RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1146

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0720900; Gene model (P.falciparum): PF3D7_0418800; Gene product: MOLO1 domain-containing protein, putative Name tag: GFP
Phenotype	Gametocyte/Gamete; Fertilization and ookinete;

Last modified: 2 December 2014, 09:28

*RMgm-1146

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene tagging
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 25418785
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 cl15cy1
Other information parent line	A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/Researcher	Guerreiro A; Mair GR
Name Group/Department	Instituto de Medicina Molecular
Name Institute	Faculdade de Medicina da Universidade de Lisboa
City	Lisbon
Country	Portugal
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Name of the mutant parasite
RMgm number	RMgm-1146
Principal name	2182
Alternative name	PBANKA_072090::GFP
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	mRNA present but absence of the GFP-tagged protein (translationally repressed)
Fertilization and ookinete	Expression of the GFP-tagged protein in developing zygotes/ookinetes.The fluorescence pattern showed evidence for localisation of the protein in the crystalloid body of ookinetes.
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal GFP-tagged form of PBANKA_072090 Protein (function) This gene was identified in a screen of mRNAs in female gametocytes that are bound to the translational repressor proteins DOZI and CITH. These genes are transcribed in female gametecytes but are translationally repressed. Translation occurs after fertilisation of the female gamete. Phenotype mRNA present in gametocytes but absence of the GFP-tagged protein (translationally repressed). Expression of the GFP-tagged protein occurs in developing zygotes/ookinetes. The fluorescence pattern showed evidence for localisation of the protein in the crystalloid body of ookinetes. Analysis of mutants lacking expression of PBANKA_072090 (RMgm-1147) showed the production of normal numbers of oocysts but the lack of formation of sporozoites within these (empty) oocysts Additional information mRNA were translationally repressed by virtue of their 5′ UTRs or elements within its open reading frame (ORF) as the 3′ UTR in the constructs was taken from the P. berghei dhfr/ts gene known to allow translation of P28 in gametocytes. Previously, only p25 was shown to be repressed in a 5′ UTR-dependent manner. The results here suggest that this mechanism may frequently be employed. In this study 5 (translationally repressed) genes were C-terminally tagged with GFP (PBANKA_082120, PBANKA_133470, PBANKA_111410, PBANKA_010770, PBANKA_072090. All genes are transcribed in gametocytes but expression of the GFP-tagged proteins only occurs after fertilization in the developing zygote/ookinete Other mutants Mutants lacking expression of PBANKA_072090 (RMgm-1147)

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_0720900

Gene Model P. falciparum ortholog PF3D7_0418800

Gene product MOLO1 domain-containing protein, putative

Gene product: Alternative name

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Details of the genetic modification

Name of the tag GFP

Details of tagging C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct (Linear) plasmid single cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

ATTTCGATATCGGGGAATTCCATATATTAGAGTATTGGAGATTGGGTAACACAAGTTGTA

ACGATGGAATTTTACTTCTTTTTGTAAAGGATGATATAAATTTTGTTTTGAAATGGAAAA

AAGGTTATCAAAGATTTTTATTTTAATAAAACAAAGAATATTTTATATATGCAACTATAT

ATATGTATGGACTGATGGGAGTAGTAGCTAAAATGAAAACGGTTTTTAATTTATTAACAC

AGGAGCGCAATCGATAATTAATTTCAGAACAGCTAGTTCTATGAACAAAACTTTTAAACA

ATATATAAGACGATACTCTTTGGAATATTCTATTTTGAGAGGTAAAAAAATGGAAATAAT

AATTTCTTAAGAATTTATAATACTATCTATATTGAAGAAAAAATGTATAATTTTTGTGGC

CATTTTATATAGCAAATAACATTCGATCACCTTAGATATAATTGCTATATCTATACATTA

ATACTCATATATTTTTTTTCTTTCATTTTTTGCAGCTGTGAAACTGACATCTCAGGTATA

TGTTTACGTTGTTTTACTTTTTTAAATAAATAATTATATTATTATTATTATTATTATTAT

TATTATTATTATTATTATTATTATTATTATTATATATATTTTTTTAACCTTGCGTAGTAT

TTAACAGAGGAAATAATACCGTCCACACAAACGGCCCAAATGTAATTAAGAAGACAGACA

CGTCTTAATAAAAATCCAAATAATATATACGTTTATAAGCATACATATATAGTGAGAAAA

ATGAATAACATAAAACATTTCGTTCTTGCATATTTAGGGTTGTTGCTCTTACTATTGCCG

TCATAGTGGGACTCAGTTATTTGGCTTGCATTTTAATAGGTATTCCTCAAGAGCATATGT

ATTTTTGCATTACTCAATTTATTATTTCATAAAAATTAATAATAGGGTTACACATGTTTT

TTTTTTATTTTTCTTAGTTTTCTCCGATGCACAAAAGGCAAATTGCGGCCGCGGATCCAT

GAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGA

TGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAA

ACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGT

CACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCA

TGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAA

AGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAA

TAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATT

GGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAAT

CAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCA

TTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCT

GTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCT

TGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAATTCT

AGAAGATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAA

AAATGTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAA

ATTATGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAG

TATGAATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATT

TAGATATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTAT

TATTTTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATT

TTTAAAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAA

AAACATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGAATTAAT

TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAAT

CGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT

CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTC

CTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCT

GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGG

GCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG

TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGC

CTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTT

CGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT

CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATG

AGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT

TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA

GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAA

GAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGT

ATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTT

GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGC

AGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGA

GGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGAT

CGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCT

GTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCC

CGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG

GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGC

GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACG

ACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCA

CTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA

AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACC

AAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAA

GGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA

CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTA

ACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC

CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA

GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTA

CCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAG

CGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTT

CCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC

ACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC

CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAAC

GCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTC

TTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT

ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAG

CGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCAC

GACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTC

ACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATT

GTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTAAAT

TATACATTTTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATA

TAAATAATTTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGAT

GTGTTTTGAARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGT

TTTATGTTTATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAA

TTAAAGCCAATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTT

TTATGGTAAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATT

TTGAATTACACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATT

TAGGGAAATTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACA

CAATATTGATATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAA

AAAAAATGTATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCAT

TTTTCCCCTGTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAA

ATGGATATATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATT

ATATTACCYTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTA

TATATTAAAATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGC

AAAGCTGTTTAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAA

TTTATATATTTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTG

TGTGTGTCAATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTA

AAATATAAAGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTAT

AATTTTTATTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGG

AATATATATAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTT

TTTAAGGGGTTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTT

TTTATATTAAGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTT

TTTCCAATTTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTA

CACATTCATTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAAT

AGTATTTTCATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGAT

CAATGCATAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCA

ACAACGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAA

AAACGACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGA

CGGGCGACTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGG

GACGGAAAACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACA

TCGTCGTTTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAG

GCCAGCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAG

AGTACAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAG

CGCTGTCTCTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGT

GCGACGTTTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTG

CGCAGGCTGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAG

AGAAGGACAATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACG

GGGTTCCCTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTG

CGGAACCGAGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACG

GGTTGCAGGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAG

AAGACCGGAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTA

GAGGCCATGAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGA

CAATGGATGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACT

CGCTGGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCG

AAGAGTTGCTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCG

TGAAGATCTGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACC

GAGAGGTCGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCAT

ACAAAGACATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCC

AGATGCTGAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAG

CGCTGGACGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACC

AGAAGGAGCTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCT

TCAACATCGCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAAC

CTAAGGAGTTCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTT

TAAAAGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGG

AACGCATCAAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCC

CGCACGGACGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGAT

CCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTG

TATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGA

ACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAAT

AGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATAT

TTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTT

ACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAA

TGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATA

TAAACACAAATGATGTTTTTTCCTTCA

Restriction sites to linearize plasmid

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 In situ C-terminal GFP-tagging was performed by single crossover homologous recombination into the corresponding locus using construct pLIS0081. The construct contain the tgdhfr/ts selectable marker under the control of P. berghei dhfr/ts 5′ and 3′ UTRs. Primers used to amplify the targeting regions corresponding to the 3′ end of the ORF excluding the stop codon are listed in Additional file 6: Table S4. Targeting regions were cloned upstream and in frame with the GFP. The plasmid was linearized and transfected into line cl15cy1.

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
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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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