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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
Transgene name | 2 zinc-finger nucleases (ZFNL, ZFNLR) |
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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 | This description below describes vectors for mutants Rmgm-1357, Rmgm-1358 and Rmgm-1359.
All vectors used for this study were derived from Pb237. The following modifications were made. A 565-bp upstream region of ef1α was amplified from P. berghei ANKA WT gDNA with P5/P6 and cloned with AgeI/ApaI to replace the promoter sequence driving the selection marker. The hdhfr gene was amplified from human cDNA with P7/P8 and cloned with AgeI/NheI. Next egfp was amplified with P9/P10 and cloned upstream in frame with hdhfr with AgeI to result in an egfp-hdhfr fusion gene (Pb238). To generate the expression box for the ZFNs, subcloning was performed in pGEM. The promoter of CSP was amplified with P11/P12 and cloned into pGEM. zfnL was amplified with P13/P14 and inserted with KpnI/PshAI, followed by insertion of the 3’ UTR of csp (P15/P16) with PshAI/SwaI. In parallel, the fragments of the 3’ UTR of dhfs, zfnR and TRAP promoter were amplified with P17/P18, P19/20 and P21/P22, respectively, cloned into pGEM with direct ligation, PshAI/KpnI and SwaI/PshAI, and cloned with EcoRV/SwaI into the first pGEM vector. The whole fragment was cloned with NotI/EvoRV into Pb238, resulting in the vector SpZFN. To generate LsZFN, the promoter of lisp2 was amplified with P23/P24, zfnL was amplified with P25/P26, introducing the 2A skip peptide via P26. Both PCR products were fused with overlap extension PCR using P23/P26 and cloned into SpZFN with NotI/PshAI. To generate mgfp for the following vectors, two fragments of egfp were amplified with P27/P28 and P29/P30, fused with overlap extension PCR P27/P30 and cloned into LsZFN with SwaI/PstI. The zfnL gene was codon-modified with the codon usage table from P. berghei (http://www.kazusa.or.jp.codon/) that was applied with OPTIMIZER (http://genomes.urv.es/OPTIMIZER/). The resulting coding sequence was aligned with zfnR and in all identical codons a silent mutation was introduced wherever possible, resulting in an additional 21 bp changed. The resulting sequence, zfnLcm with fused 2A skip peptide, was ordered from GeneArt (Regensburg). The promoter of csp/lisp2 was amplified with P31/P32 and P33/P34, cloned via NotI/HindIII into zfnLcm and together with zfnLcm cloned into LsZFN (mgfp) with NotI PshAI, resulting in Sp2ZFN and Ls2ZFN, respectively. To generate TrapZFN and Uis4ZFN, the respective promoter regions were amplified with P35/P36 and P37/P38, and cloned with NotI/NdeI into Sp2ZFN.
All vectors were linearised with PvuI prior to transfection and integrated into chromosome 12 between bases 846,483 and 847,711 using two homology regions, Chr12a and Chr12b, with lengths of 481 and 431 bp, respectively. |
Additional remarks selection procedure | |
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Other details transgene |
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Promoter |
Gene Model of Parasite |
PBANKA_1003000
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Gene Model P. falciparum ortholog |
PF3D7_0405300
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Gene product | liver specific protein 2, putative | sequestrin | 6-cysteine protein |
Gene product: Alternative name | LISP2 |
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_1340000
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Gene product | dihydrofolate synthase/folylpolyglutamate synthase, putative |
Gene product: Alternative name | PbDHFS-FPGS |
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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