RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1144

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1114100; Gene model (P.falciparum): PF3D7_0514500; Gene product: conserved Plasmodium membrane protein, unknown function Name tag: GFP
Phenotype	Gametocyte/Gamete; Fertilization and ookinete;

Last modified: 2 December 2014, 09:23

*RMgm-1144

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
top of page
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).
top of page
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
top of page
Name of the mutant parasite
RMgm number	RMgm-1144
Principal name	2184
Alternative name	PBANKA_111410::GFP
Standardized name
Is the mutant parasite cloned after genetic modification	No
top of page
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_111410 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

top of page

Details of the target gene

Gene Model of Rodent Parasite PBANKA_1114100

Gene Model P. falciparum ortholog PF3D7_0514500

Gene product conserved Plasmodium membrane protein, unknown function

Gene product: Alternative name

top of page

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

ATTTCGATATCGGGGAATTCCAATTATTTTCATTGTCTTCATTGATATTATCATGTTTAT

CTTTCATTATATTAAAAAGAAAAAATGGGACATATATTTTAACAAAGATTAAGATGTATC

CTTTTTCTTTCAAAGATTTAGTTGATGTGTCTTTGCTAACGGGGGTTGTTACGATTTCTA

TCTTTGATATAATTTTGGTTTGTGTGCACGTATGTGTATATATATAACTTTTTATAATTC

ATTGGTATATATTATTATAATTTACATTTTCACATATGCCCATATATATACATGCATACA

TTATATATGAGCATATTTCCTCCCTTTAGTGCTATCTTTACCTACTTGTAGCATTTTCTG

TTTTTGTAATAGGTATGTTTAGTGTTTTTATTTTATATACTTTTGCTATAATTATATTTC

TATTCATAATTATGTATTTTCAGAAAATATTATTTTAAAATTTTTTATTCATCTCATAGC

AACATTTTCTATCTACGAATGCCTAGACAATCCTTACATGAGGGACAATGAAAACATTAG

CAGTTTTAGTAATTAGCATTTATCCTTGTTCATGTTTTTTAAAACTCAAAAAATATTGCC

GTTTTATTTGTATATTTCTACATTTTTTTTTAACCATAGGCTTATCATTCATTTCAGTTT

TATACATGGGGTTATATGTAATTTCCAATGGAATAATTTTTAGAAATCTTCCATACAACC

CTTTAGAATATATGTGCGTAAAAAAAAAAAAATAAATAGTATATTTATAAGTTTGTATGC

GCATTTATTATAATAATCCCCAATTATTTTGTTTTATATTTTCCACATTTCTTTTTTTTT

TTTTCCTTTTTTTTTTCTTTTTAATTCAGAGGGTATATAATAAAAAATATTGCAGAAGAC

ATTTGTTCAGTCACAAAAAAATTAACAAAATTGATTATTTGCGGCCGCGGATCCATGAGT

AAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTT

AATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTT

ACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACT

ACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGAC

TTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGAT

GACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGA

ATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAA

TACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAA

GTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTAT

CAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCC

ACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAG

TTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAATTCTAGAA

GATCCCGTTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAAT

GTGTATGTTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTA

TGAACATTTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATG

AATAGCCATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGA

TATTTATGGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATT

TTTACTGTTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTA

AAATGTTTATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAAC

ATATAAACACAAATGATGTTTTTTCCTTCAATTTCGGGTACCGAGCTCGAATTAATTCAC

TGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCC

TTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCC

CTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTA

CGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATG

CCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTT

GTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTC

AGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTAT

TTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGG

GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC

TCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA

TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG

CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG

GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC

GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTG

ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGT

ACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG

CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGAC

CGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT

GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAG

CAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC

AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC

TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTA

TCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG

GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA

TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAAC

TTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAA

TCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGAT

CTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC

TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTG

GCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACC

ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGG

CTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG

ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA

CGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCG

AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGA

GGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT

GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA

GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC

CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG

CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC

CAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACA

GGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTC

ATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGA

GCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTAAATTATA

CATTTTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAA

TAATTTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGT

TTTGAARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTA

TGTTTATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAA

AGCCAATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTAT

GGTAAGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGA

ATTACACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGG

GAAATTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAAT

ATTGATATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAA

AATGTATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTT

CCCCTGTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGG

ATATATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATAT

TACCYTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATA

TTAAAATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAG

CTGTTTAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTA

TATATTTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTG

TGTCAATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAAT

ATAAAGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATT

TTTATTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATA

TATATAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTA

AGGGGTTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTA

TATTAAGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTC

CAATTTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACA

TTCATTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTA

TTTTCATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAAT

GCATAAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAA

CGGCCTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAAC

GACGCCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGG

CGACTCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACG

GAAAACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGT

CGTTTCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCA

GCAGCGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTA

CAAGGATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCT

GTCTCTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGA

CGTTTTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCA

GGCTGCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAA

GGACAATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGT

TCCCTACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGA

ACCGAGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTT

GCAGGCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGA

CCGGAAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGG

CCATGAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAAT

GGATGACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCT

GGATCAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGA

GTTGCTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAA

GATCTGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGA

GGTCGGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAA

AGACATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGAT

GCTGAGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCT

GGACGAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAA

GGAGCTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAA

CATCGCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAA

GGAGTTCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAA

AGAGCAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACG

CATCAAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCA

CGGACGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCG

TTTTTCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATG

TTGTGTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACAT

TTTATTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCC

ATATTTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTAT

GGAACATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTG

TTATAATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTT

TATAATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAA

CACAAATGATGTTTTTTCCTTCA

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 pLIS0085. 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

top of page