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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: Plasmodium |
Gene Model of Parasite |
PF3D7_1335900
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Gene Model P. falciparum ortholog |
PF3D7_1335900
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Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 |
Gene product: Alternative name | TRAP; SSP2 |
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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 | Generation of Pb-Pf double chimeric parasites expressing both PfUIS3 and PfTRAP. To generate the Double Additional Gene (DAGs) chimeric parasite PbANKA-PfTRAP+PfUIS3@Pbuis4 DAG (line 2395cl1) we used the previously generated single additional gene (SAG) PbANKA-PfTRAPPbuis4 line (line 2281cl1) as the background parent line. This line expresses the PfTRAP (PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the constitutive Pbeef1a promoter. This transgenic parasite is selectable marker (SM) free. Both the trap and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. In this line the PfUIS3 CDS (PfUIS3; PF3D7_1302200) was stably integrated into a neutral s1 gene locus (Pbs1; PBANKA_120680), through double cross-over recombination using a 2-step GIMO transfection protocol. In the first step we deleted the Pbs1 CDS and replaced it with the positive negative selectable marker, to create a Pbs1 deletion GIMO line (PbANKA-PfTRAP+PbΔs1 GIMO; line 2353cl2). In order to do this we generated the pL1928 construct that is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative (hdhfr::yfcu) SM cassette, and was used to insert both the Pbs1 5’ and 3’ gene targeting regions (TR). The linear pL1928 DNA construct was introduced into PbANKA-PfTRAPPbuis4 parasite (2281cl1) using standard methods of transfection. Transfected parasites were selected in mice through addition of pyrimethamine in the drinking water. Transfected parasites were cloned by limiting dilution, resulting in the PbANKA-PfTRAP+PbΔs1 GIMO; line 2353cl2. Correct deletion of the Pbs1 CDS and its replacement with the SM cassette were confirmed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel separated chromosomes as described.
In the second step we replaced the positive-negative SM in the genome of the PbANKA PfTRAP+PbΔs1 line (line 2353cl2) with the PfUIS3 CDS by GIMO transfection to create the Pb DAGs chimeric line expressing PfTRAP and PfUIS3. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with PfUIS3 CDS expression cassette, generating plasmid pL2043. The expression cassette in pL2043 contained the PfUIS3 CDS flanked by the 5’ and 3’ regulatory sequences of Pbuis4, which were amplified from Pb ANKA WT genomic DNA. The PfUIS3 CDS was amplified from genomic DNA of the NF54 strain of Pf. The pL2043 construct was sequenced to ensure there were no mutations in the PfUIS3 CDS. The construct was linearized using HindIII restriction enzymes outside of the 5’ and 3’ Pbs1 TRs before transfection. The construct was used to transfect parasites of the PbANKA-PfTRAP+PbΔs1 GIMO (line 2353cl2) 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. This resulted in selection of chimeric parasites where the hdhfr::yfcu SM in the Pbs1 locus of PbANKA-PfTRAP+PbΔs1 GIMO line was replaced by the PfUIS3 CDS expression cassette. Selected chimeric parasites were cloned by limiting dilution. |
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
|
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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