RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1145

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1334700; Gene model (P.falciparum): PF3D7_1471500; Gene product: transmembrane protein 43, putative Name tag: GFP
Phenotype	Gametocyte/Gamete; Fertilization and ookinete;

Last modified: 2 December 2014, 09:26

*RMgm-1145

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-1145
Principal name	2183
Alternative name	PBANKA_133470::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_133470 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_1334700

Gene Model P. falciparum ortholog PF3D7_1471500

Gene product transmembrane protein 43, 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

ATTTCGATATCGGGGAATTGAATACTAATGTTTATGACAACTATTTGTATACAGGAGATC

CATTAAATCCCCAAGTATAATTCTTATCAAAATATTTTCTTCACAATTATTTAATTTACA

TTATGGAAAATATTATATAATAAATATCCATTAATTAAATTCATAATTTAATTTTGTGAA

TAAAATAAGAAATATATACATATATACACTATTATATGATATACAGATAGGTGACATAAG

GGTTTCGTTTTATGGCAACGCGTCTACTCATGCAACAGCTATAGGAATTCAGAAAGCAAG

ATTACTAAATACCATATTCGAAATTGATACAGTTAATATCATGAATAAAGATGTAATTTT

ACTATCTGAAGATGACAAAATAATGACTAATCATACAAAAAATTTTATTTATAAAAATTA

TGGTAATAAACGAGCATTATGGGTATTTCGTATAATAGCTTATATCTTTTTATTTATTCA

AATTTTTATTTTCCTTGAAAATAGTTCTAAGGTAAGATAATGGCTATGTATATATAGCCA

TATATGTATATTATTAACATTACGTACTTATATGATTATGCACAAAATAAATAAACAACA

TTGTTAATATTCTTTAAATAATTTTTTTTTTCATTTCATTATTTTTTTAGAATGTCACAT

GGAAAGTAATTGTGAGTTCGATTGTGTCAATGATTATATTATCAATATTCCCTTGTGTTC

TATGGCTTTTTTGTGACACTGCTGTCTTTTTATGTTTATTTGTATTCATTGTTTTTTTGT

CAATAGCGTTATTATTTATATATAATAACGAAGTAGATAATGGATATACTGAGATGAAAA

ATTATATGAAAAGAGCAAATACAGAACCAACAAATTATACATTTTTAAACGTAGAAGATA

ATTGTACAGATATATATAAAGAATATACTAATGAAAAAGACAAGATGGTCACAATATTAG

ATTCAAGTAGCGATGCATCATTTTATAATTCTATAAATAAAAATGATGGACATAAATTTG

AATCATCGCCAGCTGGAGTATATCATTATAGATGCGGCCGCGGATCCATGAGTAAAGGAG

AAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGC

ACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTA

AATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCG

GTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCA

AGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGA

ACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGT

TAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACT

ATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACT

TCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAA

ATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAAT

CTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAA

CAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAATTCTAGAAGATCCCG

TTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATG

TTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACAT

TTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCC

ATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTAT

GGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTG

TTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTT

TATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAA

CACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGAATTAATTCACTGGCCGT

CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC

ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA

ACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCT

GTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA

GTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT

CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT

TTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATA

GGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGT

GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG

ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACA

TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCC

AGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACAT

CGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCC

AATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGG

GCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACC

AGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCAT

AACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGA

GCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC

GGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC

AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT

AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGC

TGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGC

AGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA

GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCA

TTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTT

TTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA

ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG

AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGC

GGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

CAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA

GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC

CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGC

GCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTA

CACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAG

AAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT

TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA

GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC

GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTT

ATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG

CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACG

CAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCC

CGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGC

ACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA

ACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTAAATTATACATTTTA

TATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTT

TTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAAR

GTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTAT

ACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAAT

TTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGT

GCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACAC

ATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTC

TACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATA

TATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTAT

AGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGT

TTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATA

GGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTG

GGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAAT

TAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTA

ATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTT

ATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAAT

TCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGR

ATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTT

AGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAA

TACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTT

ATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAG

ATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTT

TATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTT

TAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCAT

TTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAA

CCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTC

CCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCC

GAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCT

GGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACC

TGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCC

TCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGC

GTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGAT

TCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTG

GGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTC

TTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCA

GCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAAT

GAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTAC

GACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGC

AACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCT

CCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAA

AAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAA

GAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGAC

CGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAG

GCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTG

TGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGG

GACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGA

GACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATG

CACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGA

ACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAA

ATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTG

TCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCT

TCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTC

ATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAG

CTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAG

GAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGA

ATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCT

TACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTG

CATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTT

TTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTT

ATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACAT

AATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAAT

TATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATA

TGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAAT

GATGTTTTTTCCTTCA

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 pLIS0084. 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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