RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1161

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_0608300; Gene model (P.falciparum): PF3D7_1209900; Gene product: ABC transporter B family member 7, putative (MDR7 (multidrug resistance protein 7); ABCB7)
Phenotype	No phenotype has been described

Last modified: 20 March 2016, 14:33

*RMgm-1161

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) : 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_0608300

Gene Model P. falciparum ortholog PF3D7_1209900

Gene product ABC transporter B family member 7, putative

Gene product: Alternative name MDR7 (multidrug resistance protein 7); ABCB7

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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

AATGATGTTTTTTCCTTCAATTTCGATATCGAATTCGGATCATTTTTATCGGGTGGTCAA

AAACAAAGAATTTATCTTGCACAAAATTTATGTAAAGATAATAAAATATTAATTTTAGAT

GAACCTACATCTTCTTTAGATAAATTGTCAGAAAAAATTATAAATGAGGCTTTATATAAA

TATATGAAAGGAAAGACTACTGTTATATTTACTCATAGACTAGATTTACTAAATTTTGTT

GATTATATTGGCGTATTAAATGATGGAAATATGATACAATTTGATTTAAGAAATAAAGTG

CTACAAAACCCATGTTATATACTTAAACAAATTCTTAATCAGAATAAACATTAAAAAATG

TAATCATATATATATATATTATGTTTCACATTTTGTTTTTTTATCAGTATATAACAAAAT

GTGTGTTTTTATTGTTTCCCATTTGAATATATACATTTATTGCTTGTAACTGGGGATACC

CTTTTTTGTGCCTCAAAAAAATATGACCATGCAGGTTAAAATGTGTCTTTTTAATTATAC

AAATATATACCTAGATATATGTGAAATGTTTTCATCTTTTTGTCTTATACTTTCATGATA

TGATTTGTATACATGTGTTTTTTTTTAATTTTTTATACATACTTTTTCAAAATTAAAGAT

AAAAATTTTTTACATCCTAAAATGAAATTTTTGTTTATGTAAACTATTTTGTGTACATGT

ATATATGCACTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGT

CGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTA

CCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG

CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCT

GTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG

CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG

GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC

GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCT

GATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGT

TCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTT

TGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT

TTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC

CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC

CTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGT

CGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCT

GGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA

TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAG

CACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCA

ACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA

AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAG

TGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGC

TTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAA

TGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTT

GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG

GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTT

TATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG

GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTAT

GGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT

GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA

AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT

TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTG

TTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA

GATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGT

AGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGA

TAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC

GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACT

GAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA

CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG

AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT

TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTT

ACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGA

TTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAAC

GACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC

TCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAA

AGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC

TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCA

CACAGGAAACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGA

ACAAAAGCTGGTACCGTGATGGAATACACTTAGAAATTTGATTAATTCGAAATGATTACA

ATATGAATATATAATTTCTTTTTTATATTCAATTACAATTATACCACATGTTTATATTGA

ACATACACATTGGAAGTTTTTATTTTGTTATTTCTAAAGTTATCAGAAAAATAATTTTAT

AAAATTTTTTACAGACATTTTTGATTTTAAACATGTTTTGGATTATAAAGGTACATAGAA

GTTTAATTTAACGAAAATAAATAATTTTTTCTTATTTTTGTTATTTATGATTTGTTTAAA

GATGTTAAAAAAAAAACGAGCAGTAAATTTATATGTGCTATTGAATGCACACAGATGCTT

ACTAAGTAGTGGTTTGAATGTTAATATGTATAAAGATCTAATCGTATATACATCAATTAA

TAATTTAAGTTTTATAAATATTACAAAAAGTTTCTATAGTAATAATACAAAGAAATATAT

TAAATATCTGAATAATATTAATCCTAATAAATGTGAAAATAATAAAAGAAGCATATCACA

AATTTTTTTAAATAATAAAAATATATCAATAAATCATAATGAAACTAATGTCCGGAAATT

CA

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 Three unsuccessful transfection experiments using DNA construct pL1629 (exp. 1665, 2103, 1728).
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 MDR7 (multidrug resistance protein 7).

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	GGGGGTACCGTGATGGAATACACTTAG
Additional information primer 1	5’- MDR7 targeting region F (Asp718I)
Sequence Primer 2	GGGAAGCTTGCTACTAAGCATGCGAAGC
Additional information primer 2	5’- MDR7 targeting region R (HindIII)
Sequence Primer 3	GGGGAATTCGGATCATTTTTATCGGGTGGTC
Additional information primer 3	3’- MDR7 targeting region F (EcoRI)
Sequence Primer 4	GGGTCTAGAGTGCATATATACATGTACAC
Additional information primer 4	3’- MDR7 targeting region R (XbaI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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