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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_1359600
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| Gene Model P. falciparum ortholog |
PF3D7_1346700
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| Gene product | 6-cysteine protein | transmission blocking target antigen precursor |
| Gene product: Alternative name | P48/45 |
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| Details of the genetic modification |
| Short description of the mutation | P. berghei P48/45 replaced by P48/45 of P. vivax |
| Inducable system used | No |
| Short description of the conditional mutagenesis | Not available |
| 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 (see RMgm-4520).
The complete sequence of pvs48/45 from P. vivax Sal-I genomic DNA extracted from dried blood spots on filter paper (BEI Resources, Inc.) was amplified using primers 866/887 and a QIAamp DNA Blood Minikit (Qiagen) and was inserted between the SacII and BglII restriction sites in the KO plasmid. The resulting plasmid was used to obtain transgenic P. berghei expressing full-length Pvs48/45 (Tr-F) instead of endogenous Pbs48/45.
To generate the chimeric sequence of pvs48/45 flanked by pbs48/45 signal and anchor sequences, the truncated pvs48/45 fragment without N-terminal signal and C-terminal anchor sequences was amplified using primers 872/874. The 110-bp signal sequence of pbs48/45 was created by PCR using primers 874/892, and the resulting sequence contained 15 bp overlapping the 5' end of the truncated pvs48/45 fragment. A synthetic sequence (60 bp) representing the pbs48/45 anchor region (5' ttatagccacataaaaaataagggaataaatataataaacaaCTTGGCGAGGAAGCCAAA 3') was synthesized by Genescript, Inc. (NJ, USA). The synthetic anchor sequence contained an 18-bp overlap (uppercase in the sequence) with the 3= end of truncated pvs48/45 fragment. The truncated pvs48/45 fragment, secretory signal sequence, and anchor sequence were all used as the templates in PCR amplification using primers 874/889 to obtain the chimeric pvs48/45 sequence containing the internal pvs48/45 sequence flanked by the pbs48/45 signal and anchor sequences, and the PCR fragment was inserted into the KO plasmid using SacII and BglII restriction sites to generate the plasmid used to produce transgenic P. berghei expressing chimeric Pvs48/45 (Tr-C). |
| Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences  Primer information: 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 | |
| Sequence Primer 5 | |
| Additional information primer 5 | |
| Sequence Primer 6 | |
| Additional information primer 6 | |
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*RMgm-4521