RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4932
Malaria parasiteP. yoelii
Genotype
Transgene
Transgene not Plasmodium: GFPmut2
Promoter: Gene model: PY17X_1004400; Gene model (P.falciparum): PF3D7_0405300; Gene product: liver specific protein 2, putative (LISP2)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p)
Phenotype Gametocyte/Gamete; Liver stage;
Last modified: 1 December 2020, 11:12
  *RMgm-4932
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 33228734
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/ResearcherBowman LM, Lindner SE
Name Group/DepartmentDepartment of Biochemistry and Molecular Biology, The Huck Center for Malaria Research
Name InstitutePennsylvania State University
CityUniversity Park, PA
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4932
Principal namelisp2-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteSome female gametocytes with moderate GFP expression were observed.
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageStrong GFP expression in mid-to-late liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under control of the Lisp2 promoter

Protein (function)

Phenotype
The lisp2 promoter in rodent malaria species is a well-defined, mid-to-late liver stage promoter that produces a protein that is essential for late liver stage parasites. Robust activity in mid-to-late liver stages was detected, but also some female gametocytes with moderate expression were also observed.

Additional information
From the literature, promoters that have been described to be strong and constitutive or stage specific as observed in other Plasmodium species were selected for study. To produce as diverse of a promoter collection as possible, individual promoters with expression profiles that span the complete life cycle of Plasmodium parasites were chosen. The clag-a promoter (PY17X_1402200) has been defined as restricting transcription to the asexual blood stage of the related rodent malaria parasite P. berghei. The dynein heavy chain delta (“dd”, male, PY17X_0418900) and lap40 (also called ccp2, female, PY17X_1323300) promoters have been commonly used in the “820” male/female fluorescent protein reporter line (820cl1m1cl1). The trap promoter (PY17X_1354800) is known to be active throughout sporozoite development and is critical for salivary gland invasion, gliding motility, and infectivity, whereas the uis4 promoter (PY17X_0502200) flips from being weakly active in oocyst sporozoites to becoming one of the strongest promoters in salivary gland sporozoites. Finally, the lisp2 promoter (PY17X_1004400) becomes active in midliver stage and remains so throughout liver stage development [34]. Finally, the bip promoter (PY17X_0822200) was  also selected, as it is known to be a strong and constitutive promoter in eukaryotes due to the integral role that the BiP protein plays in the translocation of nascent, unfolded proteins into the ER. In previous work, a 1.5 kb portion of sequence upstream of the bip coding sequence was used to serve as a promoter for CRISPRbased gene editing. Here, two truncated variants were created in an attempt to produce a strong, minimal constitutive promoter. Promoters tested here typically included 1.6–1.8 kb of sequence upstream of (and including) the start codon, and were designed to place a unique restriction site 3′ of the start codon to avoid effects upon translation that may occur if placed upstream of it. 

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFPmut2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double 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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPrimers were designed to amplify portions of the pybip promoter consisting of either ~ 300 or ~ 500 bp upstream of the translational start site. Alternatively, promoter regions of pyclag-a, pydynein heavy chain delta (pydd), pylap4 (also called pyccp2), pytrap, pyuis4, or pylisp2 were also designed by selecting 1500–1800 bp upstream of their translational start sites. PCR amplicons were produced by Phusion polymerase (NEB), and were gel extracted (QIAquick Gel Extraction Kit, Qiagen, Cat# 28706), precipitated with ethanol, and ligated into pCR-Blunt (Life Technologies). Promoter sequences were verified via Sanger Sequencing (Penn State Sequencing Core), digested with restriction enzymes, and ligated into pSL0489 to replace the P. berghei eef1a (pbeef1a) promoter. This plasmid backbone contains a Green Fluorescence Protein mutant 2 (GFPmut2) cassette for visualization and a human dihydrofolate reductase (HsDHFR) cassette for drug selection. Plasmids were linearized by cutting between the two arms of the p230p targeting sequences.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PY17X_1004400
Gene Model P. falciparum ortholog PF3D7_0405300
Gene productliver specific protein 2, putative
Gene product: Alternative nameLISP2
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY17X_0306600
Gene product6-cysteine protein
Gene product: Alternative nameP230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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