SummaryRMgm-4575
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Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
The following genetic modifications were attempted | Gene disruption |
Number of attempts to introduce the genetic modification | 4 |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30596727 |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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Attempts to generate the mutant parasite were performed by | |
Name PI/Researcher | Kooistra RL, Kanzok SM |
Name Group/Department | Department of Biology |
Name Institute | Loyola University Chicago |
City | Chicago |
Country | USA |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1204800 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1006600 | ||||||||||||||||||||||||
Gene product | phosducin-like protein, putative | ||||||||||||||||||||||||
Gene product: Alternative name | PhLP3 (previous PhLP1) | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) PCR construct double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | Multiple attempts to disrupt PhLP3 were unsuccessful, indicating an essential role during blood stage growth/multiplication. From the paper: 'We previously identified three putative Trx-like genes in the P. berghei genome as members of the phosducin-like family of proteins (PhLP), specifically PBANKA_1204800, PBANKA_0519700, and PBANKA_1231200. PBANKA_1204800 was designated as PhLP-1 as it was the first PhLP we identified in a Plasmodium species. However, re-analysis of sequence data from multiple organisms suggests that PBANKA_1204800 belongs to the PhLP-3 subgroup, while PBANKA_1231200 is more closely related to the PhLP-1 subgroup. PbPhLP-3 shows the highest conservation among eukaryotes. A targeting vector was constructed in plasmid pBS-DHFR. A 516 bp fragment comprising the 5’ flanking sequence and a 348 bp fragment of the 3’ flanking sequence of pbphlp-3 were amplified from P. berghei genomic DNA. The 5’ fragment was inserted using the ApaI and HindIII restriction sites immediately upstream of the tgdhfr/ts cassette while the 3’ fragment was inserted into the downstream BamH1 and XbaI restriction sites. Alignment of recently characterized PhLP-3 homologues, ranging from fungi to mammals, coupled with secondary structure prediction, revealed that PbPhLP-3 consists of an N-terminal helix domain, followed by a thioredoxin-domain and a short C-terminus, an overall organization characteristic for members of the PhLP family.' | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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