RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1345
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS, CSP)
Details mutation: deletion of entire N terminus excluding the signal sequence (including region I)
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4, up-regulated in infective sporozoites; ETRAMP10.3)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (UIS4, up-regulated in infective sporozoites; ETRAMP10.3)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 25 October 2015, 14:22
  *RMgm-1345
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26271010
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-1339
Other information parent lineA P.berghei ANKA transgenic reporter line expressing mCherry under control of the (sporozoite and liver-specific) UIS4 promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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The mutant parasite was generated by
Name PI/ResearcherHopp CS; Sinnis P
Name Group/DepartmentDepartment of Molecular Microbiology and Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-1345
Principal nameCSΔN; ∆Nfull
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystMutant oocysts produced between 50 and 100% more sporozoites per oocyst compared with wild type oocysts.
SporozoiteMutant oocysts produced between 50 and 100% more sporozoites per oocyst compared with wild type oocysts. However, salivary glands contained 10-fold lower numbers of salivary gland sporozoites compared to wild type.
Mutant salivary gland sporozoites showed a higher invasion efficiency of hepatocytes in vitro compared with wild type. Mutant salivary gland sporozoites showed a higher infectivity in vivo after intravenous inoculation. After intradermal inoculation, most sporozoites did not reach the liver but were trapped in the skin.
Liver stageMutant salivary gland sporozoites showed a higher invasion efficiency of hepatocytes in vitro compared with wild type. Mutant salivary gland sporozoites showed a higher infectivity in vivo after intravenous inoculation. After intradermal inoculation, most sporozoites did not reach the liver but were trapped in the skin.
Additional remarks phenotype

Mutant/mutation
In the mutant the wild type csp is replaced with a mutated csp containing a specific deletion of entire N terminus, (including region I) and  excluding the signal sequence (amino acids NKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYNRNTVNRLLADAPEGKKNEKKNEKIERNNKLKQP)
This mutant is a similar mutant as mutant RMgm-609. The difference with RMgm-609 is the reporter protein expressed (mCherry).

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCS, CSP
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Details of the genetic modification
Short description of the mutationdeletion of entire N terminus excluding the signal sequence (including region I)
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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn the mutant the wild type csp is replaced with a mutated csp containing a specific deletion of entire N terminus, (including region I) and excluding the signal sequence (amino acids NKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYNRNTVNRLLADAPEGKKNEKKNEKIERNNKLKQP.

The mutant CSP gene was generated using a PCR-based approach. Two gene fragments flanking the region to be deleted and including engineered or endogenous restriction sites were amplified and cut to yield fragments that when ligated make a CSP mutant containing the desired deletion.

For the ∆Nfull mutant, the amino acids NKSIQAQRNLNELCYNEGNDNKLYHVLNSKNGKIYNRNTVNRLLADAPEGKKNEKKNEKIERNNKLKQP, which encompass the entire N terminus excluding the signal sequence, were deleted as follows: a 5’ 722-bp fragment was amplified using forward primer P5 (5’-AAAAAAGGTACCAAATATTATATGC-3’; existing KpnI site underlined) and reverse primer P6 (5’-AGAGCAGCTCGCCATATCCTGGAAGTAGAG-3’; introduced PvuII site underlined). Next, a 776-bp 3’ CSP fragment was amplified using forward primer P3 (5’- AGCGTAATAATAAATTGAAACAAAGGCCTCCACCACCAAACCC- 3’; introduced StuI site underlined) and reverse primer P4 (5’- TTATTTAATTAAAGAATACTAATAC-3’; existing PacI site underlined). Both PCR products were gel purified, digested SspI and StuI (resp.) to remove region 1, and ligated. The correct PmlI-PacI ligation product was determined by pcr and subsequently sub-cloned, sequenced and cloned into the pCSComp transfection plasmid.

To generate the final pCSRep targeting construct that would replace the endogenous CSP locus, a CSP 5’UTR targeting region was cloned upstream of the selection cassette in pCSComp. The 5’UTR targeting region was obtained from p9.5∆E by PCR using p9.5∆E specific primers (see article).
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 1AAAAAAGGTACCAAATATTATATGC
Additional information primer 1P5 (KpnI); 5'CSP forward
Sequence Primer 2AGAGCAGCTCGCCATATCCTGGAAGTAGAG
Additional information primer 2P6 (PvuII); 5'CSP reverse
Sequence Primer 3GAGCGTAATAATAAATTGAAACAAAGGCCTCCACCACCAAACCC
Additional information primer 3P3 (StuI); 3'CSP ORF forward
Sequence Primer 4GTTTATTTAATTAAAGAATACTAATAC
Additional information primer 4P4 (PacI); 3'CSP ORF reverse
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
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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 procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites; ETRAMP10.3
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
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3'-UTR
Gene Model of Parasite PBANKA_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites; ETRAMP10.3
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
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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