RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-758

Malaria parasite	P. berghei
Genotype
Genetic modification not successful
Disrupted	Gene model (rodent): PBANKA_1358100; Gene model (P.falciparum): PF3D7_1345200; Gene product: rhomboid protease ROM6, putative (ROM6)
Phenotype	No phenotype has been described

Last modified: 16 March 2013, 12:09

*RMgm-758

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) : 23490234
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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	Not applicable
Other information parent line	Two attempts to disrupt rom6 were made in P. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32). This 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). A third attempt to disrupt rom6 was made in P. berghei ANKA 676m1cl1 (RMgm-29). This 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	J. Lin, G.R. Mair, C.J. Janse, S.M. Khan
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_1358100

Gene Model P. falciparum ortholog PF3D7_1345200

Gene product rhomboid protease ROM6, putative

Gene product: Alternative name ROM6

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used PCR construct

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

GAACTCGTACTCCTTGGTGACGTCGCGACTATTTTTTATAGGCACATTTTGTGTGATATC

ATGCCATCATTCAATAGATGAGTTGCCTAACTGTTTTATCACTTATATATTTATATTTTT

TTTGTAATTTCAAAATTGCATGATCAAACATTAAACATAACACACGATATTAATGAAAAG

TAAACTAAAAAAATGTAAAAACATAAATTTTTTTGTGCCAAAAACGAATAAATAAATTAT

AACTTATAATATTTTGTAGAAACGATATTTCCCATGCATACACCTTACTAATATATATGT

ATATTTTTTTTAATTGGCATGTACATGTCTTATCATTCAATTTGTTCTTTATTTGGAAGT

CTAAACAAATAATTCTACGCCTTTGTTATTATTCTCATTCTTTTTAAAACAGGGAATAAA

TGTGTAATGTCACTATTTTTAACCCCCCAAAAAAGAAATAATAAATATCCATATAAAACT

AAATAACCAAATTTTTCAATTTCAAATGATTTTTTTAATATGTATATTTATATATTACAC

TACACTCCTCATTTATTAATATATTTTTTTCTAATAAAATTAACTATTATGGTAGTATAT

ATATTGATACAAAAAATTATAAAAAAATTGTTTATATATATTTAGATAATTATCAACGTT

AGTTATTGTAGTTATTACTATTTATATCTACATTTTTTTTTAAACGATACGATTATCTTT

CCTCCAACGTTAGTACTATATTTTAATTTCGCCATTTTTTTATAATTTTTATTTTTCAAT

AAGTAGTGTTTCGCTTATAAGGTGTAACACTATTGTAAAAAAAACGCTATACGAGGTCGA

CGATGCTTGTAGATGGCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTAAGTTTACAAT

TTAATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTTTAAATGCTCA

CCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTTTCAAAAAAAT

ACTATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAAAAAATATATA

AAAATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATAAAAATTAAAG

TGCCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAAATCCTCATAT

AATAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATATAAAATAATA

TGTAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTAATTTTTTCAT

TTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACTTTGTGTATAA

ATATATAAAAATATATATTTGCTTATAAATAAATAAAAAATTTTATAAAACATAGGGGGA

TCCATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAG

AACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATATTTCCAGAGAATGACC

ACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTC

TCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAA

CTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAA

CTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGT

TCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATC

ATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTT

CTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAA

TTTGAAGTATATGAGAAGAATGATGCTAGCGGAGGAGGTGGATCTGGTGGAGGTGGAAGT

GCTAGCGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTAT

GAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAAT

AACAAAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCC

ACACTACATGGTGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTAC

AAAGATACCACTTTGTATACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATC

ATGTATGGTATTCCACGCTGTGTTGTCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAG

AAATATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGATGAGAGGTGTAAAAAG

ATCATGAAACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGATATTGGTGAGGCT

TCGGAACCATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTAC

ACCATCATCAGAAATAAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATC

ATCAGATTGTTGGTTGAAGAAGGTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAA

ACTGACACCAACGAAAACTTCGAAGGTGTCTCATTCATGGGTAAAATCTGTGGTGTTTCC

ATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACTGTTGTAGGTCTGTGCGT

ATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTTATTCTACGAA

AAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCCACC

GGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGA

ATTTACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTC

CCAGAGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTA

GTTCCAGGGTTGGGTGACTTTGGTGACAGATACTACTGTGTTTAACTCGATCCCGTTTTT

CTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTG

TGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTAT

TTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATT

TTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAAC

ATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATA

ATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAA

TATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAA

ATGATGTTTTTTCCTTCAACCTTCAATTTCGGATCCACTAGAAAAACATAACCACCATAT

GCATGATATTATTTTGCATGGGGAACATATATAATTTTTGTGTGCAATTTCATTTTTACG

TATTATTTATTTGGTTTGAATCATATAAAACTCATTATATATATATCCATATTGTATTCA

TATATACTTAATATATTATTTTCTTGCTTCCTTTTTGTGTATTTTGTTTACCAAAACATG

TTACGTCACCTTGCATTTGTTTCACTATTTTTTTTTCAATTATACGATAAAACATGTTAT

ATATATGTATAGCATACACAATTAACTAATGAAAAACCCTAAACACATTTTGTGCAATTC

CTAGTAAAATTACGACCTTATCATTTTATCGATTTTTTTAAAACCTTAAAATATAGGAAA

GGTGTTTTATGTAATTTAAAATATAATCAATATATAATAAAAAAATGTAACCATAGAAAA

CTTTTTCCCTTTGCCTTTTTTCAAAGGGCCTTACAACATAATTATATATAAAAAATATAT

ATATACTTTTTCTTCATGGCAATATATATATATAAACAATATGTAATGAATATTTTAATG

TGGATTTTTCCCGTTCTAATTTGGAATAACAACGTTTTGGAGCGGATGAAGTTATCACTA

ATACACCCTTTTTCGCATGCACAAAAAGCATTTTATTCTTAGTTGTTTATATATTGGGGT

TCAAACGTATAAAAAGCTTAACGAAATAGTATATATTTAAAAACGAAAACAAGAGGGGAA

AAAGGAAAAACAGAAAAAAAAAGAAAAAGAAAAAAAAAAAAAGAAAAAAAAGAAAAAGAA

AAAAAAAAGAAAAAAAAGAAAAAGAAAAAGAAAAAGAAGAAAAAAGCAAATTTTAAGAAT

AGGTATAGCAAAGCAGGATGTCGCGAGCTGAGTGTCAATGACCAACCT

Restriction sites to linearize plasmid NruI

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 5-fluorocytosine (5-FC)

Additional remarks genetic modification The unsuccessful attempts to disrupt the rom6 gene suggest that ROM6 is essential for asexual blood stage development (exp. 2118, 2119, 2140).

Rhomboid proteins are intra-membrane proteases that play a role in multiple processes. They belong to a family of serine proteases that cleave cell-surface proteins within their transmembrane domains. The Plasmodium genome encodes a total of 8 rhomboid proteases (ROM1, 3, 4, 6, 7, 8, 9 and 10).

Plasmodium ROM6 is the ortholog of T. gondii ROM6, which clusters with the PARL-like mitochondrial rhomboids and localizes to the single mitochondrion. These ROM6 proteins share common features in their TM domains and catalytic sites, which are characteristic for PARL-type rhomboids and contain predicted mitochondrial-targeting sequences.

Attempts to disrupt the gene were made 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 respectively. Primers 2 and 3 have 5’- extensions homologues to the hdhfr::yfcu selectable marker cassette (CATCTACAAGCATCGTCGACCTC in primer 2 and CCTTCAATTTCGGATCCACTAG in primer 3). This selectable marker cassette was excised by digestion with XhoI and NotI from a plasmid (pL0048) that contains the P. berghei eef1a-hdfhr::yfcu-3’dhfr/ts (i.e. promoter-drug selectable marker-3’ terminator sequence) selection cassette. Primers 1 and 4 have 5’-terminal extensions with an anchor-tag suitable for the second PCR reaction. In the second PCR reaction, the amplified 5’- and 3’- targeting sequences were annealed to either side of the selectable marker cassette, and the joint fragment was amplified by the external anchor-tag primers 5/6, resulting in the PCR-based targeting construct

Before transfection, the PCR-based construct was digested with NruI (in primers 1 and 4) to remove the ‘anchor-tag’ and with DpnI that digests any residual pL0048 plasmid.

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	GAACTCGTACTCCTTGGTGACGTCGCGACTATTTTTTATAGGCACATTTTG
Additional information primer 1	7055 (NruI); 5’- rom6 targeting region F
Sequence Primer 2	CATCTACAAGCATCGTCGACCTCGTATAGCGTTTTTTTTACAATAGTG
Additional information primer 2	7056; 5’- rom6 targeting region R
Sequence Primer 3	CCTTCAATTTCGGATCCACTAGAAAAACATAACCACCATATGC
Additional information primer 3	7057; 3’- rom6 targeting region F
Sequence Primer 4	AGGTTGGTCATTGACACTCAGCTCGCGACATCCTGCTTTGCTATACC
Additional information primer 4	7058 (NruI); 3’- rom6 targeting region R
Sequence Primer 5	GAACTCGTACTCCTTGGTGACG
Additional information primer 5	4661; anchor-tag primer, F
Sequence Primer 6	AGGTTGGTCATTGACACTCAGC
Additional information primer 6	4662: anchor-tag primer, R

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