RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-911
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0524200; Gene model (P.falciparum): Not available; Gene product: small exported protein early transcribed membrane protein (SEP2; ETRAMP; Pbsep2; ETRAMP4)
Name tag: mCherry
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 11 July 2013, 09:31
  *RMgm-911
Successful modificationThe 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) : 23840634
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone clone 8417HP
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherCurrà, C.; Pace, T.; Ponzi, M.
Name Group/DepartmentDipartimento di Malattie Infettive, Parassitarie ed Immunomediate
Name InstituteIstituto Superiore di Sanita
CityRome
CountryItaly
Name of the mutant parasite
RMgm numberRMgm-911
Principal namePbsep2- mcherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagePbsep2- mcherry expression in blood stages
Gametocyte/GametePbsep2- mcherry expression in gametocytes
Fertilization and ookinetePbsep2- mcherry expression in ookinetes
OocystPbsep2- mcherry expression in oocysts
SporozoitePbsep2- mcherry expression in sporozoites
Liver stagePbsep2- mcherry expression in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry tagged version of SEP2. The tagged version is integrated into the c/d-ssurRNA gene locus.

Protein (function)
Early transcribed membrane protein (ETRAMP) family member. Plasmodium conserved family with greater than ten members in P. falciparum. ETRAMPs are abundantly expressed early in the intraerythrocytic cycle and are small (frequently less than 200 aa) integral membrane proteins that are localized within the parasitophorous vacuolar membrane (PVM). All members have signal peptides plus a transmembrane domain. The ETRAMP/SEP proteins of P. yoelii and P. berghei (8-11 genes) show homology to members of the ETRAMP family of proteins of P. falciparum (14 genes) but the orthologous relationship of the different members is not completely resolved.

P. berghei sep genes, Pbsep1, Pbsep2 and Pbsep3 (PBANKA_052480, PBANKA_052420 and PBANKA_050110, respectively) are 3 ETRAMP members which reside in the subtelomeric regions of chromosome 5. The three genes share the upstream regulatory region and differ in their 3’UTRs. The encoded proteins (13-16 KDa) are nearly identical in the first 81 amino acids, which include a predicted signal peptide, a lysine-rich domain and a TM region, while differ in their C-terminal portion.
Mutants lacking expression of SEP1 have been generated (RMgm-16, RMgm-18) which show a normal phenotype during the complete life cycle, comparable to wild type parasites.
Multiple unsuccessful attempts to disrupt sep2 en sep3 (RMgm-64, RMgm-65) indicate that these proteins are essential for blood stage proliferation.

In asexual blood stages SEP2 and SEP3 localize to the PVM and to vesicle-like structures exported to the erythrocyte cytosol (RMgm-680, RMgm-681), while SEP1 is mainly confined to the PVM.

Phenotype:
SEP2 is expressed in all life cycle stages. Evidence is provided for a PVM location in blood stages and liver stages and for a surface location in ookinetes and sporozoites. SEP2 is shed/released during gliding motility of sporozoites

Specific antibodies detect SEP2 in dot-like structures inside the ookinete but also close to the parasite periphery, as shown by partial co-localization with the ookinete surface protein P28. SEP2 does not co-localize with the micronemal protein SOAP

Additional information
In order to establish whether the downstream regulatory regions have a role in the control of sep2 and sep3 expression level of these genes,  A. stephensi mosquitoes were infected with two transgenic lines, gfp-3'utr-sep2 and gfp-3'utrsep3, containing a stably integrated gfp reporter under the control of the 1.2-kb upstream regulatory region common to sep genes and the 3'UTR specific for either sep2 or sep3. In asexual blood stages these transgenic lines express GFP at a comparable level, indicating that the specific 3'UTRs have no significant regulatory role. In the gfp-3'utr-sep2 line, GFP fluorescence was clearly detected during oocyst maturation (8, 11, 13 days after the blood meal) with an increased intensity in mature oocysts (13 days). A strong GFP-specific signal was also detected in salivary gland sporozoites (22 days). When the GFP was expressed under the control of the 3'-UTR specific for sep3 (gfp-3'utr-sep3 line), only mature oocysts and salivary gland sporozoites displayed a faint fluorescence. These results show that the 3'UTRs of sep2 and sep3 modulate the expression level of GFP.

Other mutants
Mutants lacking expression of SEP1  (RMgm-16, RMgm-18)
Multiple unsuccessful attempts to disrupt sep2 en sep3 (RMgm-64, RMgm-65)
Mutants expressing a flag-tagged version of SEP2 and SEP3 (RMgm-680, RMgm-681)
A mutant expressing an mCherry-tagged version of SEP3 (RMgm-912)


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0524200
Gene Model P. falciparum ortholog Not available
Gene productsmall exported protein early transcribed membrane protein
Gene product: Alternative nameSEP2; ETRAMP; Pbsep2; ETRAMP4
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutant expresses a C-terminal mCherry tagged version of SEP2. The tagged version is integrated into the c/d-ssurRNA gene locus.
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 1Gaccactagtgacgtctataattatccaaaccaataagacg
Additional information primer 1Sep2 3’UTR: 3’-bis-spe
Sequence Primer 2Gaccgcggccgcattgtagtactattattgcg
Additional information primer 2Sep2 3’UTR: 3’-bis-not
Sequence Primer 3GaccctcgagGTGTATGAATTTAAGAATTTC
Additional information primer 3Sep2 5’UTR: Bis-prom-si
Sequence Primer 4Gaccaagcttttttgtataagtaaaaaaattataat
Additional information primer 4Sep2 5’UTR: 1&bisprom-Hind-rev
Sequence Primer 5gaccgaattcaagcttATGAAACTAGCAAAAGCATT
Additional information primer 5Sep2 coding:sep1&2Hind-cod-for
Sequence Primer 6gacccccgggattcaattttacagtatatgg
Additional information primer 6Sep2 coding: sepbisSma-cod-rev