RMgmDB - Rodent Malaria genetically modified Parasites

Back to search results

Summary

RMgm-4187
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: FlpL recombinase of yeast (FlpL)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: up-regulated in infective sporozoites (UIS4, ETRAMP10.3)
3'UTR: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Sporozoite;
Last modified: 20 April 2018, 10:27
  *RMgm-4187
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone GIMO-PbANKA (RMgm-687)
Other information parent lineGIMO-PbANKA (RMgm-687; line 1596cl1) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
The mutant parasite was generated by
Name PI/ResearcherC.J. Janse, S.M. Khan, P. Sinnis
Name Group/DepartmentDepartment of Molecular Microbiology and Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health, Baltimore
CityBaltimore
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4187
Principal name1808cl1 clone A1
Alternative nameUIS4@Flp
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteMidgut and salivary gland sporozoites express Flp
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain a selectable marker. The Flp/uis4 expression cassette was introduced in the GIMO mother line using GIMO transfection (PubMed: PMID: 22216235)

Protein (function)
In the Flp/FRT site-specific recombination (SSR) system of yeast, the Flp recombinase recognizes the 34 bp FRT sites and excises any DNA located between two directly oriented sites (referred to as the FRTed sequence). The activity of Flp recombinase is greatly reduced at 37°C and above - temperatures in erythrocytic stages in the vertebrate host - and is maintained at 20°C –25°C, temperatures permissive for parasite development in the mosquito.

Phenotype
The phenotype has not been analysed in detail


Additional information
This mutant expressing Flp can be used for introducing additional DNA sequences (genes) that are flanked by FRT sequences. Subsequently these sequences can be removed by mosquito transmission. This 'conditional mutagenesis' approach allows for the analysis of the effect of removal of genes essential for blood stage development. For an example of using this approach see mutant RMgm-270

Multiple P. berghei lines have been generated that express Flp/FlpL under promoters that are active in mosquito stages. This mutant and mutant RMgm-4186 are made in a ANKA line of P. berghei. Most other mutants have been made in P. berghei NK65

See also mutant TRAP@FlpL (RMgm-4186)  for a mutant expressing FlpL under the control of the uis4 promoter that is specifically switched on in sporozoites after salivary gland invasion.

The mutants has been generated in Leiden (LUMC), the Netherlands and cloned in the Sinnis lab.

Other mutants
See Flp/FRT


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameFlpL recombinase of yeast (FlpL)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double 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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe FlpL coding sequence was flanked by 1.6 kb of the UIS4 promoter and 0.55 kb of the 3′ regulatory sequence of trap, respectively.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog Not available
Gene productup-regulated in infective sporozoites
Gene product: Alternative nameUIS4, ETRAMP10.3
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_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
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 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