RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1142

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0821200; Gene model (P.falciparum): PF3D7_0920300; Gene product: conserved Plasmodium protein, unknown function Name tag: GFP
Phenotype	Gametocyte/Gamete; Fertilization and ookinete;

Last modified: 2 December 2014, 09:18

*RMgm-1142

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-1142
Principal name	2181
Alternative name	PBANKA_082120::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.
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_082120 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. 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

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_0821200

Gene Model P. falciparum ortholog PF3D7_0920300

Gene product conserved Plasmodium protein, unknown function

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

ATTTCGATATCGGGGAATTCCTTTTAGTTTCATTTAATGAAAATTTTTTTAATAAAGGGA

AAATAACACACAGCCCTATAAATGTATATTATAAAAATTATTCTGACCATTTGCCAAATG

ATCAAAGAGATAGACTAATGGATATACTTTTACACAATCCCGAAAATGACACAAAGTTTG

ATATGTTCAATTCCATCGATATAAAAAAAATTGAGGAAAATGAAAAAGGACTAGCCACGT

TATTGAAAAAGCAAGAAGAGTTATCAAATAAAATAAAGAAGGAGTTAAATGGATATAATA

AAAGAAGTAATTTTTTTATTAACAACGAAAAGAAAACGAAAAAATTATCAATAAAATAGT

ATGAATACAATGGTAATACTTATTAAATTACATTTTTTTGATAGGTGAAAAAATGTCCAT

AAAATATAATCATGATACCAAAAAAATAAATTCGGATACAGAGAAAAACATGGAAAAGGA

AAAAAATGTATTTGATGTCGATACTGAAATTGATGGTTAGTAATTTCCAAACGTAAATAT

ATTTCATTTTTTATGCTCGTACATTTAGTGATCGGTTTAATATGTATATATATATACCAG

AAATATATGTATGATTTTTTTTATAAAAACATAATTTTCCGTATTTTATATAGTATCTGC

TAATACACTGAGCGAACCAATAATTGTTATTAAAGTACCATGGATAAATCTATATGATCA

ACTTGAAGTTAGTAAATATTTTGAAACGTATTATATTAATTTTGTCTAAGTTATTTTAAA

CCGTATAATATTTTATTATTTATTTGTTTATATATATATTGTTAATGTGTTTTTATTTTA

TACACACATATGTATATTTTTTACGCGAACCAATTTCCATAGATGGAGCATGAAATAGGA

AAATTAATAGATTTAGATAATGAAGAGTTATTCTATTGCGGCCGCGGATCCATGAGTAAA

GGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAAT

GGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACC

CTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACT

TTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTT

TTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGAC

GGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATC

GAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATAC

AACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTT

AACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAA

CAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACA

CAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTT

GTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAATTCTAGAAGAT

CCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTG

TATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGA

ACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAAT

AGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATAT

TTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTT

ACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAA

TGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATA

TAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGAATTAATTCACTGG

CCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTG

CAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTT

CCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGC

ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCG

CATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC

TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGA

GGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTT

TATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAA

ATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCA

TGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC

AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC

ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT

ACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT

TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACG

CCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACT

CACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTG

CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGA

AGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGG

AACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAA

TGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAAC

AATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC

CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCA

TTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA

GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTA

AGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTC

ATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCC

CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTT

CTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTAC

CAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCT

TCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT

TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG

CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATA

AGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA

CCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAG

GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGG

AGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGAC

TTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCA

ACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTG

CGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC

GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAA

TACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGT

TTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATT

AGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCG

GATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTAAATTATACAT

TTTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAA

TTTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTT

GAARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGT

TTATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGC

CAATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGT

AAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATT

ACACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAA

ATTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATT

GATATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAAT

GTATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCC

CTGTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATA

TATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTAC

CYTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTA

AAATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTG

TTTAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATAT

ATTTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGT

CAATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATA

AAGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTT

ATTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATAT

ATAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGG

GGTTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATAT

TAAGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAA

TTTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTC

ATTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTT

TCATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCA

TAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGG

CCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGAC

GCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGA

CTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAA

AACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGT

TTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCA

GCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAA

GGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTC

TCTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGT

TTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGC

TGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGA

CAATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCC

CTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACC

GAGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCA

GGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCG

GAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCA

TGAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGA

TGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGA

TCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTT

GCTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGAT

CTGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGT

CGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGA

CATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCT

GAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGA

CGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGA

GCTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACAT

CGCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGA

GTTCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGA

GCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCAT

CAAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGG

ACGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTT

TTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTG

TGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTT

ATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATA

TTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGA

ACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTA

TAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTAT

AATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACAC

AAATGATGTTTTTTCCTTCA

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 pLIS0080. 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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