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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
Transgene name | tdTomato |
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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 |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | Lines expressing mCherry, GFP or tdTomato in the absence of a selectable marker were created in a two-step process. 5’ and 3’ flanks of the intergenic region of chromosome 6 (see RMgm-757) were generated by PCR using primers D331-D332 and D326-D327, respectively and cloned into the EcoRI/NotI (5’) and ApaI/AflII (3’) sites of pDONR-P2/P3 (Invitrogen) to create the plasmid pINT65’3’P2-P3. The hDHR/yfcu positive-negative selectable marker cassette from pL0034 (MRA-849) was cloned into pINT65’3’P2-P3 using XmaI/PstI. The construct was linearized with NotI/AscI and transfected into Pb ANKA parasites. Transfectants were cloned and correct integration confirmed by PCR.
GFP flanked by the HSP70 5’ and 3’ UTRs in pbc-GFP@hsp70 (a kind gift of Robert Menard, Institute Pasteur) was inserted into the SacI/XmaI sites of pBluescript (Agilent) to create pBSKHG. The mCherry sequence was cut out of pLChSKD with AvrII/BamHI and cloned into the NheI/BglII site of pBSKHG, replacing the GFP to create pBSKHCh. The tdTomato sequence was amplified and cloned into pL0006 as mCherry for pLChSKD, cut with AvrII/BamHI and cloned into the NheI/BglII site of pBSKHG to create pBSKHTm. pBSKHG, pBSKHTm and pBSKHCh were cut with SacI/XmaI to liberate the HSP705’-FP-HSP703’ cassette, which was inserted into pINT65’3’P2-P3 to create pINT65’3’HG, pINT65’3’HTm, and pINT65’3’HCh. These plasmids were linearized and transfected as above, with selection on 5-FC, and cloned. Correct gene insertion was confirmed by PCR. All parasite lines were tested in the complete life-cycle to confirm previous findings that HSP70 5’ driven expression of GFP, tdTomato and mCherry in this locus does not impair parasite development. The fidelity of all PCR amplified products was confirmed by sequencing. |
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 |
Not available
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Gene product | Not available |
Gene product: Alternative name | |
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 |
Not available
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Gene product | Not available |
Gene product: Alternative name | |
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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