SummaryRMgm-4372
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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 | ≥ 5 |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 29233900 |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone | Not applicable |
Other information parent line | |
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Attempts to generate the mutant parasite were performed by | |
Name PI/Researcher | Zhang C; Yuan J |
Name Group/Department | State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, Schoo |
Name Institute | Xiamen University |
City | Xiamen, Fujian |
Country | China |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_0934300 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1115500 | ||||||||||||||||||||||||
Gene product | AP2 domain transcription factor, putative | ||||||||||||||||||||||||
Gene product: Alternative name | ApiAP2 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||
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 | Partial | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
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 | The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth. See also P. berghei mutant RMgm-2638 and RMgm-5582 for successful knocking out PF3D7_1115500 and P. berghei/P. yoelii mutants RMgm-4100 and RMgm-4372 for unsuccessful attempts to knockout this gene. The gene has been tried to disrupt using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095). To construct the vectors to disrupt the PyApiAP2 genes, we first amplified the 5'- and 3'-flanking genomic regions (400 to 700 bp) as left and right homologous arms. The left and right arms were inserted into the restriction sites (HindIII/KpnI and NcoI for the left arm and XhoI and AflII/EcoRI for the right arm) in the pYC plasmid (RMgm-1095). Sequences for single guide RNAs (sgRNAs) were similarly annealed and ligated into the pYC plasmid. From the paper: 'To investigate the functions of the PyApiAP2 gene family in parasite development, we attempted to disrupt 24 of the 26 PyApiAP2 genes in the parasite genome, excluding the orthologs of Pbap2-sp and Pbap2-l, whose functions were described when the project was initiated. We were able to knock out 12 of the 24 genes, including three PyApiAP2 genes (PY17X_1317000, PY17X_1417400, and PY17X_0523100) whose orthologs in P. berghei were either resistant to disruption or not attempted. We carefully evaluated the morphologies of asexual/sexual stages in ICR mice and sexual stages in A. stephensi mosquitoes for the 12 gene KO mutants. We also successfully tagged six PyApiAP2 proteins (PyAP2-G, PyAP2-G3, PyAP2-O2, PyAP2-O3, PyAP2-O4, and PyAP2-O5) with 6XHA tags and PyAP2-O with mCherry to investigate protein expression and localization in the parasites. There were 12 PyApiAP2 genes (PY17X_0104500, PY17X_1231600, PY17X_1209100, PY17X_0941600, PY17X_0911000, PY17X_1361700, PY17X_1456200, PY17X_0111100, PY17X_0113700, PY17X_0838600, PY17X_0934300, and PY17X_1405400) that could not be disrupted even after 4 to 12 independent transfections and selections. The orthologs of these 12 genes in P. berghei also resisted disruption attempts and are likely to be essential for parasite viability or affect the growth of these rodent parasites in the mouse'. | ||||||||||||||||||||||||
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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