RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5365
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1322200; Gene model (P.falciparum): PF3D7_1458500; Gene product: spindle assembly abnormal protein 4, putative (SAS4)
Name tag: GFP
TaggedGene model (rodent): PBANKA_0202700; Gene model (P.falciparum): PF3D7_0111000; Gene product: kinesin-8B, putative
Name tag: mCherry
Phenotype Gametocyte/Gamete;
Last modified: 14 December 2023, 23:12
  *RMgm-5365
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 38092766
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherHair M, Tewari R, Vaughan S
Name Group/DepartmentDepartment of Biological and Medical Sciences
Name InstituteOxford Brookes University
CityOxford
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5365
Principal nameSAS4-GFP/Kinesin-8B-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteFrom the paper:
A hallmark stage post-activation of gametocytes is the assembly of axonemes which are internally coiled around the nucleus and can clearly be seen at ~3-4 mins after activation using live cell imaging microscopy and basal body marker SAS-GFP with an axoneme marker Kinesin8B-mcherry.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
Parasites expressing GFP-tagged SAS4 (RMgm-5132) were crossed with parasites expressing mCherry-tagged kinesin-8B (RMgm-4844). After crossing gametocytes were analysed that express both SAS4-GFP and kinesin-8B-mCherry (SAS4-GFP/kinesin-8B-mCherry).

Protein (function)
SAS4 is a marker of the basal body (BB). SAS4/SAS6 is an ancestral core proteins involved in BB biogenesis.

kinesin-8B: Kinesins are microtubule (MT)-based motor proteins that use energy from the hydrolysis of ATP and function in various cellular processes including intracellular transport, mitotic spindle formation and chromosome segregation during cell division, and the organisation of cell polarity and cytoskeleton associated with motility.
There are 14-16 kinesin subfamilies identified in eukaryotes, and categorised based on analysis of the motor domain, with similar biological roles established by in vitro studies, and in vivo phenotypes in these organisms . Kinesin subfamilies that regulate MT dynamics, such as kinesin-8 and -13, are found in most eukaryotes including primitive and evolutionarily divergent eukaryotes. 
In a bioinformatic analysis of kinesins in Apicomplexa, nine kinesins encoded in the Plasmodium berghei genome were identified, with members of three conserved kinesin subfamilies (kinesin-5, -8B, -8X and -13); kinesin-4, -15 and -20; and two Apicomplexa-specific kinesins: kinesin-X3 and -X4 (Zeeshan et al., 2019).

Phenotype
From the paper
A hallmark stage post-activation of gametocytes is the assembly of axonemes which are internally coiled around the nucleus and can clearly be seen at ~3-4 mins after activation using live cell imaging microscopy and basal body marker SAS-GFP with an axoneme marker Kinesin8B-mcherry.

Additional information
We analysed the positioning of the 8 NPs within the nuclear envelope. As the cell undergoes three rapid rounds of DNA replication, spindle microtubules nucleate from each NP and chromosome segregate as observed with kinetochore marker NDC80. The basal body marker SAS4-GFP is found associated with the kinetochore marker NDC80-Cherry even at 8 minutes after activation when nuclear content is octoploid as observed by live cell imaging. (see RMgm-5364).

From the Abstract:
Upon activation, the male P. berghei gametocyte undergoes three rounds of inter- nuclear mitosis and assembles eight basal bodies and axonemes around the nucleus prior to ex-flagellation, resulting in 8 flagellated male gametes in 12-15 minutes. In this study we used serial block face scanning electron microscopy (SBF-SEM) and cellular electron tomography (ssET) of P. berghei microgametocytes to examine the 3D architecture of key structures during male gamete formation. Our data has revealed an exquisite organisation of axonemes coiling around the nucleus in opposite directions forming a central ‘axoneme band’ in microgametocytes. Furthermore, we discovered that the nucleus of microgametes is tightly coiled around the axoneme in an exquisitely complex structure whose formation starts before microgamete emergence during ex-flagellation.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1322200
Gene Model P. falciparum ortholog PF3D7_1458500
Gene productspindle assembly abnormal protein 4, putative
Gene product: Alternative nameSAS4
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
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 parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe C-terminus of SAS4 was tagged with GFP by single crossover homologous recombination in the parasite. To generate the SAS4-GFP line, a region of the sas4 gene downstream of the ATG start codon was amplified using primers T2011 and T2012, ligated to p277 vector.
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0202700
Gene Model P. falciparum ortholog PF3D7_0111000
Gene productkinesin-8B, putative
Gene product: Alternative name
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
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 modificationThe C-terminus was tagged with mCherry sequence by single crossover homologous recombination at the 3’end of the gene. To generate the tagged line, a region of these genes downstream of the ATG start codon was amplified, ligated to p277 vector, and transfected as described previously (Guttery et al., 2012). The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine.
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