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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
Transgene name | FRB::mCherry::TM |
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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 | pyrimethamine |
Selection (negative) procedure | 5-fluorocytosine (5-FC) |
Additional remarks genetic modification | Two parental lines for KS were generated to express a high level of FRB anchor protein localised to a defined, but accessible, subcellular localisation in P. berghei. The FRB domain was fused to a transmembrane (TM) protein (PBANKA_110790), which is the P. berghei homologue of triose phosphate transporter protein PfoTPT, an apicoplast outer membrane protein predicted to have 10 TM domains with both N- and C-termini cytoplasmically exposed. mCherry was also included generating the fusion protein FRB::mCherry::TM.
High expression was achieved using 1.7 kbp of HSP70 promoter (PBANKA_0711190) and the construct was integrated into the 230p locus to generate the KS parental line. Two similar parental lines were generated, KnockSideways Parental 1 (KSP1), in which the FRB::mCherry::TM protein regulated with a P45/48 stable 3’ UTR, and KSP2 with the FRB::mCherry::TM regulated by a P28 3’ UTR (RMgm-4142). KSP2 gave approximately 50% reduced expression of mCherry signal, but resulted better conversion rate to ookinetes.
Plasmids used to generate the KS parental lines were created by modification of plasmid pG073 (Sinha et al., 2014) by addition of the FRB rapamycin dependent heterodimerisation partner domain and MCherry (red fluorescence; Clontech) domains fused to PbOTPT (PBANKA_110790; transmembrane domain protein) downstream of 1.6 kbp of HSP70 (PBANKA_071190) 5’ promoter/intergenic region to generate plasmid pG0089, which was used to generate the Knock-Sideways Parental line 1 (KSP1). This parasite line was negatively selected to generate line KSP1-M0 and cloned using conventional single cell cloning to generate line KSP1-m0cl2, which was used as a parental line for further transfections (referred to as KSP1 in the text). The plasmid pG0089 was then modified by replacement of the 3’ UTR region. The first ~500 bp of the p45/48 3’ UTR was excised with SmaI/AfeI and replaced with 492 bp of the translationally repressive 3’ UTR from P28 (PBANKA_051490) to generate plasmid PG0089-P28. This plasmid was digested with SacII prior to transfection into a WT P. berghei line to generate the parental parasite line KSP2. KSP2 was cloned to generate KSP2cl2, negatively selected to generate KSP2Cl2M1 and cloned again to generate KSP2cl2m1cl3 which was used as a parental line for further transfections (referred to as KSP2). |
Additional remarks selection procedure | |
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Other details transgene |
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Promoter |
Gene Model of Parasite |
PBANKA_0711900
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Gene Model P. falciparum ortholog |
PF3D7_0818900
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Gene product | heat shock protein 70 |
Gene product: Alternative name | HSP70 |
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_0514900
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Gene product | 28 kDa ookinete surface protein |
Gene product: Alternative name | Pbs21; P28 |
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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