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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: Plasmodium |
Gene Model of Parasite |
PF3D7_0304600
|
Gene Model P. falciparum ortholog |
PF3D7_0304600
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Gene product | circumsporozoite (CS) protein |
Gene product: Alternative name | CS; CSP |
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Details of the genetic modification |
Inducable system used | No |
Additional remarks inducable system |
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Type of plasmid/construct | (Linear) plasmid double cross-over |
PlasmoGEM (Sanger) construct/vector used | No |
Modified PlasmoGEM construct/vector used | No
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Plasmid/construct map |
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Plasmid/construct sequence |
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Restriction sites to linearize plasmid |
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Selectable marker used to select the mutant parasite | No selectable marker |
Promoter of the selectable marker | No |
Selection (positive) procedure | No |
Selection (negative) procedure | 5-fluorocytosine (5-FC) |
Additional remarks genetic modification | Chimeric P. berghei parasites containing the P. falciparum gene of interest at the neutral 230p locus were generated following the ‘gene insertion/marker out’ (GIMO) technology as previously described, using the standard GIMO DNA construct pL0043. This construct contains 5′ and 3′ targeting sequences for the 230p locus as well as a multiple-cloning site for integration of transgene-expression cassettes. These constructs integrate by double crossover homologous recombination and replace the positive-negative selectable marker (SM) (human dihydrofolate reductase:: yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu)) cassette with the transgene-expression cassette. The expression cassette contained the transgene flanked by the 5′ and 3′ promoter and transcription terminator sequences of P. berghei UIS4, which were amplified from P. berghei ANKA wild-type (WT) genomic DNA. The coding sequence of the various P. falciparum genes were PCR amplified from P. falciparum genomic DNA (primer sequences are available upon request), apart from LSA1 and LSA3. Due to the large size of these open reading frames the coding sequence was amplified from plasmids used in the generation of the vaccine constructs (and hence codon optimized for expression in mammalian cells). In addition, a reporter-cassette containing GFP::luciferase52, driven by the constitutive P. berghei elongation factor 1 alpha (ef1α ) promoter, was also cloned into each transgene construct. The coding sequence and promoter region of all constructs was confirmed by sequencing. Linearized constructs were introduced into the GIMO parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice through addition of 5-fluorocytosine (5-FC) in drinking water, resulting in negative selection of parasites where the SM in the 230p locus was replaced by the expression/reporter-cassette. |
Additional remarks selection procedure | This transgenic parasite does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice. |
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Other details transgene |
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Promoter |
Gene Model of Parasite |
PBANKA_0501200
|
Gene Model P. falciparum ortholog |
Not available
|
Gene product | early transcribed membrane protein up-regulated in infective sporozoites |
Gene product: Alternative name | ETRAMP10.3 |
Primer information details of the primers used for amplification of the promoter sequence
Primer information details of the primers used for amplification of the promoter sequence
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
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3'-UTR |
Gene Model of Parasite |
PBANKA_0501200
|
Gene product | early transcribed membrane protein up-regulated in infective sporozoites |
Gene product: Alternative name | ETRAMP10.3 |
Primer information details of the primers used for amplification the 3'-UTR sequences
Primer information details of the primers used for amplification the 3'-UTR sequences
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
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Insertion/Replacement locus |
Replacement / Insertion | Replacement locus |
Gene Model of Parasite |
PBANKA_0306000
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Gene product | 6-cysteine protein |
Gene product: Alternative name | 230p |
Primer information details of the primers used for amplification of the target sequences
Primer information details of the primers used for amplification of the target sequences
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 | |
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