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| Type and details of transgene |
| Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
| Transgene name | puromycin-N-acetyltransferase (pac) |
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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 | puromycin-N-acetyltransferase (pac) |
| Promoter of the selectable marker | hsp70 |
| Selection (positive) procedure | puromycin in vitro |
| Selection (negative) procedure | No |
| Additional remarks genetic modification | The pac expression cassette (containing the pac gene under the 5'- and 3'-regulatory sequences of HSP70) was introduced in a locus of mutant RMgmDB-166 which contains a locus containing GFP under conontrol of the the 5'- and 3'-regulatory sequences of HSP70. Selection in vitro with puromycin selects for parasites in which the GFP expression cassette is replaced with the pac expression cassette by double cross-over integration (and loss of GFP expression) |
| Additional remarks selection procedure | In the first selection of double crossover homologous recombination experiments, 200 μl of infected blood was collected in 5 ml of culture medium by heart puncture under anesthesia, using a syringe containing heparin solution. After centrifugation at 500 × g for 8 min at RT, the supernatant was discarded. The blood was resuspended in 10.8 ml of culture medium. The suspension was put into a 25 cm2 flask (Thermo Fisher Scientific) with 1.2 ml of puromycin (10 μg/ml, diluted by culture medium) solution to make a final concentration of 1.0 μg/ml and a total volume of 12 ml. This suspension was then incubated for 20 h. After drug selection, the sample was centrifuged at 500 × g for 8 min at RT and resuspended in 100 μl of PBS. The suspension was injected intravenously into a naive ICR mouse. When parasitemia reached 0.5–3.0% (about 5 days post-injection), the selection procedure was repeated. In the 2nd and 3rd selection, about 7 μl of infected tail blood were collected in 0.5 ml of culture medium (described above) with 2 μl of heparin solution (143 units/ml), and centrifuged at 500 × g for 5 min at room temperature (RT). The supernatant was discarded and the parasites were resuspended in 0.5 ml of culture medium. The suspension (450 μl) was put into a 24-well plate (Thermo Fisher Scientific), and either 50 μl of either puromycin solution (10 μg/ml, diluted by culture medium; Wako, Osaka, Japan; stock: 50 mg/ml in distilled water) to a final concentration of 1.0 μg/ml (1.8 μM). The parasites were incubated for 20 h (36.5 °C, 5% CO2, 5% O2, 90% N2). After incubation, the parasites were separated by centrifuge at 500 × g for 5 min at RT, and resuspended in 100 μl of PBS. They were then injected intravenously into a naive ICR mouse.
Three selections were performed after transfection: the first after 2 days, the second after 8 days, and the third after 12 days. After the second selection, more than 99% of parasites were GFP-negative. The mutant ratio increased significantly from first to second selections, but there was no significant ifference between second and third selections. A typical transfectant line was also analyzed using flow cytometry. More than 99% of parasites were GFP- negative after the second selection |
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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_0711900
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| Gene product | heat shock protein 70 |
| Gene product: Alternative name | HSP70 |
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 | Insertion locus |
| Gene Model of Parasite |
PBANKA_0719300
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| Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative |
| Gene product: Alternative name | dhfr/ts |
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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*RMgm-4180