RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4929
Malaria parasiteP. yoelii
Genotype
Transgene
Transgene not Plasmodium: GFPmut2
Promoter: Gene model: PY17X_1323300; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (CCp2, LAP4)
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 Asexual bloodstage; Gametocyte/Gamete;
Last modified: 30 November 2020, 17:44
  *RMgm-4929
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-4929
Principal namelap4-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageLow level of GFP expression in blood stages (see below)
Gametocyte/GameteGFP expression in female gametocytes (see below)
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under control of the lap4 promoter

Protein (function)

Phenotype
The dynein heavy chain delta (dd) and lap4 promoters have been used for male-specific or female-specific expression respectively, such as in the 820 line of P. berghei. In agreement with this, it was observed here that the strongest expression does occur in these stages, but that some low-level expression is also present in asexual blood stages

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_1323300
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameCCp2, LAP4
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