top of page |
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
|
Gene product | circumsporozoite (CS) protein |
Gene product: Alternative name | CSP |
top of page |
Details of the genetic modification |
Inducable system used | No |
Additional remarks inducable system |
|
Type of plasmid/construct | (Linear) plasmid double cross-over |
PlasmoGEM (Sanger) construct/vector used | No |
Modified PlasmoGEM construct/vector used | No
|
Plasmid/construct map |
|
Plasmid/construct sequence |
|
Restriction sites to linearize plasmid |
|
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 | A transgenic Pb parasite line containing a Pfcs expression cassette in the neutral 230p locus was generated using the ‘gene insertion/marker out’ (GIMO) technology as previously described. The Pfcs expression cassette was introduced into the neutral 230p locus of the GIMO mother line 1596cl1, using construct pL1988. The pL1988 construct contains the Pfcs coding sequence (CDS) under the control of the Pbuis4 5′ and 3′ UTR regulatory sequences flanked by the 5′ and 3′ targeting sequences for the 230p locus. This construct integrates by double crossover homologous recombination and replaces the positive–negative selectable marker (SM) (human dihydrofolate reductase:: yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu)) cassette in the GIMO mother line 1596cl1 with the Pfcs expression cassette. The expression cassette contains the Pfcs CDS, which was amplified by PCR from Pf NF54 genomic DNA.
The CDS is flanked by the 5′ and 3′ promoter and transcription terminator sequences of Pb UIS4, which were amplified from Pb ANKA WT genomic DNA. The coding sequence of the Pfcs gene was confirmed by sequencing. The construct pL1988 was linearized by digestion with by SacII before introduction into parasites of the GIMO mother line 1596cl1 using standard methods of GIMO transfection.
Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection results in selection of chimeric parasites where the hdhfr::yfcu SM in the 230p locus is replaced by the Pfcs expression cassette. |
Additional remarks selection procedure | The construct containing the PfCSP expression cassette is integrated into the p230p locus and thereby removing the yfcu::hdhfr (negative) selectable marker cassette |
top of page |
Other details transgene |
top of page |
Promoter |
Gene Model of Parasite |
PBANKA_0501200
|
Gene Model P. falciparum ortholog |
PF3D7_1016900
|
Gene product | early transcribed membrane protein 10.3 | protein of early gametocyte 4 |
Gene product: Alternative name | UIS4; 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 | |
|
|
top of page |
3'-UTR |
Gene Model of Parasite |
PBANKA_0501200
|
Gene product | early transcribed membrane protein 10.3 | protein of early gametocyte 4 |
Gene product: Alternative name | UIS4; 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 | |
|
|
Insertion/Replacement locus |
Replacement / Insertion | Replacement locus |
Gene Model of Parasite |
PBANKA_0306000
|
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 | |
|
|
top of page |