RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4934
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0800500; Gene model (P.falciparum): PF3D7_1252200; Gene product: chitinase (CHT1)
Details mutation: The P. berghei cht1 gene replaced by the cht1 gene of P. falciparum
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene not Plasmodium: Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 25 January 2021, 14:10
  *RMgm-4934
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 33489941
MR4 number
top of page
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4933
Other information parent lineIn this CHT1 GIMO line (3152cl1; RMgm-4933) the endogenous P. berghei cht1 gene has been deleted by replacing the cht1 gene with the drug-selectable marker cassette hdhfr-yfcu. It also expresses mCherry and luciferase under constitutive promoters
top of page
The mutant parasite was generated by
Name PI/ResearcherPatra KP, Kaur H, Kolli SK, Janse CJ, Vinetz JM
Name Group/DepartmentSection of Infectious Diseases, Department of Internal Medicine
Name InstituteYale School of Medicine
CityNew 7 Haven, Connecticut
CountryUSA
top of page
Name of the mutant parasite
RMgm numberRMgm-4934
Principal name3165cl1
Alternative namePb-PfCHT1(r)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
top of page
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the mutant the P. berghei cht1 gene has been replaced by the cht1 gene of P. falciparum (using GIMO transfection using the CHT1 GIMO line 3152cl1; RMgm-4933). It also expresses mCherry and luciferase under constitutive promoters

Protein (function)
CHT1, a secreted protein, is expressed in the ookinete stage and located in the micronemes. It contains putative proenzyme and chitin-binding domains. Chitinases are enzymes that hydrolize chitin, a (1-4)-β homopolymer of N-acetylglucosamine.

The Plasmodium ookinete secreted chitinase(s) are endochitinases of family 18 glycohydrolases that enable the parasite to dissolve the chitin (N-acetylglucosamine polymer)-containing peritrophic matrix (PM) within the mosquito midgut, en route crossing the midgut epithelium. Plasmodium ookinete-secreted chitinases have been classified as short (PfCHT1, PgCHT2 and PrCHT1) and long form (PgCHT1, PvCHT1 and PbCHT1) based on the presence or absence of predicted proenzyme and chitin binding domains (CBD) at the N- and C-terminal of the protein, respectively where short forms do not possess either domain.

Phenotype
The phenotype has not been analysed in detail. Pb-PfCHT1(r) produced ookinete and oocyst numbers that were in the range of wild type parasites.

Additional information
From the Abstract: 
'We show that the chimeric P. berghei ookinetes expressed monomeric PfCHT1 but a high molecular weight complex (HMW) complex containing PfCHT1 was absent.'

Other mutants

  Mutated: Mutant parasite with a mutated gene
top of page
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0800500
Gene Model P. falciparum ortholog PF3D7_1252200
Gene productchitinase
Gene product: Alternative nameCHT1
top of page
Details of the genetic modification
Short description of the mutationThe P. berghei cht1 gene replaced by the cht1 gene of P. falciparum
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationIn the mutant the P. berghei cht1 gene has been replaced by the cht1 gene of P. falciparum (using GIMO transfection using the CHT1 GIMO line 3152cl1; RMgm-4933).

We used a 2-step gene insertion/marker out (GIMO) transfection protocol.
We deleted the Pbcht1 CDS by replacing it with the positive-negative selectable marker (SM) to create a P. berghei cht1 deletion GIMO line (PbΔcht1). To do this, we generated DNA construct pL2321, which is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative human dihydrofolate reductase::yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu) selection marker (SM) cassette. The 5’ and 3’ targeting regions of Pbcht1 were amplified from P. berghei ANKA genomic DNA. Fragments were digested (with ApaI/SacII and KpnI/NotI, respectively) and ligated into vector pL0034 to obtain pL2321. For transfection, pL2321 was linearized with ApaI/NotI and the linear construct was introduced in parasites of the P. berghei ANKA reference line 1868cl1 using standard methods of transfection. Transfected parasites were cloned by limiting dilution, resulting in cloned line PbΔcht1 (line 3152cl1).

In the second step, we replaced the positive-negative SM cassette in PbΔcht1 with the Pfcht1 CDS by GIMO transfection to create the P. berghei chimeric Pb-PfCHT1(r) replacement line. This was achieved by modifying the construct used in the first step (pL2321); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the Pfcht1 CDS sequence, generating plasmid pL2322. The Pfcht1 CDS was amplified from DNA of P. falciparum NF54 (PF3D7_1252200). Pfcht1 CDS fragment was digested with SacII/KpnI and ligated into vector pL2321 to obtain pL2322. The pL2322 construct was sequenced to ensure that there were no mutations in the Pfcht1 CDS. The construct was linearized using ApaI and NotI restriction enzymes. outside the 5’ and 3’ targeting regions before transfection. The construct was used to transfect parasites of PbΔcht1 (line 3152cl1) using standard methods of GIMO transfection. Transfected parasites (exp 3165) were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of the mice. Negative selection results in the selection of chimeric parasites where the hdhfr::yfcu SM in the cht1 locus of PbΔcht1 is replaced by the CDS of Pfcht1. Selected chimeric parasites were cloned by the method of limiting dilution resulting in cloned line 3165cl1 (Pb-PfCHT1(r)).
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
top of page
  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
top of page
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
top of page
Other details transgene
top of page
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
top of page
3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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
top of page
  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
top of page
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
top of page
Other details transgene
top of page
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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
top of page
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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
top of page
Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren