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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
Transgene name | GFP |
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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 | Plasmid pL0006 (MRA-775; BEI Resources, Inc.) was modified by replacing pbef 5' UTR-hdhfr with a cassette consisting of pbef 5' UTR-hdhfr-gfp. Using pL0006 as the template, pbef 5' UTR and hdhfr regions were amplified using primers 858/859 and 860/861, respectively. PCR-amplified pbef 5' UTR was cloned by TA cloning in pCR2.1-TOPO vector (Invitrogen), followed by insertion of PCR-amplified hdhfr into the MluI and SalI sites. Next, the PCR-amplified gfp gene (amplified using primers 862/863 from plasmid pL0021 [MRA-790; BEI Resources, Inc.]) was cloned into pCR2.1-TOPO vector downstream of hdhfr using SalI and BamHI sites. Forward primer 862, used for amplification, also contained 5 codons to insert a linker of five alanine residues to separate hDHFR and GFP polypeptide domains within the fusion protein. The gene cassette containing pbef 5' UTR and a fused hdhfr-gfp gene in the pCR2.1-TOPO vector was used to replace the fragment of pbef 5' UTR-hdhfr in pL0006 using PstI and BamHI to obtain plasmid pL0006-gfp.
To generate the knockout (KO) plasmid for homologous recombination by double crossover, pbs48/45 5' UTR and pbs48/45 3= UTR fragments were obtained by PCR amplification using genomic DNA as the template and primers 864/865 and 870/886, respectively. The pbs48/45 5' UTR fragment was inserted into plasmid pL0006-gfp using ApaI and SacII sites (Fig. S1). Likewise, the pbs48/45 3' UTR fragment was inserted in the Xhol and NotI sites. In addition, the pbdhfr-ts 3' UTR (pbdt 3' UTR) gene was amplified from plasmid pL0006 using primers 884/885 and inserted between the BglII and PstI restriction sites to obtain the KO plasmid for pbs48/45 knockout in P. berghei parasites. |
Additional remarks selection procedure | |
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Other details transgene |
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Promoter |
Gene Model of Parasite |
PBANKA_1133300
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Gene Model P. falciparum ortholog |
PF3D7_1357100
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Gene product | elongation factor 1-alpha |
Gene product: Alternative name | eef1a |
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 |
PBANKA_1359600
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Gene product | 6-cysteine protein | transmission blocking target antigen precursor |
Gene product: Alternative name | P48/45 |
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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