABCB4

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

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

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

AGCTTGCATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAAC

ATATAATTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAA

AATATATAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTT

TGTATGTTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAG

ACACAAATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATA

TTTTCTTTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTT

ATTTTGTCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAA

TAATTTTAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTA

ATACACATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATAT

ATATATATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAA

TAATATATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCA

TACATGCAATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATT

ATGGATATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATT

TTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAAT

TTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTG

AARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTT

TATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCC

AATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTA

AGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTA

CACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAA

TTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTG

ATATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATG

TATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCC

TGTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATAT

ATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACC

YTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAA

AATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGT

TTAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATA

TTTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTC

AATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAA

AGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTA

TTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATA

TAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGG

GTTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATT

AAGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAAT

TTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCA

TTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTT

CATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCAT

AAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGC

CTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACG

CCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGAC

TCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAA

ACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTT

TCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAG

CGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAG

GATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCT

CTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTT

TTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCT

GCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGAC

AATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCC

TACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCG

AGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAG

GCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGG

AAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCAT

GAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGAT

GACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGAT

CAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTG

CTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATC

TGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTC

GGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGAC

ATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTG

AGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGAC

GAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAG

CTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATC

GCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAG

TTCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAG

CAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATC

AAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGA

CGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTT

TCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGT

GTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTA

TTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATAT

TTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAA

CATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTAT

AATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATA

ATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACA

AATGATGTTTTTTCCTTCAATTTCGATATCGAATTCCCACAATTAAGCATTGTGATTATA

TTTATGTTTTAAAAAATGGAAAAATTATTGAACAAGGGTTACGAACAAAATTACAAGAAG

ACAAAAACAGTGAATATTCGAAAAAGCTCAACGAATTTTAGTGTTAACAAATATTTTATC

GCTATACAATTCATAGAAAAATAAAAGAAAATAAATCCATTCTCAAAAAAATAGCTATAT

ATACCTATATCGCATATAGATAATTGCCGTATATATCCCTATCCTATTCATTTGCGTTTT

TTAACAAAACAATAGACCATTTCTCAAATAAAATGACACATACTCATACTTTATATTTTT

ATTGGACATAAATATAGCTCTATGTATGCATTTACTTTATATAAACAAAATCGCATTTTT

ATTTTTTCGTAAAGATAAGAAGAAAAATAATTCACATTATTTATTTCATCATTCCTTCTT

TAATTTTTTGTTTTTTTTCTTTATTAAGAAAATACAATTTGTACTTTAGTTATTTCTTAT

TTGTTATTTTATTTTCATATAGAAAAGGTTATACTTACAAAATTATATATGTCACATGTA

TGTGTGTACATATGCTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAG

TGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGG

CGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGA

AGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGC

GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTAC

ACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTT

CGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGC

TTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATC

GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT

CTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGG

GATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC

GAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGC

GGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAA

TAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTC

CGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAA

ACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA

CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG

ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAA

GAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTC

ACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC

ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTA

ACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAG

CTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATA

GACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC

TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCA

ACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGG

TAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAA

TTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGT

GAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT

CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTG

GTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA

GCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC

TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGT

GGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAG

CGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC

GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAG

GCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCA

GGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGT

CGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCC

TTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCC

CCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGC

CGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAA

CCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGAC

TGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCC

CAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAA

TTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAA

AGGGAACAAAAGCTGGTACCGTTGCTTATCGTATTATGGGATATATTTTGTTTATTAGAT

CATAAATTTTTATTAATTTTATGAAAATAAACTAAAGGAGAAAAAGAACAAAAAAACAAA

ATGCCCAAGGAATGAACTTAATTTACTTTAAAAAGTATATAACACAGAATATTATAAAAA

TAAAGAGCTGAAATATTCAAAGGAAAAAAATACTTATTACAGTAAAATATTTTATAATAA

GGCTCATATAATTTATATAAGTATAAAAATGTATGAAAAAAGGTGTAATGAATATAAATA

TATAATTTAAAGCTGATGAAAGTAGGGTGATGGATAAAAATAGATAATGATGAATACGCC

ATGTTACTGAAATAGAAAAGGAAAACAAAGAGAAACAAAAAAAAATATATATCAAAATTA

GTCAATATATCTAAGTTACTAAATTTTGCAAAACTAAAAGCTGATAAAAAAACTAAAAGA

AAACTTAACATACTAATTTGCGAAATACAACATACATCCAAAGAAAAAAAGTATTTATTA

TTTCATAGAAAAAAGAAGACATAATATGCGATCTTAATATGTAATTATATAGAGAAAGAA

AAAGGGTGATTTAGAAACATGAAACTGCTGGGA

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 tgdhfr

Promoter of the selectable marker pbdhfr

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification Four unsuccessful transfection experiments using DNA construct pL1725 (exp. 1820, 1881, 1929, 2040).
The unsuccessful attempts to delete the gene indicate an essential role during blood stage growth/multiplication.

In different Plasmodium parasites 14-16 ABC-transporter genes have been identified (16 in P. falciparum, 14 in P. berghei). Based on phylogenetic analysis of the conserved nucleotide-binding domains, seven of these are recognized as members of the B family of ABC transporters (both in P. falciparum and in P. berghei). This includes MDR4 (multidrug resistance protein 4).

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	GGGGGTACCGTTGCTTATCGTATTATGGG
Additional information primer 1	5’- MDR4 targeting region F (Asp718I)
Sequence Primer 2	GGGAAGCTTCCCAGCAGTTTCATGTTTC
Additional information primer 2	5’- MDR4 targeting region R (HindIII)
Sequence Primer 3	GGGGAATTCCCACAATTAAGCATTGTG
Additional information primer 3	3’- MDR4 targeting region F (EcoRI)
Sequence Primer 4	GGGTCTAGAGCATATGTACACACATACATGTG
Additional information primer 4	3’- MDR4 targeting region R (XbaI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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