RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-255

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1232200; Gene model (P.falciparum): PF3D7_0517400; Gene product: FACT complex subunit SPT16, putative (FACT-L; FACT)
Phenotype	No phenotype has been described

Last modified: 22 September 2011, 11:16

*RMgm-255

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)	Not published (yet)
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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 507cl1 (RMgm-7)
Other information parent line	P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter (PubMed: PMID: 16242190).
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Attempts to generate the mutant parasite were performed by
Name PI/Researcher	E. Laurentino, C.J. Janse
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_1232200

Gene Model P. falciparum ortholog PF3D7_0517400

Gene product FACT complex subunit SPT16, putative

Gene product: Alternative name FACT-L; FACT

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT

AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC

GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT

CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC

TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA

CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC

ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA

CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT

TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT

ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT

GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA

CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC

GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC

CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG

GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA

TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC

GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG

CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT

TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC

TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT

CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC

ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC

TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT

TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG

ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC

AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA

CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG

GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTA

GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA

CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG

TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG

GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG

CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC

CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG

TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT

GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG

CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG

CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA

ATTGTGAGCGGTAAACAATTTCACACAGGAAACAGCTTGACCATGATTACGCCAAGCTTG

GGCCCCCGCGGCATAAGGATATAAAAGGGGTTTATATATATACTAGCCATAAGTGTGCAT

ATAATTTTTGAACTTTGTAAAAACAAGCTAGCAAAAATGAGTGAAGGATTAGATGTAGAT

AACGCCAAAGAAAAGATAAACTTCATGTTTACATATTGGAAAAATATTAATAATAAGGAT

TTTGAAAATAGTAATGCATTCTGTATTTTATCGGGTAAATCTAGCAAAGATGATAATGCA

ACGATTCAAGAACAATTTCAGATGTGGCTTTTAGGATACCAACTAACAGAAACGTTTTTT

TTATTTTGTAAAAAAAATGAAAAACTAATAATACTCACAAGTGATAAAAAAAAAAAATTT

TTACAACCATTATTAGATAAAATGAATAATATAACAATTTTAGAAAGAAATAATAATGAT

AATAGTGAAAATTTTGAAAAAATAAAAAATGAAATAAATATGTTTAATGATAAAGAACTA

TTAATTTTAAAAGATAAAGATTCTACTGGAAGTTTTTTTGAAGCATGCTATGATTTTATT

AAAAATTTGAATAAAACTGAAATAGATGTAAATAATAATATAAAAAGTTTGTTAAATCTT

AGATCTAAATCAGATGTAAAATTACAAAAATCAGCTAGCGATATAGCTAGCATTATAATG

AAAAATGTTTTGATAACTACCATTGAAAATTCATTGGATAGTGAAGAATATGAAAGTCAT

AATAAAATAAAAGAAAAAGTTTTAAAATTTAATGAAAATAAAAAATGTGTTGTTAAGATA

AAGGATAAATTAAAAGCAGATATAGATGATATAGATGTTATATATAGTAGTGTACAAAGT

GGTAATAATTTTTTGTTAAATTTCAAAAATACTAATGATAATAATTATTTATCACAAAAT

GATGGTACTATAGTTATAGGTATAGGTGTCAAATATAAAGAATTGTGTGCAAATATAAAT

AGAACCTTATTATTAAATGCTAAAGAATATCATAAAGAACTTTATAATTTTACGTTTTCT

ATACAAAAATACATAATTAACGATTGTTTAAAATATAATACATCTTTTTCAGATGTATAT

AAAAAGGCAATGCAATATATTAAAGATAATAAACGAAACTATCAAACAATTGGAAATATA

AATTTAGAAAATTATTTTATAAAATGTTTGGGTCATGTTATAGGCTAGGCCTCTGCAGCC

CAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAATTTAATATTCATACTTTAA

GTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCACCCCTCAAAGCACCAGTA

ATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAATACTATGCATATAATGTTA

TACATATAAACATAAAACGCCATGTAAATCAAAAAATATATAAAAATATGTATAAAAATA

AATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAGTGCCCTTTATTAACTAGT

CGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATATAATAATATAATTAATATA

TGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATATGTAAATTAATTCAAAAA

ATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCATTTGCCTTCAAAATTTAAA

TTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAAATATATAAAAATATATAT

TTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGATCCATGGTTGGTTCGCTA

AACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACCTGCCCTGG

CCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAA

GGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAAT

CGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAA

GGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAA

TTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCC

ATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGT

GACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGT

GTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAG

AATGATTAAGGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAA

CTGTAAAAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATA

GCTAAAATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAAT

GGCTAGTATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTA

AAAATTTAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTAT

TATTATTATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGAC

TTGATTTTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATT

AAAAAAAAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGATGGGTACCCTCGA

GGCTAGCGATATCTGATCACCCGGGGCGGCCGCGAATTCGAAATACCATATCCAGAATTA

ACATTTTCAGGTGTTCCAAACAAAAGTAATGTTGAAATATTTGTTACTGCCAATACTATT

AATCATTTAATTGAATGGCCACCTTTTATATTATCTGTAGAAGATATTGAAATTGCATCT

CTAGAAAGAGTACATCATGGTTTAAGAAACTTTGATATGATTTTTGTTTTTAAGGATTAT

ACTAAGCCTGTAAAAAGAATTGATGTTATACCTGTCGAATATATAGACACAATTAAAAAA

TGGCTAACTACTATTGACATAGTATATTATGAAGGAAAAAATAATCTTCAATGGGGAAAT

ATACTCAAAACTATTCTTGCAGATATTGAATCTTTTGTTAATTCCAAGGGATTTGATGGA

TTTTTAGGAGAAGATGATGATGAAGAAGAACAGTCTGCTGAAGATGAAGATGAGGACGAC

GAATATGAAGTTGATGAATCAGAAATGGTAAGTCAATCTCTATCTTTTAACTTTATCATC

ATGTTCTCTAATTTTGTTATTTTTCTAATTTTTTTGCATATTTACATTCCCTTTACTTCT

TTTTTTATTTTTCAGAGTGCAGAAGAAGACAGTGAATATGAAGACAGTGAAGAAGAAAGT

TTAGCCACAGAAAGTGATGGGGATGAAGAAGTTGAAGAAGACTCAGATGATGAAGGATTA

TCTTGGGACGAGCTTGAGGAAAGAGCTAAAAAAGGTAAACCCCAATATATACAGTAAAAT

GAGATGATTTTACTTTGGCTACTAATTTTAATTCCTGTTTATTGAAAACATAAATAGTAT

CTCCACATGTATCATGTATTTTTTTTTCTTTATTATTTTCTATTTCTTAGACGACAAAAA

ACGATTTGCATATCAAAGTGACGATGGTGATGATAGTGAAGGATACAATAAAAGAAAGAG

GAAAAAAAATTAATTTTACAAATCATTTATATTTTTTTTTTTCAATTATTAATTTTTGGC

TATTTTCATAACACTTTATTTTTATTTCATTCTGTTTATCATAATAAGCATATTATTACA

CACCCAATTGAAGTTCAAATATAAATATAGAAATAATCTTATTATTATTTGCAACTCGCT

TTTTTTTAGCATACATATTAATTATATTTTATTAGAGTAAGAAAATAAACATATATATGT

ACATTTATTTATATATATATCATTTTATACGTCTTTTGGCCTTTTTTACAAATGCCAAAC

AAAATTTTTGCAAATGTACACATACGTACGTCTATAATG

Restriction sites to linearize plasmid

Partial or complete disruption of the gene Partial

Additional remarks partial/complete disruption PB000119.00.0 reads 3231 bp (PlasmoDB). The selectable marker was inserted between bp positions 1090 and 2502, replacing everything in between.

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 The unsuccessful ttempts to disrupt the fact-l gene in P. berghei indicates an essential role of FACT-L for blood stage development/multiplication.

See also RMgm-652, RMgm-653 and RMgm-654 for mutants in which the promoter of fact-l is replaced by an 'asexual blood stage specific’ promoter that is silent in gametocytes (the promoter of PBANKA_140060).

Disruption experiments were performed in the Leiden Malaria Research Group (Exp. 902, 917; pl1230)
An additional attempt to disrupt the gene was performed using a different plasmid targeting a bigger part of the gene (double cross-over integration; exp. 1147; pl1147). Primers used to amplify the target sequences are
L3576 (SacII): gcCCGCGGATGCAATCCTTATTAGTG; L3576 (StuI): gcAGGCCTCATCTGATTTAGATCTAAG; L3577 (XhoI): cCTCGAGCTAGAAAGAGTACATCATGG; L3578 (EcoRV): gcGATATCGCGTTCATTTCATATATAAC.
The selectable marker was inserted between bp positions 519 and 2742 of the PB000119.00.0 ORF, replacing everything in between.

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	GCCCCGCGGCATAAGGATATAAAAGGG
Additional information primer 1	L2819 (SacII); 5'
Sequence Primer 2	TCAAGGCCTAAGCCTATAACATGACCC
Additional information primer 2	L2820 (StuI); 5'
Sequence Primer 3	CTGATGAAttcATGCATGATGAAAT
Additional information primer 3	L2779 (EcoRI); 3'
Sequence Primer 4	ATTATAGACGTACGTATGTGTACA
Additional information primer 4	L2780; 3'
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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