RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-801

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_0714000; Gene model (P.falciparum): PF3D7_0816800; Gene product: meiotic recombination protein DMC1, putative
Phenotype	Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;

Last modified: 10 January 2013, 21:29

*RMgm-801

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 23285059
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	not reported
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	G. Mlambo; I. Coppens; N. Kumar
Name Group/Department	Department of Molecular Microbiology and Immunology
Name Institute	Bloomberg School of Public Health, Johns Hopkins University
City	Baltimore
Country	US
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Name of the mutant parasite
RMgm number	RMgm-801
Principal name	PbDmc1 KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Normal ookinete production in vitro. Light microscope morphology of mature ookinetes normal. Nuclei of mutant ookintes were different from those of wild type ookinetes at the electron microscope level (see 'Additional information phenotype')
Oocyst	Normal ookinete production in vitro. Mutant parasites produced 50-80% less oocysts. Mutant oocysts were smaller compared to wild type oocysts (see also 'Additional information phenotype'). Mutant oocysts produced 30-fold lower midgut sporozoites.
Sporozoite	Mutant parasites produced 50-80% less oocysts. Mutant oocysts were smaller compared to wild type oocysts. Mutant oocysts produced 30-fold lower midgut sporozoites and 20-fold lower numbers of salivary gland sporozoites (day 21 after infection). Mutant sporozoites are not infective to mice.
Liver stage	Mutant oocysts produced 30-fold lower midgut sporozoites and 20-fold lower numbers of salivary gland sporozoites (day 21 after infection). Mutant sporozoites are not infective to mice.
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of meiotic recombination protein DMC1, putative. Protein (function) Dmc1 (disrupted meiotic cDNA) and Rad51 are bacterial RecA homologs and play a role in mitosis and meiosis. Both Dmc1 and Rad51 are structurally similar and are widely conserved among several species. Dmc1 and Rad51 colocalize during meiosis to form nuclear complexes before meiotic chromosome synapses that is essential to stabilize ssDNA and promote recombination. During bacterial recombination, RecA binds to ssDNA to form a nucleoprotein filament. The RecA-ssDNA complex allows the search for homologous sequences and catalyzes exchange of the DNA strand. Rad51 and Dmc1 are important in meiotic recombination and Dmc1 was first described in Saccharomyces cerevisiae when mutants were screened for the ability to form spores. ScDmc1 mutants were deficient in chromosome synapses and arrested during meiotic prophase suggesting that Dmc1 plays a central function during meiotic recombination Phenotype Mutant parasites produced 50-80% less oocysts. Mutant oocysts were smaller compared to wild type oocysts. Mutant oocysts produced 30-fold lower midgut sporozoites and 20-fold lower numbers of salivary gland sporozoites (day 21 after infection). Mutant sporozoites are not infective to mice. Transmission electron microscopy analyses indicate aberrant nuclei of mutant ookinetes and oocysts and aberrant sporozoite formation Additional information Other mutants

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0714000

Gene Model P. falciparum ortholog

PF3D7_0816800

Gene product

meiotic recombination protein DMC1, putative

Gene product: Alternative name

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Partial

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Initially a plasmid construct was designed containing 5' UTR and 3'UTR sequences of PbDmc1 to delete the entire gene, and after at least three failed attempts to generate transformed parasites, the targeting plasmid construct was revised to disrupt the gene by using other 3' and 5' that are located inside the ORF.
The 5' targeting sequence comprised of exons 1 to 3 and at the end of the exon 3, a stop codon (TAA) was added. The 3' targeting sequence comprised of exons 4 to 6.

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	5'-ggtaccCAGCCAGCAAAGTTGCAT-3'
Additional information primer 1	primer 587: 567 bp fragment consisting of the 5' region of PbDmc1, nt position +26 to +593
Sequence Primer 2	5'-aagcttCTACACTTGAGTTTTTCCGCA-3'
Additional information primer 2	primer 588: 567 bp fragment consisting of the 5' region of PbDmc1, nt position +26 to +593
Sequence Primer 3	5'-gaattcCGAGTAGACTTTAGTGGGC-3'
Additional information primer 3	promer 589: 3' targeting sequence 490 bp in size, nt position +980 to nt +1470 (comprising of exons 4 through 6 and the spanning introns)
Sequence Primer 4	5'-gcggccgcCGGGAAGGTTTGGAG-3'
Additional information primer 4	promer 590: 3' targeting sequence 490 bp in size, nt position +980 to nt +1470 (comprising of exons 4 through 6 and the spanning introns)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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