RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-785
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1329500; Gene model (P.falciparum): PF3D7_1466100; Gene product: protein phosphatase containing kelch-like domains (PPKL; PPalpha)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 11 November 2012, 16:46
  *RMgm-785
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22957089
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherN. Philip; A.P. Waters
Name Group/DepartmentWellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation
Name InstituteUniversity of Glasgow
CityGlasgow
CountryUK
Name of the mutant parasite
RMgm numberRMgm-785
Principal nameppkl-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal gametocyte production. Normal gamete formation (exflagellation). Aberrant development of ookinetes.

Differentiation of female gametes into ookinete stages was severely impaired producing abnormal forms, with 63% appearing as retort-shaped forms (the Type III form).

Ookinetes are affected in motility
OocystNo oocyst formation. Ookinetes are affected in motility and migration through the midgut wall
SporozoiteNo oocyst formation and sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PPKL (protein phosphatase containing kelch-like domains) and expresses GFP under control of the constitutive eef1a promoter.

Protein (function)
PPKL is a distinctive PP1-related enzyme belonging to the PPP family of phosphatases comprising an N-terminal kelch repeat domain and a C-terminal PP1-like phosphatase domain (named PPKL: Protein Phosphatase with Kelch-Like domains). It has been detected in the apicomplexans Cryptosporidium hominis, Toxoplasma gondii and Theileria parva (one gene per genome), as well as in the land plants Arabidopsis thaliana and Oryza sativa (4 and 5 genes respectively). The kelch motif is widespread and involved in many cellular functions, particularly in actin-based cytoskeleton formation and transcriptional regulation. PPKL has only been studied in detail in Arabidopsis thaliana (where it is known as BSU1 or bri1 suppressor1) and along with the kinase BIN2, is involved in brassinosteroid hormone signalling and phosphorylation of the transcription factors BZR1 and BES1

In contrast to the human phosphatome (comprising approximately 156 phosphatases the Plasmodium genome codes for one of the smallest phosphatomes of all the eukaryotic phyla known to date, with 27 putative protein phosphatases falling into four major classes: phosphoprotein phosphatases (PPPs), metallo-dependent protein phosphatases (PPMs), protein tyrosine phosphatases (PTPs) and NLI interacting factor-like phosphatases (NIFs).

Phenotype
Phenotype analyses indicate an essential role of PPKL in maturation of ookinetes.
Deletion of the ppkl gene caused abnormal ookinete development, loss of ookinete motility and failure of ookinetes to invade the mosquito midgut epithelium. Evidence is presented for aberrant formation of the apical end and disorganization of microtubules.

Additional information
Pbppkl was disrupted in 2 independent experiments and transgenic parasites were generated in both non-fluorescent background (P. berghei ANKA) and a wild type line which constitutively expresses a gfp transgene under the control of the eef1a promoter (P. berghei ANKA 507cl1) described here.

While, ppkl mRNA was found in all asexual blood stages, gametocytes and ookinetes, PPKL protein expression was detected only in schizonts, female gametocytes and ookinetes (see also mutants RMgm-786 and RMgm-788 that express an endogenous GFP-tagged version of PPKL). Immunofluorescence studies using anti-PPKL polyclonal antibody showed expression in merozoites, female gametocytes, zygotes, ookinete, and sporozoites. Only female gametocytes and not males expressed PPKL. The phosphatase is localized predominantly in the cytoplasm with a particularly intense expression at the apical tip of the developing and mature ookinete.

Since the effect of PPKL absence was visible post-fertilization it was examined whether the defect was male or female specific by performing genetic crosses between ppkl– parasites and parasites lines which are specifically male fertile (line 270, p47-) or female fertile (line 137, p48/45-). Consistent with the observed wild type pattern of PPKL expression only crosses with wild type females from p48/45- (line 137) generated normal ookinetes (26%) revealing the requirement of PPKL to be female specific.

Other mutants
RMgm-786: A mutant expressing an endogenous GFP-tagged version of PPKL
RMgm-787: An independent mutant lacking expression of PPKL
RMgm-788: An independent mutant expressing an endogenous GFP-tagged version of PPKL


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1329500
Gene Model P. falciparum ortholog PF3D7_1466100
Gene productprotein phosphatase containing kelch-like domains
Gene product: Alternative namePPKL; PPalpha
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbppkl (PBANKA_132950) was targeted for genetic disruption by double-crossover homologous recombination
with PL0035 vector containing the human dhfr/ts:yfcu selection cassette flanked by 1 kb targeting regions
both upstream (KpnI-GU150, XhoI-GU151) and downstream (HindIII-GU148, SacII-GU149) of the ORF.
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 1CATAAAGCAAAAAAAAGACAAAGGAGAAATATACAATG
Additional information primer 1GU150 (KpnI); Rev-Upstream arm for ppkl KO
Sequence Primer 2GAGGTATATATATTTTTTTTGATTGAATTCCACTATATATTTTTTTGTTCATAATAAG
Additional information primer 2GU151 (XhoI); For-Upstream arm for ppkl KO
Sequence Primer 3GGAAATAATTCAAATTAGAGAAAAAAAATTCATGAGTTTC
Additional information primer 3GU148 (HindIII); Rev-Downstream arm for ppkl KO
Sequence Primer 4TCCCTTTATTATTATTATGCCTTTATATTATGGAAATTTAC
Additional information primer 4GU149 (SacII); For-Downstream arm for ppkl KO
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Click to view information
Click to hide information
Plasmid/construct sequence
Click to view information
Click to hide information
" 1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
51 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
101 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
151 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
201 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
251 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
301 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
351 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
401 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
451 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
501 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
551 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
651 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
701 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
751 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
801 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
851 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
901 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
951 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
1001 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
1051 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
1101 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
1151 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
1251 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
1301 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
1351 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
1401 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
1451 agcattggta actgtcagac caagtttact catatatact ttagattgat
1501 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
1551 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
1601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
1651 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
1701 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
1751 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
1851 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
1901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
1951 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
2001 actgagatac ctacagcgtg agcattgaga aagcgccacg cttcccgaag
2051 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
2101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
2151 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
2201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
2251 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
2301 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
2351 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
2401 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
2451 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
2501 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
2551 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
2601 tcacacagga aacagctatg accatgatta cgccaagctt ccgcgggtat
2651 atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct
2701 ataaataaat ttacagattt attttttaat acaaaagata tagatatacc
2751 agaaataaat gatcagttta aaggttttaa attctttatg acatcattta
2801 taaatcatgg atcatatcca ctaacaatag aatgtggtgt aacaaatggt
2851 ggaactagtt ataaaagagc aattatttta ttgcatgttc gaactgattt
2901 aaaagataga ccagtttcat tttgtgattt tcgaaaagga gaattatata
2951 attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa
3001 tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc
3051 aaaaaattgt tttacgcaag tatatgaaaa agggtatcta aatgacgcca
3101 ataaaattaa tactaaaaat gttattaact attcatttga aaatccagaa
3151 tatgcgctag ctggttytaa ttatacatta acaaaatcgt atcaatttga
3201 atgtcattgt gtagataaag aaacagaaca aattgtaaaa acggttttag
3251 tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg
3301 gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg
3351 catgcctgca gcccagctta attcttttcg agctctttat gcttaagttt
3401 acaatttaat attcatactt taagtatttt ttgtagtatc ctagatattg
3451 tgctttaaat gctcacccct caaagcacca gtaatatttt catccactga
3501 aataccatta aattttcaaa aaaatactat gcatataatg ttatacatat
3551 aaacataaaa cgccatgtaa atcaaaaaat atataaaaat atgtataaaa
3601 ataaatatgc actaaatata agctaattat gcataaaaat taaagtgccc
3651 tttattaact agtcgtaatt atttatattt ctatgttata aaaaaatcct
3701 catataataa tataattaat atatgtaatg ttttttttat tttataattt
3751 taatataaaa taatatgtaa attaattcaa aaaataaata taattgttgt
3801 gaaacaaaaa acgtaatttt ttcatttgcc ttcaaaattt aaatttattt
3851 taatatttcc taaaatatat atactttgtg tataaatata taaaaatata
3901 tatttgctta taaataaata aaaaatttta taaaacatag ggggatccat
3951 gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
4001 tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa
4051 ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg
4101 aaaactacct gttccatggc caacacttgt cactactttc ggttatggtg
4151 ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc
4201 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa
4251 agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
4301 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga
4351 aacattcttg gacacaaatt ggaatacaac tataactcac acaatgtata
4401 catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta
4451 gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
4501 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
4551 gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
4601 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat
4651 gaactataca aataaatgga tcccgttttt cttacttata tatttatacc
4701 aattgattgt atttataact gtaaaaatgt gtatgttgtg tgcatatttt
4751 tttttgtgca tgcacatgca tgtaaatagc taaaattatg aacattttat
4801 tttttgttca gaaaaaaaaa actttacaca cataaaatgg ctagtatgaa
4851 tagccatatt ttatataaat taaatcctat gaatttatga ccatattaaa
4901 aatttagata tttatggaac ataatatgtt tgaaacaata agacaaaatt
4951 attattatta ttattatttt tactgttata attatgttgt ctcttcaatg
5001 attcataaat agttggactt gatttttaaa atgtttataa tatgattagc
5051 atagttaaat aaaaaaagtt gaaaaattaa aaaaaaacat ataaacacaa
5101 atgatgtttt ttccttcaat ttcgggtacc gagctcgaat tctcttgagc
5151 ccgttaatga aatagataca attcattcat gttatataca tctagaacat
5201 aatctgaata tggttcaagt taaatgtcca aaaattataa aaagtgatga
5251 tatttttgat ggtaatacca taatagacac caaggtaaca tcacgaagta
5301 gtcaacaaaa taatttttat ttagaaaata cagatgttga accagaagaa
5351 atagagaaat ataaaaatat agaatacata ccagaaaacg atgaagtaat
5401 gcatctagac aaaaaagaaa agctagatga tatattacca ggtgttatca
5451 tattagataa aaataaaatg ttcaaagaaa aaggacattt cacttttgtt
5501 actccattaa ttgtagaaaa ggtattaata ttaaaaatat attgtgataa
5551 tactaaaaca ataattaata atatgaaagg gaaaaaaggt attacagtaa
5601 taaggatttc tcaaaataca acaaaaaata aattttatgg atgtgacttt
5651 tcaggtaatt ctaaaaaaac attttactat tccaatgttt atgatttaga
5701 aaaaaaaaat gagttttgtg aaatagaatt aaaagaaaat atagtagtta
5751 gcttaaattg tccaactggt aaaattaatc caaaaaattg ttttagaaat
5801 gtatatataa aaagtaatat gaatgaacaa acaaccgaaa atatagaaaa
5851 tatatttaac gaaataaaag ttatagatgc agattatttt ataaataatt
5901 catcaacctt tttgatgatt tccaaaatta caaaaaaaga gtttgatttt
5951 tattgtacat gtgaagatta taaaaccaaa aatataggaa caatatatat
6001 taaaaattat gaatatctag attcaaaacc taaatataaa aataaacaaa
6051 tttcctatat agatgtagtt ccatacccgc ggggaaaggg cg"
Restriction sites to linearize plasmid KspI (SacII)
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 15'-cccaagcttccgcgggtatatggtaaagaacctactaacac
Additional information primer 1primer L1345: 0.7kb 5'region of PBANKA_030600 (230p gene)
Sequence Primer 25'-cccaagcttgatgtgttttatttggatgtgc
Additional information primer 2primer L1346: 0.7kb 5'region of PBANKA_030600 (230p gene)
Sequence Primer 35'-ccggaattctcttgagcccgttaatg
Additional information primer 3primer L1347: 1kb 3'region of PBANKA_030600 (230p gene)
Sequence Primer 45'-tccccgcgggtatggaactacatctatatagg
Additional information primer 4primer L1348: 1kb 3'region of PBANKA_030600 (230p gene)