Summary

RMgm-4382
Malaria parasiteP. yoelii
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PY17X_1334500; Gene model (P.falciparum): PF3D7_1466400; Gene product: AP2 domain transcription factor AP2-EXP; AP2 domain transcription factor AP2-SP, putative (ApiAP2; AP2-EXP; AP2-SP)
PhenotypeNo phenotype has been described
Last modified: 15 December 2017, 13:12
  *RMgm-4382
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherZhang C; Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, Schoo
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1334500
Gene Model P. falciparum ortholog PF3D7_1466400
Gene productAP2 domain transcription factor AP2-EXP; AP2 domain transcription factor AP2-SP, putative
Gene product: Alternative nameApiAP2; AP2-EXP; AP2-SP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth (in P. berghei this gene has been successfully deleted).

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  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
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