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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: Plasmodium |
Gene Model of Parasite |
Not available
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Gene Model P. falciparum ortholog |
Not available
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Gene product | 'P. vivax like' csp (locus PVU09738; Accession U09738) |
Gene product: Alternative name | PvCSP, G10 |
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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 | hdhfr/yfcu |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | No |
Selection (negative) procedure | 5-fluorocytosine (5-FC) |
Additional remarks genetic modification | The PvCSP-P. vivax-like Gabon Clone G10 CDS (Locus PVU09738, Accession U09738) gene was introduced into the genome as an additional copy of the gene in the neutral 230p locus using the previously described ‘gene insertion/marker out’ (GIMO) technology and the standard GIMO DNA construct pL0043 to generate the chimeric P. berghei additional copy line. This construct contains 5’ and 3’ targeting sequences for the 230p locus, as well as a multiple-cloning site for the integration of transgene expression cassettes. This construct integrates transgenes by double crossover homologous recombination and replaces the positive–negative SM (human dihydrofolate reductase:: yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu)) cassette with the transgene expression cassette. The expression cassette contained the PvCSP-P. vivax-like CDS flanked by the 5’ and 3’ promoter and transcription terminator sequences of the P. berghei uis4 gene (PBANKA_0501200), which were amplified from P. berghei ANKA WT genomic DNA. The coding sequence of the P. vivax CSP-like G10 gene (Locus PVU09738, Accession U09738) was ordered from GeneArt Gene Synthesis—Thermo Fisher Scientific. In addition, a reporter cassette containing GFP::luciferase30 driven by the constitutive P. berghei elongation factor 1 alpha (ef1α) promoter was also cloned into the transgene construct to generate the gene insertion construct pL2163 (PvCSP-Like G10@Pbuis4 + GFP::Luc@Pbeef1a_230p) targeting the neutral 230p locus on chromosome 3. The coding sequence and promoter region of the construct were confirmed by sequencing. The pL2163 construct was linearized by SacII restriction digestion and introduced into parasites of the GIMO motherline 1596cl1 using standard methods of GIMO transfection. Transfected parasites were selected in mice through the addition of 5-fluorocytosine (5-FC) to the drinking water, resulting in negative selection of parasites in which the SM in the 230p locus was replaced by the PvCSP-P. vivax-like expression/reporter cassette. The selected chimeric parasites were cloned using the limiting dilution method. Correct integration of the PvCSP-P. vivax-like coding sequence (under control of the Pbuis4 promoter) into the genome of clones of the chimeric line (Pb-PvCSP-like G10, 2700 cl1) was analyzed by performing a diagnostic PCR analysis of gDNA and Southern analysis of pulsed field gel (PFG)-separated chromosomes. |
Additional remarks selection procedure | |
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Other details transgene |
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Promoter |
Gene Model of Parasite |
PBANKA_0501200
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Gene Model P. falciparum ortholog |
Not available
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Gene product | early transcribed membrane protein up-regulated in infective sporozoites |
Gene product: Alternative name | ETRAMP10.3; UIS4 |
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
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Gene product | early transcribed membrane protein up-regulated in infective sporozoites |
Gene product: Alternative name | ETRAMP10.3, UIS4 |
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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