RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4399
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1017000; Gene model (P.falciparum): PF3D7_1429200; Gene product: AP2 domain transcription factor AP2-O3, putative (ApiAP2; AP2-O3)
Name tag: HA
Phenotype Gametocyte/Gamete; Oocyst;
Last modified: 16 December 2017, 22:44
  *RMgm-4399
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29233900
MR4 number
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
The mutant parasite was generated 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
Name of the mutant parasite
RMgm numberRMgm-4399
Principal namePyap2-o3 HA-tagged
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteWe tagged the gene with the sequence encoding 66HA and performed IFA to investigate protein expression in various developmental stages. The protein was expressed in the nuclei of gametocytes (females, not males), zygotes, and oocysts but not in asexual stages and ookinetes
Fertilization and ookineteNot different from wild type
OocystWe tagged the gene with the sequence encoding 66HA and performed IFA to investigate protein expression in various developmental stages. The protein was expressed in the nuclei of gametocytes (females, not males), zygotes, and oocysts but not in asexual stages and ookinetes
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses of 6x-HA tagged version of AP2-O3
The gene has been tagged using CRISPR/cas9 genome editing (see mutant RMgm-1096 for details).

Protein (function)
Most sequence-specific transcription factor families found in other eukaryotes seem to be absent from Plasmodium. Instead, an expansion of a protein family containing one or more apetala2 (AP2) DNA-binding domains was observed across the phylum apicomplexa. In total, 27 members of this family have been found in the human malaria parasite Plasmodium falciparum (although a possible 28th member of the family may be present. In total, 26 of these have syntenic orthologs in rodent malaria species, each with its unique stage-specific expression profile.
The paper presents a systematic knockout (KO) screen targeting the ApiAP2 family in the rodent malaria parasite P.yoelii

Phenotype
We tagged the gene with the sequence encoding 66HA and performed IFA to investigate protein expression in various developmental stages. The protein was expressed in the nuclei of gametocytes (females, not males), zygotes, and oocysts but not in asexual stages and ookinetes

Additional information
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

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'.

Other mutants
see ApiAP2


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1017000
Gene Model P. falciparum ortholog PF3D7_1429200
Gene productAP2 domain transcription factor AP2-O3, putative
Gene product: Alternative nameApiAP2; AP2-O3
Details of the genetic modification
Name of the tagHA
Details of tagging6xHA
Additional remarks: taggingTo construct vectors for tagging PyApiAP2 genes with sequences coding for mCherry or 6xHA, we first amplified the C- or N-terminal part (300 to 800 bp) of the coding region as the left or right arm and 400 to 800 bp from the 5= UTR or 3' UTR following the translation stop codon as the right or left arm.
A DNA fragment encoding the mCherry or 6xHA tag was inserted between the left and right arms in frame with the gene of interest. For each gene, one sgRNA was designed to target the site close to the C- or N-terminal part of the coding region.
(see mutant RMgm-1096 for details).
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo construct vectors for tagging PyApiAP2 genes with sequences coding for mCherry or 6xHA, we first amplified the C- or N-terminal part (300 to 800 bp) of the coding region as the left or right arm and 400 to 800 bp from the 5= UTR or 3' UTR following the translation stop codon as the right or left arm.
A DNA fragment encoding the mCherry or 6xHA tag was inserted between the left and right arms in frame with the gene of interest. For each gene, one sgRNA was designed to target the site close to the C- or N-terminal part of the coding region.
(see mutant RMgm-1096 for details).
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